Fetal Medicine: Basic Science and Clinical Practice [3rd Edition] 9780702072864, 9780702072871

Table of contents :
Fetal Medicine: BASIC SCIENCE AND CLINICAL PRACTICE......Page 2
Copyright......Page 3
Foreword......Page 4
Preface......Page 5
List of Contributors......Page 6
Origin of the Early Embryological Terms......Page 13
Embryonic Cells in Culture......Page 14
Computing Sciences and Embryological Terminology......Page 15
Conclusion......Page 16
References......Page 17
The Cytoskeleton......Page 18
Epithelia......Page 20
Mesenchymal Cells......Page 22
Embryonic Induction and Cell Division......Page 24
The Cell Cycle......Page 25
Instructive Interactions......Page 26
Neural Ectoderm and Neural Crest Mesenchyme Interactions: The ‘Fly-Paper Model’ of Skull Development......Page 27
Surface Ectoderm and Somatopleuric Mesenchymal Interactions in the Limb......Page 29
Intraembryonic Mesoderm and Intermediate Mesenchyme Interactions......Page 31
Other Cells Types Affecting or Affected by Local Interactions......Page 32
Conclusion......Page 34
References......Page 35
Human Stage Series......Page 36
The Stage 11 Embryo, Body Plan Stage......Page 37
Obstetric Timing and Staging of Embryos and Fetuses......Page 40
Conclusion......Page 42
References......Page 43
Mechanisms of Teratogenicity......Page 44
Underlying Principles......Page 45
Medication......Page 46
Environmental Agents......Page 48
Selected Infections......Page 49
Selected Recreational Exposures......Page 50
Conclusion......Page 51
References......Page 52
Congenital Uterine Anomaly......Page 55
Normal Intrauterine Pregnancy......Page 56
Early Embryonic Demise......Page 57
Ultrasound Features......Page 58
Recurrent Miscarriage......Page 59
Risk Factors for Ectopic Pregnancy......Page 60
Tubal Ectopic Pregnancy......Page 61
Surgical management......Page 62
Conclusion......Page 63
References......Page 64
Nidation......Page 67
Trophoblast Interaction with Extracellular Matrix......Page 68
Matrix Degradation by Trophoblast......Page 69
Leukocyte Populations in Decidua......Page 70
Maternal KIR–Fetal HLA-C Combinations Influence Reproductive Success......Page 71
Conclusion......Page 73
References......Page 74
The Placenta at Delivery......Page 75
Haemochorial Placental Blood Flow......Page 76
Phenotypes of Extravillous Trophoblast......Page 77
Small Spindle-Shaped Extravillous Trophoblast Cells......Page 79
Transformation of the Uteroplacental Arteries......Page 80
Flow of Maternal Blood into the Intervillous Space......Page 82
Architecture of the Villous Trees......Page 83
Villous Development......Page 84
The Placental Barrier......Page 86
Physiology of Fetoplacental Blood Flow......Page 87
Conclusions......Page 88
References......Page 89
The Placental Exchange Barrier......Page 91
Types of Exchange Mechanisms......Page 92
Factors Affecting Maternofetal Exchange......Page 93
Ions......Page 94
Amino Acids......Page 96
Immunoglobulin G......Page 97
Clinical Considerations: Maternofetal Exchange and Fetal Growth Restriction......Page 98
Conclusions......Page 99
References......Page 100
Placental Pathological Assessment......Page 102
Prenatal Assessment of Specific Placental Pathologies......Page 103
Categories of Placental Pathologies......Page 105
Abnormalities of Placental Perfusion......Page 106
Primary Abnormalities of Villous Development......Page 107
Inflammatory Lesions......Page 108
Tumours and Tumourlike Lesions......Page 109
Conclusion......Page 110
References......Page 111
Brief Overview of Heart Development......Page 112
The Heart Fields......Page 113
Contraction......Page 114
Development of the Chambers and Outflows......Page 115
The Interventricular Septum......Page 116
The Atrioventricular Junction......Page 118
Outflow Tract......Page 119
Conduction Tissue......Page 120
Umbilical Veins......Page 121
The Arterial System......Page 122
Conclusion......Page 126
References......Page 127
Pseudoglandular Stage (5–17 Weeks)......Page 128
Structural Development......Page 129
Functional Development of the Pulmonary Circulation......Page 131
Changes at Birth......Page 132
Control of Fetal Lung Liquid Secretion......Page 133
Control of Fetal Lung Liquid Volume......Page 134
Regulation of Fetal Lung Growth......Page 135
Fetal Lung Hypoplasia......Page 136
Epithelial Cell Differentiation......Page 137
Conclusions......Page 138
References......Page 139
Timeline of Kidney Development......Page 141
The Metanephros......Page 142
Final Nephron Number......Page 143
Causes of Human Renal Anomalies......Page 144
Genetic Factors......Page 145
Potential Adverse Effects of Prematurity......Page 146
Conclusions......Page 147
References......Page 148
Introduction......Page 150
Legal Aspects, Aims and Types of Autopsy......Page 151
Internal Examination......Page 152
Retention of Organs......Page 153
Postmortem Imaging......Page 154
Hypoxic Ischaemic Changes......Page 155
Respiratory Anomalies......Page 156
Musculoskeletal Anomalies......Page 157
Postfetal Intervention......Page 158
Conclusion......Page 159
References......Page 160
Randomised Controlled Trials......Page 161
Evaluation of Screening and Diagnostic Tests......Page 162
Analysing Databases and Survey Data......Page 164
Meta-analysis......Page 165
Conclusion......Page 166
References......Page 167
The Fetus as a Patient......Page 168
The McCullough and Chervenak Model......Page 169
Why the McCullough and Chervenak Model Is Flawed......Page 170
Rationale of Offering Fetal Treatment......Page 171
Saving the Fetus at What Price to the Child......Page 172
Giving the Child a Better Life......Page 173
Ethical Challenges of Fetal Therapy Research......Page 174
Reproductive Autonomy and Parental Responsibility......Page 175
Conclusion......Page 176
References......Page 177
Basic Parameters of Diagnostic and Screening Tests......Page 179
Likelihood Ratios......Page 180
Receiver Operator Characteristic Curves......Page 181
Cost Effectiveness of Prenatal Screening......Page 182
Conclusions......Page 183
References......Page 184
Why Good Practice in Information-Giving Is so Important......Page 185
Conveying Information About Risk......Page 186
Noninvasive Prenatal Testing Using Cell-Free DNA......Page 187
Prenatal Diagnosis of Fetal Anomaly......Page 188
Pregnancy Management After Diagnosis......Page 189
Information Needs When Parents Decide to Continue With an Affected Pregnancy......Page 190
Conclusions......Page 191
References......Page 192
Historical Perspective......Page 194
Prevalence According to Gestational Age......Page 195
Principal Down Syndrome Markers......Page 196
Covariables......Page 197
Markers in More Detail......Page 198
For Localities with High-Quality Ultrasound......Page 199
Detection of Trisomy 18 and 13......Page 200
Ultrasound Markers......Page 201
Anomaly Scan Results......Page 202
Renal Transplant......Page 203
Smith-Lemli-Opitz Syndrome......Page 204
Maternal-Fetal Conditions......Page 205
Cost Effectiveness......Page 206
Risk Calculation Software......Page 207
Conclusions......Page 208
References......Page 209
Anatomical Survey at 11 to 13+6 weeks (Fig. 19.1 and Table 19.1)......Page 212
Increased Nuchal Translucency (Fig. 19.4) and Structural Anomalies......Page 214
First Trimester Fetal Brain and Spine Investigation......Page 215
Most Common Brain and Neural Tube Anomalies......Page 216
Midline Defects......Page 219
Early Markers for Congenital Heart Disease......Page 220
Accuracy of Congenital Heart Disease Detection by Early Ultrasound Investigation......Page 221
How to Perform a Complete Early Fetal Echocardiography......Page 222
Conclusions......Page 223
Thorax, Diaphragm, Abdominal Wall and Bowel......Page 224
Abdominal Wall Defects......Page 225
Rare Intraabdominal and Abdominal Wall Anomalies......Page 227
Genetic Syndromes......Page 228
Conclusion......Page 229
References......Page 230
20 - Evidence for Routine Ultrasound Screening for Fetal Abnormalities in the Second and Third Trimesters......Page 233
RADIUS Trial......Page 234
Eurofetus Study......Page 235
Better Estimate of Gestational Age......Page 237
Equipment......Page 238
Resource Utilisation......Page 239
Conclusions......Page 240
References......Page 241
Fetal Cells in the Maternal Circulation......Page 243
Future Directions......Page 245
Cell-Free Fetal DNA in the Maternal Circulation......Page 246
Massively Parallel Sequencing and Next-Generation Sequencing......Page 247
Single Nucleotide Polymorphism......Page 248
Methylation-Based Technology......Page 249
Methods for Detection of Subchromosomal Abnormalities (Microdeletions and Microduplications)......Page 250
False-Negative Noninvasive Prenatal Testing Results......Page 251
Choice of the Confirmatory Diagnostic Procedure......Page 252
Conclusion......Page 254
References......Page 255
Introduction......Page 258
Fetal Sex Determination......Page 262
Noninvasive Prenatal Diagnosis for Monogenic Disorders......Page 263
Noninvasive Prenatal Diagnosis for Autosomal Dominant Conditions and Exclusion of Paternal or De Novo Mutations......Page 264
Noninvasive Prenatal Diagnosis for Autosomal Recessive and Sex-Linked Disorders......Page 265
Ethical and Social Issues......Page 267
Conclusions......Page 268
References......Page 269
Technique of Amniocentesis......Page 271
Fetal Risks of Amniocentesis......Page 272
Pregnancy Losses After Amniocentesis......Page 273
Technique......Page 274
Safety of Chorionic Villus Sampling in Multiple Pregnancies......Page 275
Fetal Blood Sampling......Page 276
Safety of Fetal Blood Sampling......Page 277
Conclusion......Page 278
References......Page 279
Prenatal Specimens......Page 284
Abnormal Screening Result......Page 285
Noninvasive Prenatal Screening Using Cell-Free Fetal DNA......Page 286
Chromosome Rearrangement or Copy Number Variant in a Parent......Page 287
Aneuploidy......Page 289
Long Contiguous Stretches of Homozygosity......Page 290
Karyotype......Page 291
Rapid Aneuploidy Detection......Page 293
Chromosomal Microarray......Page 294
Genotype–Phenotype Correlation......Page 295
Maternal Cell Contamination......Page 296
Concluding Remarks......Page 297
References......Page 298
Introduction......Page 301
What Is Next-Generation Sequencing, and How Does It Work......Page 302
How Are Next-Generation Sequencing Data, Such as Whole-Exome Sequencing Results, Analysed and Interpreted......Page 304
Multigene Panels......Page 305
Pretest Counselling......Page 306
Conclusions......Page 307
References......Page 308
Genetic Screening Using Molecular Techniques......Page 309
Expanded Carrier Screening......Page 311
Next-Generation Sequencing......Page 312
Premarital Carrier Screening Programs......Page 313
Couple Screening......Page 314
Pretest Counselling......Page 315
Posttest Counselling and Management......Page 316
Conclusions......Page 317
References......Page 318
No Functional α-Globin Genes: Hb Bart Disease or Homozygous α0-Thalassemia......Page 320
One Functional α-Globin Gene: Hb H Disease......Page 321
Homozygous β-Thalassemia......Page 322
Haemoglobin Electrophoresis and High-Performance Liquid Chromatography or Capillary Electrophoresis......Page 323
Workup for Screen-Positive Couples......Page 324
Detection of αo- and α+-Thalassemia Deletion......Page 325
Detection of Nondeletion α+-Thalassemia Mutations......Page 326
Ultrasound Exclusion of Homozygous α0-Thalassemia......Page 327
Other Noninvasive Testing for Homozygous α0-Thalassemia......Page 328
Noninvasive Prenatal Diagnosis for Thalassemia......Page 329
Conclusion......Page 330
References......Page 331
Introduction......Page 332
Ventriculomegaly......Page 333
Anomalies Related to Dorsal Induction Failure......Page 335
The classification of spinal dysraphism has been revised recently as shown in Fig. 28.16.41......Page 339
Anomalies of Prosencephalic Development......Page 341
Posterior Fossa Anomalies......Page 346
Disorders of Cortical Development......Page 349
Congenital Infections......Page 352
Destructive Lesions......Page 354
Vascular Malformations......Page 357
Intracranial Masses: Cysts and Tumours......Page 358
Conclusion......Page 361
References......Page 362
Screening for Congenital Heart Disease......Page 367
Prenatal Therapy......Page 368
Lesion with Abnormal Four-Chamber View......Page 369
Lesions Requiring Views of the Outflow Tracts......Page 377
Lesions Difficult to Detect Prenatally......Page 382
Arrhythmia......Page 384
Conclusion......Page 385
References......Page 386
Thoracic Malformations Detected on Prenatal Ultrasound......Page 388
Congenital Pulmonary Airway Malformations......Page 389
Pulmonary Sequestration......Page 390
Pulmonary Hypoplasia and Agenesis......Page 391
Cystic Lung Lesions......Page 392
Cystic Lung Lesions......Page 393
Pulmonary Agenesis and Hypoplasia......Page 394
Conclusion......Page 395
References......Page 396
Aetiology and Pathogenesis......Page 398
Prenatal Diagnosis and Outcome Prediction......Page 399
Antenatal Therapeutic Strategies......Page 402
Experimental Antenatal Treatments......Page 404
Conclusion......Page 405
References......Page 406
Embryologic Development......Page 408
Sonographic Features at 12 Weeks’ Gestation......Page 410
Sonographic Features after 28 Weeks’ Gestation......Page 411
Gastroschisis......Page 412
Exomphalos......Page 413
Bladder and Cloacal Extrophy......Page 414
Dilated Bowel......Page 415
The Liver......Page 416
Intraabdominal Calcification......Page 417
Conclusions......Page 418
References......Page 419
Embryology......Page 421
Normal Sonographic Development of the Fetal Kidneys and Urinary Tract......Page 422
Urinary Tract Anomalies......Page 424
Prenatal Management of Fetal Obstructive Uropathies......Page 428
Renal Abnormalities......Page 434
Anomalies of Position......Page 435
Abnormalities in Renal Size, Structure and Echogenicity......Page 436
Nonhereditary Cystic Kidneys Disease......Page 440
Bladder Malformations......Page 442
References......Page 443
Embryology and Sonographic Appearance of the Normal Fetal Skeleton......Page 445
Maternal Disease......Page 446
Abnormal Findings on Routine Ultrasound......Page 452
Timing of Diagnosis......Page 455
Osteogenesis imperfecta types IIA, IIB and IIC......Page 456
Thanatophoric dysplasia......Page 459
Asphyxiating thoracic dystrophy......Page 460
Ellis-van Creveld syndrome......Page 464
Osteogenesis imperfecta type IV......Page 465
Rhizomelic chondrodysplasia punctata......Page 466
Conradi Hunermann syndrome......Page 467
Other Skeletal Dysplasias Associated With Short, Straight Limbs......Page 468
Acromesomelic dysplasia......Page 469
Kniest dysplasia......Page 470
Limb Deficiency or Congenital Amputations......Page 471
Conclusions......Page 473
References......Page 474
Ultrasound Investigation of the Fetal Face......Page 475
Three-Dimensional Ultrasound of the Fetal Face......Page 476
Cross-Sectional Imaging......Page 477
Rendered Images......Page 478
Facial Clefts......Page 479
Ultrasound Examination of Facial Clefts......Page 480
Ultrasound Examination of Micrognathia......Page 483
Forehead......Page 487
Nose......Page 489
Jaws......Page 490
Mouth......Page 491
Eyes......Page 494
Ears......Page 495
Conclusions......Page 498
References......Page 500
Diagnosis of Hydrops Fetalis by Ultrasound......Page 502
Pathophysiology......Page 503
Cardiovascular Conditions......Page 504
Chromosomal Disorders......Page 505
Haematologic Conditions......Page 507
Lymphatic Dysplasia......Page 508
Metabolic Conditions......Page 509
Clinical Evaluation......Page 510
Fetal Assessment......Page 511
Fetal Therapy......Page 513
Prognosis and Recurrence Risk Counselling......Page 516
Conclusion......Page 517
References......Page 518
Cystic Lymphangioma......Page 520
Antenatal Management......Page 522
Intrapartum Management: The EXIT Procedure......Page 524
Postnatal Management......Page 526
Antenatal Management......Page 527
Conclusion......Page 529
References......Page 531
Lethal Conditions......Page 533
Pre- and Intraoperative Management......Page 535
The Surgical Procedure......Page 536
Postoperative Management......Page 537
Outcomes of Spina Bifida Aperta......Page 542
Outcomes of Congenital Thoracic Malformations......Page 544
Outcomes of Sacrococcygeal Teratoma......Page 545
Conclusions......Page 546
References......Page 547
Epidemiology......Page 549
Classification of Fetal Growth Restriction......Page 550
Fetal Factors......Page 551
Uterine Artery Doppler......Page 552
Ultrasonographic Biometry......Page 553
Ultrasonographic Biometry......Page 554
Confirmation of Gestational Age......Page 556
Ultrasound......Page 557
Potential Interventions......Page 559
Timing of Delivery......Page 560
Summary of Recommendations for Management of Fetal Growth Restriction......Page 561
Counselling......Page 562
Conclusion......Page 563
References......Page 564
Pathogenesis......Page 568
Fetal Response......Page 569
Rh Variants......Page 570
Kell......Page 571
Other Rare Antigens......Page 572
Indirect Coombs Test......Page 574
Fetal Genotype Testing......Page 575
Management of Previously Affected Pregnancies......Page 576
Intrauterine Transfusion......Page 577
Transfusion Interval......Page 578
Conclusion......Page 579
Autoimmune or Idiopathic Thrombocytopenic Purpura......Page 584
Obstetric Management......Page 585
Natural History......Page 586
Pathophysiology......Page 587
Diagnostics......Page 589
Obstetric Management......Page 591
Conclusion......Page 594
e1References......Page 595
Virology......Page 598
Congenital Infection......Page 599
Epidemiology......Page 603
Fetal Infection......Page 604
Management......Page 605
Fetal Infection......Page 606
Prevention......Page 607
Congenital Infection......Page 608
Management Options......Page 609
Maternal Infection......Page 610
Fetal Infection......Page 611
Prevention......Page 612
Maternal Infection......Page 613
Treatment......Page 614
Conclusion......Page 615
References......Page 616
Semiquantitative Ultrasound Assessment of Amniotic Fluid Volume......Page 622
Amniotic Fluid Volume and Perinatal Outcome......Page 623
Amniotic Fluid Assessment in Multiple Pregnancies......Page 624
Oligohydramnios......Page 625
Cause of Polyhydramnios......Page 626
Conclusion......Page 627
References......Page 628
Chorionicity......Page 629
Noninvasive Prenatal Testing Performance in Twin Pregnancies......Page 630
Considerations for Aneuploidy Screening in Twin Pregnancies......Page 631
Invasive Prenatal Diagnosis......Page 632
Disorders of Fetal Growth in Multifetal Gestations......Page 633
Single Intrauterine Fetal Death......Page 635
Twin–Twin Transfusion Syndrome......Page 639
Twin-Reversed Arterial Perfusion......Page 646
Conjoined Twins......Page 647
Monoamniotic Twins......Page 648
Selective Feticide......Page 649
Conclusion......Page 650
References......Page 651
What Can We Learn From Animal Models......Page 657
In Utero Transplantation......Page 658
Drug Development......Page 660
Conclusion......Page 662
References......Page 663
The Potential Advantages and Disadvantages of Fetal Gene Therapy......Page 665
Selecting the Right Disease for Fetal Gene Therapy......Page 667
Practical Considerations for Clinical Fetal Gene Therapy......Page 670
Potential Adverse Consequences of Fetal Gene Therapy......Page 673
Clinical Translation of Fetal Gene Therapy......Page 674
Conclusion......Page 676
Kermack and Forsdahl......Page 679
Mechanisms of the Developmental Origins of Health and Disease......Page 680
Postnatal ‘Catch-up’ Growth......Page 681
The Fetal Insulin Hypothesis......Page 682
Genomic Variation and Epigenetics......Page 683
Infant Nutrition......Page 685
Conclusion......Page 686
References......Page 687
Current Status of Pharmacotherapy in Pregnancy......Page 689
Absorption......Page 690
Distribution......Page 691
Metabolism......Page 692
Placental Transport......Page 693
Functional Impact of Xenobiotics on the Fetus......Page 694
Conclusion......Page 695
References......Page 696
Delivery Room Management......Page 697
Brain Injury in the Preterm Infant......Page 698
Cerebellar Injury......Page 699
Long-Term Follow-Up in in Extreme Preterm Children......Page 702
Neurologic, Motor and Cognitive Outcome......Page 703
Conclusions......Page 704
References......Page 706
Answer 3......Page 707
Answer 1......Page 708
Question 1......Page 709
Answer 2......Page 710
Answer 1......Page 711
Answer 4......Page 712
Answer 1......Page 713
Answer 5......Page 714
Question 1......Page 715
Answer 2......Page 717
Answer 3......Page 718
Answer 4......Page 720
Answer 2......Page 722
Answer 1......Page 723
Answer 1......Page 724
Answer 2......Page 725
Question 2......Page 727
Answer 1......Page 728
Question 3......Page 729
Question 2......Page 730
Question 2......Page 731
Question 3......Page 732
Answer 4......Page 733
Answer 3......Page 734
Answer 4......Page 735
Question 2......Page 736
Answer 1......Page 737
Answer 3......Page 738
Question 3......Page 739
Answer 4......Page 740
Question 4......Page 741
Answer 1......Page 742
Answer 3......Page 743
Answer 4......Page 744
Answer 4......Page 745
Answer 1......Page 746
Answer 2......Page 747
Answer 1......Page 748
Question 4......Page 749
Answer 2......Page 750
Answer 3......Page 751
Answer 3......Page 752

Citation preview

THIRD EDITION

Fetal Medicine

BASIC SCIENCE AND CLINICAL PRACTICE Pranav P. Pandya, BSc, MBBS, MD, FRCOG Consultant and Director of Fetal Medicine University College London Hospitals London, England, UK

Dick Oepkes, MD, PhD, FRCOG

Professor of Obstetrics and Fetal Therapy Department of Obstetrics Leiden University Medical Center Leiden, The Netherlands

Neil J. Sebire, MBBS, BClinSci, MD, FRCOG, FRCPath, FFCI Professor of Paediatric and Developmental Pathology Department of Histopathology Great Ormond Street Hospital London, England, UK

Ronald J. Wapner, MD

Professor of Obstetrics and Gynecology Vice Chair of Research Director of Reproductive Genetics Columbia University Irving Medical Center New York, NY, USA

© 2020, Elsevier Limited. All rights reserved. First edition Harcourt Brace and Company 1999 Second edition Elsevier Limited 2009 No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Details on how to seek permission, further information about the Publisher’s permissions policies and our arrangements with organizations such as the Copyright Clearance Center and the Copyright Licensing Agency, can be found at our website: www.elsevier.com/permissions. This book and the individual contributions contained in it are protected under copyright by the Publisher (other than as may be noted herein).

Notices Practitioners and researchers must always rely on their own experience and knowledge in evaluating and using any information, methods, compounds or experiments described herein. Because of rapid advances in the medical sciences, in particular, independent verification of diagnoses and drug dosages should be made. To the fullest extent of the law, no responsibility is assumed by Elsevier, authors, editors or contributors for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions, or ideas contained in the material herein. ISBN: 978-0-7020-6956-7 E-ISBN: 978-0-7020-7287-1

Content Strategist: Sarah Barth Content Development Specialist: Sharon Nash Project Manager: Beula Christopher Design: Brian Salisbury Illustration Manager: Nichole Beard/Teresa McBryan Marketing Manager: Michele Milano

Printed in China Last digit is the print number: 9 8 7 6 5 4 3 2 1

Foreword

It seems difficult to believe that 20 years have passed since the first edition of Fetal Medicine was published in 1999. Over that time, many things have changed. In fact, in the Foreword to the first edition, John Hobbins charted the preceding 20 years to indicate the progress that had been made in the speciality up to 1999. John Queenan, writing the Foreword for the second edition in 2009, emphasised how an improved understanding of the basic science of fetal medicine together with the development of technologies with which to treat fetuses had brought us to the point at which we could reasonably talk of the ‘fetal patient’. Now the four new editors of this, the third edition, have taken things to a new level and are to be congratulated both in encouraging previous experts to make further updated contributions and recruiting some outstanding ‘new blood’ from experts all over the world. It is clear from the excellent chapters that in the past 10 years, there have been a consolidation of knowledge in many areas and

viii

some spectacular progress in subjects such as noninvasive genetic diagnosis and fetal imaging and in the use of randomised studies in the extremely difficult area of open fetal surgery. This beautifully produced and illustrated edition provides readers with a cornucopia of up-to-date knowledge in the field of fetal medicine which should be a close companion for anyone involved in the speciality. Professor Charles H. Rodeck, MB, BS, BSc, DSc, FRCOG, FRCPath, FMedSci Emeritus Professor, Institute of Women’s Health, Obstetrics and Gynaecology, University College London, London, UK Professor Martin J. Whittle, MD, FRCOG, FRCP(Glas) Emeritus Professor of Fetal Medicine, University of Birmingham, Birmingham, UK

Preface

The two previous editions of this book are established as authoritative textbooks in fetal medicine, with the last edition published in 2009. I was therefore delighted to be asked by Professors Rodeck and Whittle to be an editor for the third edition. There have been so many significant advances in fetal medicine during the past decade that I thought it would only be possible to do justice to a new edition with an expanded team of international expert coeditors. This would not have been possible without Professors Dick Oepkes, Neil Sebire and Ron Wapner and their monumental efforts in preparing this new edition. We were clear from the start that this edition should continue and build on the same premise as the previous, namely combining basic science with contemporary clinical practice. At the same time, we wished to thoroughly revise the content and style to reflect the rapid advances in fetal medicine and science. We have changed the appearance of chapters with a focus on major topics in fetal medicine, providing scientific evidence on recent advances, structured text, key points at the beginning of each chapter, concise chapter summaries, new images and new videos online. The aim is to allow the readers to both rapidly access relevant information to

aid management and to provide a comprehensive understanding of the topic. With these aims, there are numerous new chapters and extensive revision of existing chapters to keep pace with the dramatic innovation in fetal medicine. We are deeply indebted to the multidisciplinary international authors who are recognised experts in their field; they have generously contributed their time, knowledge and experience to this book. They have delivered up-to-date information that is practical and accessible and provides deeper understanding of complex issues within fetal medicine and basic science. We also thank all the team at Elsevier for their constant support and help, in particular Sharon Nash, Beula Christopher, Sarah Barth and Kate Dimock. Finally, we would like to thank again Charles Rodeck and Martin Whittle for trusting us to produce the third edition of this book. Pranav P. Pandya Dick Oepkes Neil J. Sebire Ronald J. Wapner

ix

List of Contributors

The editors would like to acknowledge and offer grateful thanks for the input of all previous editions’ contributors, without whom this new edition would not have been possible. Ganesh Acharya, MD, PhD Professor of Obstetrics and Gynecology Department of Clinical Sciences, Intervention and Technology Karolinska Institute Stockholm, Sweden Michael Aertsen, MD Radiologist Department of Radiology University Hospitals Leuven Leuven, Belgium Yalda Afshar, MD, PhD Maternal Fetal Medicine Fellow Division of Maternal Fetal Medicine Department of Obstetrics and Gynecology University of California Los Angeles, CA, USA Cande V. Ananth, PhD, MPH Professor and Vice-Chair for Academic Affairs Chief, Division of Epidemiology and Biostatistics Department of Obstetrics, Gynecology, and Reproductive Sciences Rutgers Robert Wood Johnson Medical School New Brunswick, NJ, USA Michael Ashworth, MD, FRCPath Consultant in Paediatric Pathology Department of Histopathology Great Ormond St Hospital for Children London, England, UK Patrick Au, MPhil, MSc Scientific Officer (Medical) Prenatal Diagnostic Laboratory Tsan Yuk Hospital Hong Kong SAR, China Spyros Bakalis, BSc, MBBS, MRCOG, MD Consultant in Obstetrics and Fetal Medicine St Thomas’ Hospital London, England, UK

x

Guillaume Benoist, MD, PhD Department of Obstetrics and Gynecology University Hospital Caen, France University of Normandy Normandy, France Colleen G. Bilancia, PhD, DABMGG Clinical Cytogeneticist and Molecular Geneticist Lineagen, Inc. Salt Lake City, UT, USA Caterina M. Bilardo, MD, PhD Professor in Fetal Medicine Department of Obstetrics and Gynaecology Amsterdam University Medical Centers VUmc Amsterdam, The Netherlands; University Medical Centre Groningen University of Groningen Groningen, The Netherlands Louise D. Bryant, BSc (Hon), PhD Associate Professor of Medical Psychology Leeds Institute of Health Sciences University of Leeds Leeds, England, UK Colin R. Butler, BSc, MBBS, MRCS Tracheal Research Fellow Great Ormond Street Hospital for Children London, England, UK Frank Van Calenbergh, MD, PhD Professor of Neurosurgery Academic Department of Neurosciences Biomedical Sciences Faculty of Medicine; Department of Neurosurgery University Hospital Gasthuisberg UZ Leuven Leuven, Belgium

List of Contributors

Steve N. Caritis, MD Professor Department of Obstetrics, Gynecology and Reproductive Sciences, School of Medicine Magee Women’s Hospital of UPMC University of Pittsburgh Pittsburgh, PA, USA Lyn S. Chitty, BSc, PhD, MBBS, MRCOG Professor of Fetal Medicine and Genetics UCL Great Ormond Street Institute of Child Health NE Thames Regional Genetics Service Great Ormond Street Hospital for Children NHS Foundation Trust London, England, UK Patricia Collins, BSc(Hon), PhD Professor of Anatomy AECC University College Bournemouth, England, UK James Cook, MBBS, MSc, MRCPCH Subspecialty Trainee in Paediatric Respiratory Medicine Department of Paediatric Respiratory Medicine Great Ormond Street Hospital for Children NHS Foundation Trust London, England, UK Howard Cuckle, BA, MSc, DPhil Adjunct Professor Department of Obstetrics and Gynecology Columbia University Medical Center New York, NY, USA Anna L. David, PhD, FRCOG, MB, ChB Professor and Consultant in Obstetrics and Maternal Fetal Medicine Institute for Women’s Health University College London London, England, UK Luc De Catte, MD, PhD Feto-maternal Specialist Fetal Medicine Department of Obstetrics and Gynecology University Hospitals Leuven Leuven, Belgium Paolo De Coppi, MD, PhD NIHR Professor of Paediatric Surgery Head of Stem Cells and Regenerative Medicine Section Developmental Biology and Cancer Programme UCL Institute of Child Health Consultant Paediatric Surgeon Great Ormond Street Hospital London, England, UK

xi

Elisabeth de Jong-Pleij, MD, PhD Physician-Sonographer Department of Obstetrics and Gynaecology St. Antonius Hospital Nieuwegein, The Netherlands; Department of Obstetrics and Gynaecology University Medical Centre Utrecht Utrecht, The Netherlands Bart De Keersmaecker, MD Feto-Maternal Specialist Fetal Medicine Department of Obstetrics and Gynecology University Hospitals Leuven Leuven, Belgium; Department of Obstetrics and Gynecology Kortrijk, Belgium Jan Deprest, MD, PhD, FRCOG Professor Head of the Department of Development and Regeneration Department of Obstetrics and Gynecology University Hospital of Leuven Gasthuisberg Leuven, Belgium; Institute for Women’s Health University College London London, England, UK Roland Devlieger, MD, PhD Professor of Obstetrics and Gynaecology Academic Department of Development and Regeneration Cluster Woman and Child, Biomedical Sciences Faculty of Medicine, KU Leuven; Centre for Surgical Technologies, Faculty of Medicine, KU Leuven; Fetal Medicine Unit, Division of Woman and Child, Department of Obstetrics and Gynaecology University Hospital Gasthuisberg, UZ Leuven Leuven, Belgium Guido M. de Wert, PhD Professor of Biomedical Ethics Department of Health, Ethics and Society Research Schools CAPHRI and GROW University of Maastricht Maastricht, The Netherlands Jan E. Dickinson, MD, FRANZCOG, DDU, CMFM Professor Maternal Fetal Medicine Division of Obstetrics and Gynaecology Faculty of Health and Medical Sciences The University of Western Australia Perth, Western Australia, Australia Mark Dilworth, PhD MRC Career Development Award Research Fellow Maternal and Fetal Health Research Centre, Faculty of Biology, Medicine and Health University of Manchester; St. Mary’s Hospital Manchester University NHS Foundation Trust Manchester Academic Health Science Centre Manchester, England, UK

xii

List of Contributors

Wybo J. Dondorp, PhD Associate Professor of Biomedical Ethics Department of Health Ethics and Society Research Schools CAPHRI and GROW University of Maastricht Maastricht, The Netherlands Caroline E. Dunk, PhD Research Associate Research Centre for Womens and Infants Health Lunenfeld Tanenbaum Research Institute Mount Sinai Hospital Toronto, Ontario, Canada Thomas R. Everett, BSc MBChB, MD, MRCOG Consultant in Fetal Medicine Leeds General Infirmary Leeds, England, UK Jane Fisher, BA (Hon), MA Director Antenatal Results and Choices London, England, UK Henry L. Galan, MD Professor Department of Obstetrics and Gynecology Colorado Fetal Care Center University of Colorado School of Medicine Aurora, CO, USA Mythily Ganapathi, PhD, FACMG Assistant Professor of Pathology and Cell Biology at CUMC College of Physicians and Surgeons Columbia University Medical Center and the New York Presbyterian Hospital New York, NY, USA Helena M. Gardiner, MD, PhD Director of the Fetal Echocardiography Fellowship and Training Program The Fetal Center UT Health McGovern Medical School Houston, TX, USA Cecilia Gotherstrom, PhD Associate Professor Department of Clinical Science Intervention and Technology Division of Obstetrics and Gynecology Karolinska Institutet Stockholm, Sweden Richard Harding, PhD, DSc Emeritus Professor Department of Anatomy and Developmental Biology Monash University Melbourne, Australia

Jenny Hewison, BA (Hon), MSc, PhD Professor of the Psychology of Healthcare Leeds Institute of Health Sciences University of Leeds Leeds, England, UK Richard J. Hewitt, BSc, DOHNS, FRCS (HNS-ORL) Consultant Paediatric ENT Head and Neck and Tracheal Surgeon Director of the National Service for Severe Tracheal Diseases Great Ormond Street Hospital for Children London, England, UK Liran Hiersch, MD Staff Physician Department of Obstetrics and Gynecology Lis Maternity Hospital Tel Aviv Sourasky Medical Center Sackler Faculty of Medicine Tel Aviv University Tel Aviv, Israel Melissa Hill, BSc, PhD Senior Social Scientist Genetics and Genomic Medicine UCL Great Ormond Street Institute of Child Health NE Thames Regional Genetics Service Great Ormond Street Hospital for Children NHS Foundation Trust London, England, UK Sara L. Hillman, BSc, MBBS, PhD, MRCOG NIHR Academic Clinical Lecturer Subspecialty Trainee in Maternal Fetal Medicine University College London London, United Kingdom An Hindryckx, MD Consultant in Obstetrics and Fetal Medicine University Hospitals Leuven Leuven, Belgium Stuart B. Hooper, PhD Centre Head The Ritchie Centre The Hudson Institute for Medical Research Department of Obstetrics and Gynaecology Monash University Melbourne, Australia Berthold Huppertz, PhD Chair Division of Cell Biology, Histology and Embryology Gottfried Schatz Research Center Medical University of Graz Graz, Austria J. Ciaran Hutchinson, MRes, MBBS, DipFMS Clinical Research Fellow Department of Histopathology Great Ormond Street Hospital London, England, UK

List of Contributors

Jon Hyett, MBBS, BSc, MD, MRCOG, FRANZCOG Clinical Professor and Head of High Risk Obstetrics RPA Women and Babies Royal Prince Alfred Hospital University of Sydney Camperdown, NSW, Australia Luc Joyeux, MD, MSc General Paediatric Surgeon and PhD Candidate in Fetal Surgery Academic Department of Development and Regeneration Cluster Woman and Child, Biomedical Sciences Faculty of Medicine Katholieke Universiteit; Centre for Surgical Technologies Faculty of Medicine KU Leuven, Belgium Leuven, Belgium Davor Jurkovic, FRCOG, MD Consultant Gynaecologist Department of Obstetrics and Gynaecology University College Hospital London, England, UK John C. Kingdom, MD Professor of Maternal-Fetal Medicine Department of Obstetrics and Gynecology University of Toronto Toronto, Ontario, Canada Sylvie Langlois, MD Professor and Clinical Geneticist Department of Medical Genetics University of British Columbia Vancouver, British Columbia, Canada Lara S. Lemon, PhD, PharmD Research Assistant Professor Department of Obstetrics, Gynecology and Reproductive Sciences University of Pittsburgh School of Medicine; Data Scientist Department of Clinical Analytics University of Pittsburgh Medical Center Pittsburgh, PA, USA Marianne Leruez-Ville, MD, PhD Department of Virology Necker Enfants Malades Hospital University of Paris Descartes Paris, France Liesbeth Lewi, MD, PhD Professor and Staff Member Department of Obstetrics and Gynecology University Hospital of Leuven Gasthuisberg Leuven, Belgium Brynn Levy, MSc (Med), PhD, FACMG Professor of Pathology and Cell Biology at CUMC College of Physicians and Surgeons Columbia University Medical Center and the New York Presbyterian Hospital New York, NY, USA

xiii

Y.W. Loke, MD, FRCOG Emeritus Professor of Reproductive Immunology Kings College University of Cambridge Cambridge, England, UK Enrico Lopriore, MD, PhD Professor and Head of the Neonatology Division Division of Neonatology Department of Pediatrics Leiden University Medical Center Leiden, The Netherlands George A. Macones, MD Professor and Chair Department of Obstetrics and Gynecology Washington University in St. Louis St. Louis, MO, USA Fergal D. Malone, MD, FACOG, FRCOG, FRCPI Master of the Rotunda Hospital, Dublin Chair and Professor of Obstetrics and Gynecology Royal College of Surgeons in Ireland; Consultant Obstetrician and Gynecologist and Maternal-Fetal Medicine Specialist Rotunda Hospital Dublin, Ireland Anahit Martirosian, RDMS Sonographer Center for Fetal Medicine and Women’s Ultrasound Los Angeles, CA, USA Fionnuala McAuliffe, MD, FRCOG, FRCPI Chair and Professor of Obstetrics and Gynecology Head, Women’s and Children’s Health University College Dublin Dublin, Ireland; Consultant Obstetrician and Gynecologist and Maternal-Fetal Medicine Specialist National Maternity Hospital Dublin, Ireland; Council Member, Royal College of Obstetricians and Gynecologists London, England, UK Annie R.A. McDougall, PhD Research Officer The Ritchie Centre The Hudson Institute of Medical Research Melbourne, Australia Kenneth J. Moise Jr., MD Professor Division of Maternal-Fetal Medicine Department of Obstetrics Gynecology and Reproductive Sciences UT Health McGovern School of Medicine; Co-Director The Fetal Center Children’s Memorial Hermann Hospital Houston, TX, USA

xiv

List of Contributors

Ashley Moffett, MD, FRCOG Emeritus Professor of Reproductive Immunology Department of Pathology University of Cambridge Cambridge, England, UK

Kuhan Rajah, MRCOG Subspecialty Trainee in Reproductive Medicine Department of Obstetrics and Gynaecology University College Hospital London, England, UK

Sieglinde M. Müllers, PhD Specialist Registrar in Obstetrics and Gynecology Royal College of Surgeons in Ireland Rotunda Hospital, Dublin, Ireland

Rashmi Rao, MD Assistant Professor Division of Maternal Fetal Medicine Department of Obstetrics and Gynecology University of California Los Angeles, CA, USA

Ran Neiger, MD Director Maternal-Fetal Medicine Unit Ma’ayanei Hayeshua Hospital Bnei Brak, Israel John P. Newnham, AM, MD, FRANZCOG, CMFM, DDU Professor of Maternal Fetal Medicine and Head Division of Obstetrics and Gynaecology The University of Western Australia Perth, Western Australia, Australia Sarah G. Obican, MD Assistant Professor University of South Florida Division of Maternal Fetal Medicine Department of Obstetrics and Gynecology Tampa, FL, USA Anthony O. Odibo, MD, MSCE Professor of Obstetrics and Gynecology Director of Ultrasound and Fetal Therapy University of South Florida Morsani College of Medicine Tampa, FL, USA Dick Oepkes, MD, PhD, FRCOG Professor of Obstetrics and Fetal Therapy Department of Obstetrics Leiden University Medical Center Leiden, The Netherlands Pranav P. Pandya, BSc, MBBS, MD, FRCOG Consultant and Director of Fetal Medicine University College London Hospitals London, England, UK Lawrence D. Platt, MD Professor Center for Fetal Medicine and Women’s Ultrasound Division of Maternal Fetal Medicine Department of Obstetrics and Gynecology University of California Los Angeles, CA, USA Rosalind Pratt, MBChB, Bsc Clinical Research Fellow University College London London, England, UK

Jute Richter, MD, PhD Professor and Staff Member Department of Obstetrics and Gynecology University Hospital of Leuven Gasthuisberg Leuven, Belgium Joshua I. Rosenbloom, MD Fellow Division of Maternal-Fetal Medicine Department of Obstetrics and Gynecology Washington University in St. Louis Francesca Maria Russo, MD Research Fellow Department of Obstetrics and Gynecology University Hospital of Leuven Gasthuisberg Leuven, Belgium Anthony R. Scialli, MD Director Reproductive Toxicology Center Washington, DC, USA Neil J. Sebire, MBBS, BClinSCi, MD, FRCOG, FRCPath, FFCI Professor of Paediatric and Developmental Pathology Department of Histopathology Great Ormond Street Hospital London, England, UK Andrew Sharkey, BA, PhD Associate Lecturer Department of Pathology University of Cambridge, UK Cambridge, England, UK Susan C. Shelmerdine, MBBS, BSc, MRCS, FRCR Clinical Research Fellow Department of Radiology Great Ormond Street Hospital London, England, UK Colin Sibley, PhD, DSc Professor of Child Health and Physiology Maternal and Fetal Health Research Centre Faculty of Biology, Medicine and Health University of Manchester; St. Mary’s Hospital Manchester University NHS Foundation Trust Manchester Academic Health Science Centre Manchester, England, UK

List of Contributors

Saul Snowise, MD Minnesota Perinatal Physicians and the Midwest Fetal Care Center Children’s Hospital Minneapolis, MN, USA Sylke Steggerda, MD, PhD Neonatologist Division of Neonatology Department of Pediatrics Leiden University Medical Center Leiden, The Netherlands Emily J. Su, MD, MSCI Associate Professor Department of Obstetrics and Gynecology Colorado Fetal Care Center University of Colorado School of Medicine Aurora, CO, USA Mary Tang, FRCOG Clinical Associate Professor Prenatal Diagnostic and Counselling Division Department of Obstetrics and Gynaecology University of Hong Kong Pokfulam, Hong Kong SAR, China Arjan B. Te Pas, MD, PhD Neonatologist Division of Neonatology Department of Pediatrics Leiden University Medical Center Leiden, The Netherlands Alan T. Tita, MD, PhD Professor and Director Center for Women’s Reproductive Health Department of Obstetrics and Gynecology University of Alabama Birmingham, AL, USA Frederick Ushakov, MD Specialist in Fetal Medicine Fetal Medicine Unit University College London Hospital London, England, UK Ignatia B. Van den Veyver, MD Professor Departments of Obstetrics and Gynecology and Molecular and Human Genetics Baylor college of Medicine Houston, TX, USA Jeanine M. van Klink, PhD Clinical Psychologist Division of Neonatology Department of Pediatrics Leiden University Medical Center Leiden, The Netherlands

Raman Venkataramanan, PhD Professor Department of Pharmaceutical Science, School of Pharmacy Department of Pathology, School of Medicine University of Pittsburgh Pittsburgh, PA, USA Yves Ville, MD, FRCOG Professor Department of Obstetrics and Fetal Medicine Necker Enfants Malades Hospital University of Paris Descartes Paris, France Magdalena Walkiewicz, PhD Assistant Professor Department of Molecular and Human Genetics Baylor college of Medicine Baylor Genetics Laboratories Houston, TX, USA Colin Wallis, FRCPCH, MD Consultant Respiratory Paediatrician Respiratory Unit Great Ormond Street Hospital London, England, UK Lilian Walther-Jallow, PhD Department of Clinical Science Intervention and Technology Division of Obstetrics and Gynecology Karolinska Institutet Stockholm, Sweden Ronald J. Wapner, MD Professor of Obstetrics and Gynecology Vice Chair of Research Director of Reproductive Genetics Columbia University Irving Medical Center New York, NY, USA Magnus Westgren, MD, PhD Professor Department of Clinical Science Intervention and Technology Division of Obstetrics and Gynecology Center for Fetal Medicine Karolinska University Hospital Stockholm, Sweden Scott W. White, MBBS, PhD, FRANZCOG, CMFM Clinical Senior Lecturer in Maternal Fetal Medicine Division of Obstetrics and Gynaecology The University of Western Australia Perth, Western Australia, Australia Louise C. Wilson, MB, BS, FRCP Consultant in Clinical Genetics Great Ormond Street Hospital NHS Foundation Trust London, England, UK

xv

xvi

List of Contributors

R. Douglas Wilson, MD, MSc Professor and Head Department of Obstetrics and Gynecology Cumming School of Medicine University of Calgary Calgary, Alberta, Canada Dian Winkelhorst, MD PhD Student and Clinical Researcher Divison of Fetal Therapy Department of Obstetrics Leiden University Medical Center Leiden, The Netherlands Paul J.D. Winyard, BM, BCh, MA, PhD, FRCPCH Professor of Paediatric Education and Honorary Consultant in Paediatric Nephrology Developmental Biology and Cancer Programme UCL Great Ormond Street Institute of Child Health London, United Kingdom Christoph Wohlmuth, MD, PhD, Priv. Doz. Department of Obstetrics and Gynecology Paracelsus Medical University Salzburg, Austria

Karen Wou, MD Clinical Genetics Fellow Department of Pediatrics, Division of Clinical Genetics Columbia University Irving Medical Center New York, NY, USA Yuval Yaron, MD Director, Prenatal Genetic Diagnosis Unit Genetic Institute, Tel Aviv Sourasky Medical Center Associate Professor, Department of Obstetrics and Gynecology Sackler Faculty of Medicine, Tel Aviv University Tel Aviv, Israel Kwok Yin Leung, MBBS, MD, FRCOG Chief of Service and Consultant Department of Obstetrics and Gynaecology, Queen Elizabeth Hospital Hong Kong SAR, China Angela Yulia, MRCOG, PhD, PGCert, Med Ed Subspecialty Trainee in Maternal and Fetal Medicine Fetal Medicine Unit University College London Hospitals NHS Foundation Trust London, England, UK

1

Early Concepts and Terminology PATRICIA COLLINS

KEY POINTS • T his chapter considers the language used within embryological research and how it is evolving. • The terms used to describe embryos, cells and tissues derive from the social constructs of science during the time they were created. • Newer terms have been added as scientific methods have increased, although there may not be a consensus on the definition of some terms. • The application of computer sciences to development has produced its own terminology. • The use of computer ontologies may impact development and the evolution of embryological concepts and terminology with which to explain the observed processes.

The Changing Concepts and Language of Embryology Embryological terminology used today is a strange and diverse mixture of terms accrued over the course of two centuries and used as a vernacular language with different dialect depending on the topic and techniques of study. The accumulated terms include: • The very old concepts generated between 1830 and 1900 • The newer understanding of cell phenotype generated in the 20th century from in vitro and in vivo experimentation and cell culture • The very modern and rapidly changing 21st-century terms which describe gene expression and metabolism within embryonic tissues The language of the latter group is driven by computer ontologies: hierarchies of embryological terms and key words programmed as algorithms, which are used to mine databases and publications. The spur for this interest is a future ability to unlock embryological pathways as a method for treating adult pathology and harnessing the regenerative potential of stem cells. 

Origin of the Early Embryological Terms Embryological terms are a product of their time and a reflection of how developmental science was explained. At a time when the theory of evolution was being formulated in the mid-1800s, Ernst Haeckel promoted a concept which stated that embryos would pass through all the previous evolutionary 2

stages, resembling a series of extant or extinct adult animals as they recapitulated evolution during development.1 Thus Haeckel designated a blastula stage of development when a sphere or bilaminar layer of embryonic cells was present and a later gastrula stage achieved after the blastula cells had invaginated to produce more than one or two layers. He also inaugurated the term gastrulation to describe the process where cells initially on the embryonic surface move inside the embryo to produce intraembryonic cell populations. At this time the instruments for examining embryos were rudimentary, and cell theory, being formulated also in the mid-1800s, was still relatively young. Embryologists of the day saw layers of tissue rather than the individual cells composing the layers and did not link the morphology of the earliest cells with differences in function. In studies in which it is clear from the publications that cells could be seen, distinctions among early embryonic cell types probably could not be made with the instruments available. The concepts thus generated by these early embryologists were products of their time, dependent on the methods of experiment and observation customary when they were formulated. For most of the 20th century, textbooks supported the notion that the tissues of the developed body were derived from one of the ‘three germ layers’. Whilst this is not untrue in simplistic terms, the accent on three layers (ignoring the cell phenotypes) moved attention away from and limited interrogation of the dynamic differentiation processes occurring in embryos. Without a full range of words with which to think about developmental processes the reflections on, and explanations of, what is seen histologically becomes obfuscated. A similar interpretive process driven by evolution theory occurred with the description of external embryonic form. In 1828, Von Baer2 noted that all vertebrate embryos pass through externally similar stages, and Haeckel published a series of drawings demonstrating remarkable similarity between embryos which go on to become very dissimilar adults. This latter concept remained unchallenged for more than a century. Recent examination of Haeckel’s pictures, together with a clear analysis of the developmental stages of various organs in each embryo, revealed a different story. Richardson3 noted that drawings by contemporaries of Haeckel show much more accurate interpretations of mammalian embryos of the same developmental stage with clear differences among them. He noted that Haeckel’s drawings had given a misleading view of embryonic development. Thus the idea of one stage of development, during which all vertebrate embryos are the same, promoted extensively at the turn of the 20th century and repeated unchallenged, hampered investigation into what really occurs in a number of vertebrate embryos. This

Chapter 1  Early Concepts and Terminology

again obfuscated the search for what is actually present in embryos by limiting the embryological concepts taught, the language used and consequently the expectations and explanations of the processes observed. Embryology was advanced in the middle and later years of the 20th century by in vitro studies of developing reptile, avian and mammalian embryos, particularly using chimeric embryos in which specific cells lines could be followed.4 These experiments provided information about the similarities and differences among species. Also at this time, the genes expressed within developing tissues were studied, and the range of genes used in basic cell functioning and at particular points of development were elucidated. The functions of many genes were studied by the experimental production of animals in which specific genes were knocked out or knocked in, and the effects of the homozygous were compared with the wild type. These experiments demonstrated the importance of some genes, with knock-out causing lethality; in other cases, the actual effect of taking one gene out within an embryo was confounded by the catch-up mechanisms in built into development: the change in one part of the system causing compensatory change in the remainder. 

TABLE 1.1 Lineage Concepts

Term

Meaning

Three cell lineages identified as zygote undergoes cleavage

• T rophectoderm—will become placenta and extraembryonic membranes • Hypoblast—sometimes called primitive endoderm; maintains the primitive streak • Inner cell mass—sometimes called primitive ectoderm; usually termed epiblast; all embryonic cell lines are derived from this lineage

Polarity genes

Cells within an early embryo form epithelia and mesenchyme. The epithelial cells exhibit polarity genes which specify the apical, basal and lateral surfaces; the position of junctional complexes; and the direction of the mitotic spindle during cell division. Not observed before compaction in morula.6

Germ layers

These were historically the ectoderm, endo­ derm and mesoderm. The terms are still widely used regardless of cell phenotype. They all derive from the epiblast of the early blastocyst.

Early Embryonic Cell Interactions Expansion of in  vitro fertilisation techniques and the selection of healthy embryos for implantation have demonstrated that the secondary oocyte has a range of genes ready for expression to ensure cleavage, morula and blastocyst formation. When the dividing cells are an appropriate size, polarity is expressed, junctions are formed and embryonic cell–cell interaction commences.5 After hatching from the zona pellucida, the blastocyst is able to interact with maternal tissue. There is no time when the genome is not being read and epigenetic consequences, because of local environmental conditions, are not part of the next process. Three cell lineages are now identified in the blastocyst, leading to extraembryonic and embryonic cell populations (Table 1.1). All of these lineages express polarity genes and form epithelia. The process of gastrulation produces cells which do not have an epithelial phenotype (i.e., mesoblast and mesenchymal cells). Recovery of embryonic cells has led to the development of embryonic stem cells which can be immortalised in two-dimensional culture conditions. 

Embryonic Cells in Culture Historically, the definitions of the terms used to describe the putative abilities of embryonic cells were easily found. Today recent papers note the difficulty of accurately defining these terms (Table 1.2). Adult cells can be induced to grow in culture and now so can embryonic cells. Early in vitro culture techniques mainly concerned the growth of adult cells within a two-dimensional physical environment. Cells are grown to confluence and then split into subcultures (passaging) and are regrown many times. The passages and new media promote expansion of the numbers of cells in the culture and prolong the life of the cells beyond that of the original donor. This methodology is still utilised. Three-dimensional environments, the norm for all body tissues, are being explored in in  vitro culture. It has been noted that cells in three-dimensional culture systems form organoids, in which the epithelial cells form spheres surrounded by

   TABLE Definitions of the Terms Used to Describe 1.2 Zygotes and Stem Cells

Term

Meaning

Totipotent

The ability of a single cell to develop into an adult organism and generate offspring. In humans, the zygote is totipotent. The loss of totipotency is now seen as a process.

Pluripotent

The ability of a single cell to develop into cells from one of the ‘three germ layers’ and ‘germ’ cells in vitro and in vivo. EpiSCs are postimplantation epiblast stem cells.

Embryonic stem cells

Human embryonic stem cells (hESCs). Origin not clear. Not quite the same as inner cell mass cells. Need specific culture conditions; grow in two dimensions. Now have been adapted to long-term in vitro culture.

Human-induced pluripotent stem cells (hiPSCs)

Cells derived from adult cells (e.g., fibroblasts) which have been cultured with specific transcription factors. They undergo transition to epithelial cells and express epithelial genes, becoming polarised. They also change their metabolism.

Human spheroids or organoids

When hiPSCs are grown in three-dimensional culture and encouraged along a particular developmental pathway, they form spheres of inner epithelial and outer mesenchymal phenotypes. Organoids have been created from hiPSCs specified as endoderm which differentiate into airway or gut phenotypes with appropriate epithelial and supporting mesenchymal cells. Self-organisation into layered tissues has also been seen in three-dimensional cultured brain cortical cells and retinal tissue.

  

3

4

SE C T I O N 1     Early Fetal Development

mesenchymal cells.7,8 These self-organising cell lines have been implanted into animals and will continue growing.9 The ultimate aim of these studies is to grow replacement portions of gut, respiratory conducting airways or kidney which ultimately can be used for transplant. Cultured cells have also been purposefully arranged in specified three-dimensional shapes by bioprinting methods and encouraged to grow and differentiate along specific lines by the addition of targeted growth factors to the media.10 The complexity of setting up all three-dimensional systems and recording cell growth, movement and interaction are particularly challenging. Further experimental methods have attempted to immortalise embryonic cell lines. By specifying the growth factors used in the culture media and the oxygen levels supplied and by forcing cells to change their phenotype (mesenchyme to epithelial), the cells have been driven along particular developmental pathways to form specific cell lines, e.g., cardiac myocytes, hepatocytes and neurons.11,12 Such cultures are used for further optimisation of culture conditions, to gain knowledge of the genes the cells are utilising, and for testing drugs on cells in culture rather than on laboratory animal species. 

Interpretation of the Genome The latter years of the 20th century and the beginning of the 21st saw an explosion of interest in embryological pathways, first because of the identification of the human genome and the genomes of the commonly used laboratory animals (Table 1.3) and second because it was thought that re-expression of embryonic genes and pathways in an adult could lead to treatment of many pathological conditions. The success of the Human Genome Project led to elucidation of the genomes of laboratory species and greater understanding of the shared genes upregulated in development. Information on the temporal and spatial regions of gene expression during development superimposed on internal and external embryonic form has been shared via the internet. Such websites also have the methodologies for demonstrating specific genes and transcription factors. 

Computing Sciences and Embryological Terminology Embryology has now become a domain of computer sciences as well as laboratory-based sciences. Powerful computing was necessary for the collation of the genomes and the proteins encoded. Two interrelated lines of research can now be noted: (1) the relationships among cells, tissues, organs and time within developing embryos and (2) the relationship between genes and the molecules they encode. Ontologies, hierarchical structures of specified vocabulary, have been created for developmental processes; embryonic cells and tissues; for genes, transcription factors and proteins. New genes, or the spatial and temporal expression of known genes, are added to specific ontologies either by a curator or by the computer ontology algorithm itself (an inferred electronic annotation). Predictions of future gene function can be gained from these methods. The laboratory techniques of mass spectrometry and microarray methods can identify proteins and metabolites in very small samples of culture media. Information concerning what genes the cells are expressing and what proteins they are making at each time

TABLE Internet Sources of Information on a Range of 1.3 Genomes

Genome and proteins

Internet site

Human Genome

National Human Genome Research Institute Has timeline for the Human Genome Project

Human ­Proteins

• Human Proteome Project; Human Proteome Map • Human Protein Atlas Human Developmental Studies Network (HUDSEN) • Electronic atlas of a developing human brain • Human spatial gene expression database • Virtual Human Embryo • Multidimensional Human Embryo Both use Carnegie staging for human embryos Brainspan Atlas of the developing human brain, developmental transcription factors

FlyBase

A database of the genome and genes expressed in Drosophila throughout development

WormBase

A database of the genome and genes expressed Caenorhabditis elegans and other species

Mouse eMAP

• E dinburgh Mouse Atlas Project • eMA: three-dimensional mouse anatomy atlas • eHistology, with serial sections of mouse embryos throughout development • eMAGE: gene expression database for mouse embryos DBTMEE • Database of transcriptome in mouse early embryos

echickatlas

• T hree-dimensional anatomical atlas of chick embryo development • e-Chick Atlas of gene expression

Geisha

Gallus Expression In Situ Hybridisation Analysis • Anatomical atlases • Chick development stage series • Bird genomes • Transgenic lines of chickens and quail

  

point of development has been shown to provide, for example, objective data in the assessment of preimplantation embryos from in vitro fertilisation.13 These methods have also been used to analyse the supernatant of embryo cultures at specific time points or to analyse the supernatant of induced pluripotential stem cells culture as the cells are induced to follow specific phenotypic pathways (Table 1.4). The techniques of high-resolution mass spectrometry are now able to identify and quantify protein in single embryonic cells.16 The language used to describe these techniques is now part of the embryological vocabulary. The use of powerful computing has also enabled threedimensional images of animal and human embryos of all stages to be made available, showing external form and serial histological sections. The spatial and temporal location of specific gene expression may also be added to these images (see Table 1.3).

Chapter 1  Early Concepts and Terminology

TABLE 1.4 Common Bioinformatics Terms

Data-driven technology term

Meaning

Gene ontology

The terms entered into a computer in a relational hierarchy. It provides a structured language to describe gene function and tools to predict gene function.

Metabolome

A range of small molecules (amino acids, adenosine triphosphate, hormones, signalling molecules) which can be measured in the culture media in which embryos are grown. These measurements give insights into the biochemical and metabolic pathways operating within the embryo at particular times of development. They can be used to assess viability of preimplantation embryos.14

Proteomics

Study of all the proteins of a cell type and their interactions15

Protein interaction network

A scheme which shows interactions between gene products of differentially expressed genes

Secretome

The collection of transmembrane proteins and proteins secreted into the extracellular space, e.g., cell–cell signalling molecules within a culture

Transcriptome

The examination of the global transcription factors expressed in cell or embryo culture by microarray methods at specified a particular time of or after cell perturbation

   Much of the painstaking work of capturing and interpreting photographs of cells in culture is now being completed by computer software programmes and their ontological algorithms (e.g., spatial expression of genes),17 and fluorescence of cytoskeletal elements.18 Metadata analyses now provide information on comparative functional genomic studies and on systemslevel analyses integrating epigenetic and functional data in development.19 

A Note of Caution For many years, the painstaking work of those who examined serial sections of embryos, elucidating and correlating the morphological changes within embryos at specified stages (e.g., Matt Kaufmann20 for mouse embryos and Ronan O’Rahilly and Fabiola Müller21,22 for their extensive and fundamental work on the Carnegie collection of human embryos) was underrated. Yet an understanding of the four-dimensional changes and the interactions between cells and tissues within an embryo is necessary for the accurate interpretation of the newer developmental techniques. The significance of the newer findings and how they relate to human development, from the embryo to adult senescence, still needs to be considered and theories and concepts re-examined. Findings from techniques in which cell lines experience very different epigenetic processes to in vivo development must be treated with care, just as differences in the timing of particular gene expression between animal species are treated.

5

In stem cell culture, changes in cell behaviour (from what might be considered ‘normal’) caused by the culture conditions may lead to the misinterpretation of experimental results and should be considered with caution. Human-induced pluripotent stem cells (hiPSCs) are generated by resetting their epigenetic landscapes.23 Although they are used as undifferentiated cells which can be pushed into developmental pathways by the culture media, undifferentiated embryonic cells are only transiently seen in a developing embryo.24 In a similar construct, the more computers use specified gene ontologies and make predictions on the results of their algorithms on the basis of inferred electronic annotation, the more distant the generated results become from the fundamental questions they may have been used to answer. Attempts are being made to make the output of mining proteomic databases easier to interpret,25 so those not familiar with embryological development or cell interactions may promote the use of these complex techniques, but they also separate further the understanding of the answer from the original question. Hutter and Moerman26 urge caution when interpreting outcomes from the use of ‘big data’ in biological sciences. If the input data used to solve a problem becomes convoluted and obfuscated, then the output may not be the answer to the question being investigated. This is an unprecedented time for embryological research. The advances in knowledge are enormous; however, what each finding means within the whole, beyond an increase in complexity that could not have been imagined in the past century, is not yet clear. The excitement of gathering an unprecedented amount of data which computers analyse does not explain which of the processes are operating in a human embryo at a particular time, even more so now that the outcomes must be genome, environmental and epigenetic specific. Although embryological language may have evolved in its origin and usage, this evolution will dwindle now that terms are fixed in computer ontologies. As the complexity of research outcomes increases and not all may be clear of the meaning ascribed to a term, it may be important that researchers define the particular meanings they attribute to concepts, cells and tissues within their studies. 

Conclusion This chapter has presented and considered the changing terminology used in the study and description of embryological development. Terms coined by researchers reflect the tools, scientific constructs and societal beliefs at the time of the study of developmental processes. Some older terms and language have been retained regardless of their limited specificity and utility to described recent advances in embryology. The use of computers to analyse the extensive data gathered on genomes and their products has contributed its own terminology. Computer algorithms used in the analysis of vast data arrays may now themselves be limiting the development of new evolutionary and conceptual terms. Careful reflection may be needed concerning the outcomes of computer algorithms and analyses in embryological research and the ways in which continued evolution of embryological concepts and language can be achieved. Access the complete reference list online at ExpertConsult.com Self-assessment questions available at ExpertConsult.com

References 1. H  aeckel E. The gastrea theory, phylogenetic classification of the animal kingdom and the homology of the germ lamellae (trans. E.O. Wright). Q J Micr Sci. 1874;14:142–165, 223–247. 2. Von Baer KE. Entwicklungsgeschichte der Tiere: Beobachtung und Reflexion. Königsberg: Bonträger; 1828. 3. Richardson MK. Heterochrony and the phylotypic period. Dev Biol. 1995;172:412–421. 4.  Le Douarin NM. The Nogent Institute—50 years of embryology. Int J Dev Biol. 2005;49: 85–103. 5. Tao H, Inoue K, Kiyonart H, et  al. Nuclear location of Prickle2 is required to establish cell polarity during early mouse embryogenesis. Dev Biol. 2012;364:138–148. 6. Gray RS, Roszko I, Solnica-Kresel L. Planar cell polarity: coordinating morphogenetic cell behaviours with embryonic polarity. Dev Cell. 2012;21:120–133. 7. Wells JM, Spence JR. How to make an intestine. Development. 2014;141:752–760. 8. Eiraku M, Sasai Y. Self-formation of layered neural structures in three dimensional culture of ES cells. Cur Opin Neurobiol. 2012;22: 768–777. 9. Lei NY, Jabaji Z, Wang J, et al. Intestinal subepithelial myofibroblasts support the growth of intestinal epithelial stem cells. PLoS One. 2014;9(1):e84651.

10. K  olesky DB, Homan KA, Skylar-Scott MA, Lewis JA. Three-dimensional bioprinting of thick vascularized tissues. Proc Natl Acad Sci U S A. 2016;113:3179–3184. 11. Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell. 2006;126:663–676. 12. Takahashi K, Yamanaka S. A developmental framework for induced pluripotency. Development. 2015;142:3274–3285. 13. Katz-Jaffe MG, McReynolds S, et al. The role of proteomics in defining the human embryonic secretome. Mol Hum Reprod. 2009;15: 271–277. 14. Botros L, Sakkas D, Seli E. Metabolomics and its application for non-invasive embryo assessment in IVF. Mol Hum Reprod. 2008;14:679– 690. 15. Graves PR, Haystead AJ. Molecular biologist’s guide to proteomics. Microbiol Mol Biol Rev. 2002;66:39–63. 16. Lombard-Banek C, Moody SA, Nemes P. Single-cell mass spectrometry for discovery proteomics: quantifying translational cell heterogeneity in the 16-cell frog (Xenopus) embryo. Angev Chem In Ed. 2016;55:2454–2458. 17. Visel A, Thaller C, Eichele G. GenePaint.org: an atlas of gene expression patterns in the mouse embryo. Nucleic Acids Res. 2004;32: D552–D556. 18. Pavie B, Rajaram S, Ouyang A, et  al. Rapid analysis and exploration of fluorescence microscopy images. J Vis Exp. 2014;(85): e51280.

19. N  ord AS, Pattabiraman K, Visel A, Rubenstein JLR. Genomic perspectives of transcriptional regulation in forebrain development. Neuron. 2015;85:27–47. 20. Kaufmann MH. The Atlas of Mouse Development. St. Louis: Elsevier; 1992. 21. O’Rahilly R, Müller F. Developmental Stages In Human Embryos. Publication 637. Carnegie Institution of Washington; 1987. 22. O’Rahilly R, Müller F. Developmental stages in human embryos: revised and new measurements. Cells Tisues Organs. 2010;192:73–84. 23. Liang G, Zhang Y. Genetic and epigenetic variations in iPSCs: potential causes and implications for application. Cell Stem Cell. 2013;13:149–159. 24. De Paepe C, Krivega M, Cauffman G, et  al. Totipotency and lineage separation in the human embryo. Mol Hum Reprod. 2014;20: 599–618. 25. Boyle J, Kreisberg R, Bressler R, Killcoyne S. Methods for visual mining of genomic and proteomic data atlases. Bioinformatics. 2012;13:58–68. 26. Hutter H, Moerman D. Big data in Caenorhabditis elegans: quo vadis? Mol Biol Cell. 2015;26:3909–3914.

5.e1

2

Cellular Mechanisms and Embryonic Tissues PATRICIA COLLINS

KEY POINTS • T his chapter describes the tissue types present in early embryos and the interactions between these tissues. • The membrane systems and cytoskeletal elements within a typical cell are reviewed. • Early embryos contain only epithelial and mesenchymal populations. • Each tissue type produces specialised extracellular matrix molecules and proteins which permit and encourage tissue interactions. • Specific interactions between developing epithelia and mesenchyme are presented, and the common cytokines and growth factors are discussed.

This chapter provides a brief overview of concepts of cells and tissues and the terminology by which developmental processes are described. It should be noted that the genetic programme of the early conceptus is now being studied by analysis of substances secreted1 and metabolites produced,2 some of the very early cell interactions between the fertilised oocyte and the maternal endothelium have been revealed,3 and the ways in which cell types interact before and during organogenesis is now being explored in human embryonic stem cell and human-induced pluripotent stem cell culture.4

General Characteristics of all Cells All cell types have a plasma membrane and internal organelles, and all are supported by a range of cytoskeletal structures. The details and arrangement of proteins within cells and within the matrices they synthesise encompass a vast region of research beyond the scope of this text. Only the main proteins and structures necessary for the appreciation of developmental processes are presented. Readers are recommended to consult other texts for further details.5

Plasma Membrane The plasma membrane of cells is composed of phospholipids molecules arranged in a bilayer. Within this layer are vast numbers of proteins which may reside entirely within the bilipid layer or 6

protrude intracellularly, extracellularly, or both. Proteins which project exteriorly are covered with carbohydrates as glycoproteins or proteoglycans and contribute to the glycocalyx of the cell (Fig. 2.1). The glycocalyx also contains glycoproteins and proteoglycans which have been secreted into the extracellular space around the cells and then absorbed onto the cell surface. Thus it is difficult to specify exactly where a cell plasma membrane ends and the surrounding extracellular matrix (ECM) begins. The glycocalyx is fundamental in cell–cell and cell–matrix communication. Cells place specific protein and carbohydrate groups into the glycocalyx when touching and adhering to other cells, when displaying cell markers to other cells, in blood clotting cascades and in inflammatory responses. The membrane systems (organelles) within the embryonic cells (i.e., nucleus, mitochondria, endoplasmic reticulum, Golgi apparatus, lysosomes and secretory vesicles) are also made of the plasma membrane; secretory vesicles can become incorporated into the external plasma membrane to release contained contents. Intracellular membrane systems are supported within the cytoplasm by a range of cytoskeletal elements which also allow cells to maintain surface specialisations (microvilli), to change shape (as in the movements of endocytosis and exocytosis), to move in specific directions (with the glycocalyx molecules) and to adhere strongly to a substrate when movement ceases. 

The Cytoskeleton The cytoskeleton is a highly dynamic network of protein filaments that extends throughout the cell. As it also allows the cell to move or move portions of its plasma membrane, the cytoskeleton is less like a bony framework and more like a moveable muscular system. Three types of protein filaments produce a diverse range of cytoskeletal elements, including actin filaments, microtubules and intermediate filaments (Fig. 2.2); these are synthesised from actin, tubulin and a range of fibrous proteins (e.g., vimentin, laminin, respectively). Actin filaments. Actin filaments are polar structures composed of globular molecules of actin arranged as a helix. They work in networks and bundles, often found just beneath the plasma membrane, where they crosslink to form the cell cortex. Actin filaments are used to change the shape of the plasma membrane, moving it outwards in projections or inwards in invaginations. Whereas discrete bundles of actin, anchored into the cortex, can produce thin spiky protrusions of the plasma

CHAPTER 2  Cellular Mechanisms and Embryonic Tissues

7

LUMEN

= Sugar residue

Adsorbed glycoprotein

Transmembrane proteoglycan

Glycocalyx

Glycolipid

Bilipid layer

Transmembrane glycoprotein

CYTOSOL

• Fig. 2.1  The glycocalyx is synthesised by epithelial cells. It is a feltwork of glycoproteins on the luminal aspect of an epithelium. It is the interface for communication between the lumen of a tube or body cavity and the cells.

Actin supporting microvilli

Cell cortex

Centrosome

Actin filaments

Microtubules

Intermediate filaments

• Fig. 2.2  Three types of cytoskeletal elements within a cell allow the cell to maintain a shape or move it. membrane, microvilli, sheet-like extensions of the membrane (lamellipodia) are supported by continuous flattened bundles of actin similarly anchored. Conversely, actin filaments can pull portions of the membrane inwards in the formation of endocytotic vesicles or in cell division. Here, contractile bundles of actin associated with the motor protein myosin form. Although myosin is most familiar in muscle fibres, nonmuscle cells contain various myosin proteins. Contractile bundles of actin filaments

and myosin filaments are synthesised for specific functions and then disassembled (e.g., during cell division after chromosomal separation, the plasma membrane constricts to the middle of the cell, allowing two daughter cells to separate). Such assemblies of actin and myosin are also found in development near the apical surface of epithelial cells where they play a role in folding of epithelial sheets and in mesenchyme where they can form stress fibres which allow the cells to exert tension on the ECM. 

8

SE C T I O N 1     Early Fetal Development

Microtubules. Microtubules are long, hollow cylinders

composed of the globular protein tubulin. They are much more rigid than actin filaments. Microtubules emanate from the centre of the cell in a region termed the centrosome. They lengthen by adding tubulin to the proximal end of each microtubule while subunits are lost from the distal end. The centrosome offers a focus and region of stabilisation for the proximal ends of the hundreds of microtubules in a cell; it also contains the two centrioles which are used by the cell when dividing. Vast numbers of microtubules extend in all directions to the plasma membrane and seem to ensure that the centrosome is at the centre of the cell. From this position, the microtubule array sites the other cellular organelles and holds them in place using a range of contact proteins. If a cell touches another cell, there may be internal movements of the organelles driven by the microtubules, resulting in repositioning of the centrosome. Microtubules display a dynamic instability, with new subgroups being added or subtracted very rapidly. The turnover of distal units can be slowed by contact with proteins close to the plasma membrane; this allows cells to maintain a particular shape and polarity. Microtubules are also used in cell-surface specialisations, where they form the basis of cilia in the familiar 9+2 arrangement of nine microtubule doublets around a pair of single microtubules.  Intermediate filaments. Intermediate filaments are made of a variety of proteins all formed from highly elongated fibrous molecules. They are arranged as rope-like fibres which span each cell often from one cell junction to another. They are termed intermediate filaments because their apparent diameter on electron microscopy is between that of actin filaments and thick myosin filaments. Specific varieties of intermediate filaments are present in epithelial and mesenchymal cells. Whereas epithelial cells contain keratin filaments, mesenchymal cells have vimentin and vimentin-related filaments, and in the cells which will develop a myogenic lineage, desmin filaments are seen. Neuroepithelial cells develop neurofilaments and glial fibrillary acidic protein filaments are seen in astrocytes. 

Embryonic Tissues Two early tissue arrangements can be seen in embryos – epithelia and mesenchyme. Individual cells within each arrangement secrete extracellular proteins which form the ECM. This structures the space around and within cell populations and provides the appropriate conditions for development.

Epithelia Cells composing epithelia are polarised with apical and basal surfaces. Whereas the apical surface commonly displays specialised features such as microvilli, the basal surface is the site of extracellular protein deposition in the form of a basal lamina. Laterally, the cells contact their neighbours via varieties of juxtaluminal junctional complexes which bridge the narrow intercellular clefts. Epithelial cell polarity factors regulate the relative size of the surfaces or domains and the internal organisation of the cytoskeleton.6 The transmembrane protein Crumbs specifies the apical domain, Baz/ PAR-3 controls the position and extent of the junctions, Scribble restricts the size of the junctional domains, an internal contractile actomyosin network which produces the planar polarity of epithelia is contiguous across the lateral borders of the cells through

E-cadherin adhesion,7 and the basal region displays integrins which link to the basal lamina. Basal lamina. Basal laminae are thin, flexible sheets of ECM which are made by and underlie epithelial cells (Fig. 2.3). Basal laminae are also found surrounding individual skeletal muscle fibres, fat cells and Schwann cells. The presence or absence of a basal lamina beneath an epithelium during development is of consequence. Basal laminae organise the proteins in adjacent cell membranes, induce cell differentiation and cell metabolism, serve as routes for cell migration and can influence cell polarity in those cells that touch them; they can change with time during development and thus can maintain a developmental impetus. The basal lamina is described in electron microscopic studies as having an electron lucent layer, the lamina lucida or rara, closest to the basal surface of the cell and an electron-dense layer, the lamina densa below. If the epithelial layer rests on underlying mesenchyme, a layer of collagen fibrils connects the basal lamina to the underlying tissue, sometimes with specialised anchoring collagen fibrils. The strength of this connection is important for development and growth. Many textbooks do not distinguish between the basal lamina as described and the basement membrane, a thicker layer which includes the basal lamina and extracellular components of the underlying connective-tissue matrix. Embryonic basal laminae are composed chiefly of laminin. Later, other extracellular molecules such as type IV collagen, perlecan and entactin (see below) contribute to the feltwork of the layer (see Fig. 2.3). In some regions of the body, basal laminae form specialised structures or units which have a specific function during development or in adult life. An example of this arrangement is seen in tooth development, where initially ameloblasts and odontoblasts are separated by a basal lamina. The ameloblasts deposit enamel directly onto one side of this basal lamina, and the odontoblasts deposit dentine onto the other (see Fig. 2.12); in this way, the tooth is formed. In both the kidneys and the lungs, the basal lamina from the specialised cells of the organ abuts directly onto the endothelial basal lamina, producing a selectively permeable barrier. In the kidneys, this is the glomerular basement membrane. In development, the basal lamina acts as a selective barrier to the movement of cells, and migrating cells will move along basal laminae but not through them. Cells beneath an epithelial layer see only the basal lamina which the overlying cells produce. Changes in the local basal laminal composition is one way by which the epithelial cells can communicate with the cells migrating beneath them. In adult tissues, the basal lamina permits the movement of macrophages, lymphocytes and nerve processes and plays an important part in tissue regeneration after injury.  Cell–Cell junctions. Juxtaposed cells usually do not touch. For contact to be established, the cells produce specific molecules which promote the development of a cell–cell junction between them (Fig. 2.4). Junctional complexes allow sheets of epithelial cells to act in concert in maintaining a barrier or in producing alterations in the overall epithelial morphology; they also permit cell–cell communication and are in this respect especially important in development. Cell junctions are classified into three main groups: (i) tight junctions, which prevent leakage of molecules between cells from one side of a sheet of cells to the other8; (ii) anchoring junctions, where the neighbouring cell membranes attach and are supported by cytoskeletal elements within the cells, either actin or intermediate filaments (this type of junction also anchors epithelial cells to the ECM); and (iii) communicating junctions, which

CHAPTER 2  Cellular Mechanisms and Embryonic Tissues

9

Key: Entactin Perlecan

Laminin Type IV collagen

•

Fig. 2.3  A basal lamina is synthesised by epithelial cells. It is a feltwork of proteins which attach to the epithelial cells and provide an attachment for underlying cells. It is the interface for communication between the extracellular space and the cells.

mediate the passage of electrical or chemical signals from one cell to another. The formation of these junctional complexes is dependent on a range of cell adhesion molecules (CAMs) (see Fig. 2.4). In cell– cell anchoring junctions (adhesion belts and desmosomes), the CAMs involved are termed cadherins; they are attached intracellularly to intermediate or actin filaments in the cell cortex.9 The latter run parallel to the plasma membrane; thus the actin bundles of adjacent cells are linked. Concomitant contraction of the actin bundles results in narrowing of the apices of the epithelial cells and rolling of the epithelial layer into a deep groove or a tube. Epithelial cells contact the underlying basal lamina they synthesise by different types of anchoring junctions (hemidesmosomes and focal contacts). In these cases, the transmembrane linker proteins belong to the integrin family of ECM receptors.10 Cytoskeletal filaments support the connection of the integrin within the cell membrane to the ECM.

Communication between adjacent epithelial cells is mediated by gap junctions. In forming a gap junction, each cell contributes six identical protein subunits (called connexins) which form a structure, similar to an old-fashioned cotton reel, termed a connexon. This is situated across the bilaminar membrane with the thicker rims extending into the extracellular and intracellular spaces. Each connexon is capable of opening and closing, thus controlling the gap. When two connexons from adjacent cells are aligned, a tubular connection is made between the cells. Each gap junction is really a cluster of apposed connexons which each permit molecules smaller than 1000 daltons to pass through them. In early embryos, most cells are electrically coupled to one another by gap junctions. Later in development, epithelial cells synthesise gap junctions at particular stages when it is inferred that information is passing from cell to cell. When gap junctions are removed, there is often a difference in differentiation in the cellular progeny. Gap junctions are seen in adult tissues (e.g., connecting cardiac

10

SE C T I O N 1     Early Fetal Development

Actin supporting microvilli

Key to cell junctions:

Junctional complex

Tight junction

Adhesion molecules:

Adhesion belt

Cadherins

Desmosome

Cadherins

Gap junction

Connexins

Actin Intermediate filaments

Hemidesmosome Integrins

Basal lamina

• Fig. 2.4  Junctional complexes form between epithelial cells, preventing passage between cells, permitting communication between cells, and joining cells to each other and to the basal lamina. Junctions are supported by adhesion molecules and the cytoskeletal elements within the cells.

myocytes to permit transmission of the electrical signals of the cardiac cycle). (For further information on CAMs, see Alberts et al.5) 

Mesenchymal Cells Mesenchymal cells, in contrast to epithelial cells, have no polarity and thus no directional surface specialisations. They have junctional complexes which are not juxtaluminal, and they produce extensive ECM molecules and fibres from the whole cell surface (Fig. 2.5). As development proceeds, proliferating mesenchymal populations begin to differentiate. This is often first seen by the upregulation of specific mRNA in the cell or in the production of different ECM molecules by selected progeny. Extracellular matrix. A substantial part of the developing embryo is made up of ECM. This name is given to the vast array of complex molecules which are secreted locally by mesenchymal cells and assembled into networks which structure the spaces between the embryonic cells. In early development, mesenchymal populations are composed of migrating epithelial cells and mesenchymal cells generated from germinal (proliferative) epithelia. It is the latter group which will give rise to the range of connective tissues seen in the adult. Connective tissues form the architectural framework of the body, and the matrices determine the tissue’s physical properties (i.e., in bone, cartilage or fascia). Variation in

• Fig. 2.5  Scanning electron microscope view of mesenchyme cells. Mesenchyme cells have no polarity. They control the space around them by their synthesis of extracellular matrix. (Photograph by Dr P. Collins.)

CHAPTER 2  Cellular Mechanisms and Embryonic Tissues

11

Basal lamina Basement membrane

Proteoglycans hooking into basal lamina

Collagen

Proteoglycan links Lamina propria Hyaluronic acid filling in gaps

Mesenchyme cell

• Fig. 2.6  Mesenchyme cells and extracellular matrix (ECM), forming a lamina propria beneath an epithelium. The mesenchyme cells synthesise collagens, proteoglycans and glycosaminoglycans as a framework and add other proteins (e.g., fibronectin) as necessary. ECM is attached to a basal lamina, forming a basement membrane.

the constituents and amount of the matrix molecules gives the diversity of connective tissues (Fig. 2.6). There are two main classes of ECM molecules, glycosaminoglycans (GAGs) which may be linked covalently to proteins as proteoglycans, and fibrous proteins, such as collagen, elastin, laminin and fibronectin. The GAG and proteoglycan molecules form a highly hydrated gel-like ground substance into which the fibrous proteins are embedded. Four main groups of GAGs have been described: (i) hyaluronic acid, (ii) chondroitin sulphate and dermatan sulphate, (iii) heparan sulphate and heparin and (iv) keratan sulphate. Hyaluronic acid is the simplest GAG. It is especially abundant in embryos, where it fills the spaces between cells and because of its level of hydration becomes turgid and generally resists compression. By synthesising hyaluronic acid, cells can open up migration pathways or support epithelia which are undergoing morphological change. The other GAGs, which are more complex than hyaluronic acid, have much shorter disaccharide chains, contain sulphated sugars and are usually bound to a protein core forming proteoglycans. Aggregates of hyaluronate and proteoglycans can make huge molecules which occupy a volume equivalent to a bacterium. Within tissues, GAGs can form gels of varying pore size and thus act as filters regulating the movement of molecules according to their size or charge. The heparan sulphate proteoglycan perlecan is found in the basal lamina of the kidney glomerulus and functions as a filter in the glomerular basement membrane.

Proteoglycans which bind various growth factors act as reservoirs for messages which can be positioned in the matrix by cells at one stage to be read by cells developing later. GAGs and proteoglycans associate with the fibrous proteins in the matrix and provide support between the fibres. Collagens form the major fibrous proteins in the matrix. Several families or groups of collagens are described, each made from a range of basic α-chains and each encoded by a separate gene. Each collagen molecule is made from three α-chains wound around one another like a rope. The main types of fibrillar collagen found in connective tissue are types I, II, III, V and XI. These types aggregate into the huge hawser-like bundles which can be seen on electron microscopy. Collagen types IX and XII are smaller and link the larger fibres to one another and to other matrix molecules. Types IV and VII are found in the basal laminae, where type IV forms the feltwork of the mature basal lamina, and VII forms the anchoring structures which attach the basal lamina to the underlying matrix. Collagen is used to provide the initial matrices for cartilage and bone and is particularly seen in tendons and ligaments. In these cases, the amount of GAGs is reduced, and the collagen fibres are aligned by the fibroblasts in response to the direction of the stresses acting on the collagen. In this way, the connective tissues of the body are responsive to the physical demands placed upon them. Anomalies of collagen synthesis can give rise to diseases in life (e.g., the condition osteogenesis imperfecta is caused by mutations in type I collagen production, leading to bones which are brittle and

12

SE C T I O N 1     Early Fetal Development

fracture with little stress; mutations in type II collagen lead to disorders of cartilage). Elastin is composed of short elastin proteins which are crosslinked so that when relaxed, the fibres are randomly coiled but when stretched each elastin molecule can expand so contributing to the overall effect. Elastic fibres are at least five times more extensible than a rubber band of the same cross-sectional area. Usually collagen fibres are interwoven with elastic fibres to prevent overstretching and damage. Laminin is one of the first extracellular proteins synthesised in the embryo, forming most of the early basal laminae. Each molecule is shaped like an asymmetric cross. Molecules join together to form a feltwork often supported by smaller entactin molecules. Fibronectin is a high-molecular-weight glycoprotein found in extracellular matrices and in blood plasma. Within the ECM, fibronectins promote cells adhesion. Generally, contact with fibronectin causes cells to move. It has been shown in culture that neural crest cells preferentially migrate along fibronectinrich substrates, and within three-dimensional cultures, migrating cells can achieve their greatest speeds migrating along fibronectin pathways. It is interesting to note that bonding with fibronectin does not necessarily fix a cell to one position within the matrix, so contacts with this protein are made and then released with ease. Biomechanical properties of the ECM. It is now appreciated that mechanical characteristics and movements of the ECM are fundamental to developmental processes and that mesenchymal cells are sensitive to the tension and stiffness of the matrix itself. The application of shear forces to matrix molecules results in fibre alignment along the axis of force and the development of tension: an individual mesenchymal cell is able to sense parts of the a matrix as soft or stiff depending on whether the tension is generated perpendicular (soft) or parallel (stiff) to a fibre.11 ECM movements occur across tissue-level scales exhibiting both vortical and convergent extension motion patterns. The physical-chemical and biochemical reactions which drive matrix fibre assembly collectively exert compression and stretching forces on embryonic cell arrangements.12,13  Cell–Matrix junctions. Cells interact with the ECM molecules via protein receptors or co-receptors in the plasma membrane. Syndecans are proteoglycan co-receptors which span the plasma membrane. The extracellular part carries chondroitin sulphate and heparan sulphate chains while the intracellular domain interacts with the cell cortex actin filaments. Integrins are receptor proteins which bind to and respond to the ECM information. An extracellular receptor site binds with matrix molecules, especially fibronectin and laminin; intracellularly, the integrin binds via vinculin to the actin cytoskeletal network or intermediate filaments. When the integrins in the cell membrane contact a matrix molecule (e.g., fibronectin), the orientation of the fibronectin will cause alignment of the actin cytoskeleton and a reorientation of the cell itself. Later, when the cells are depositing ECM molecules, the actin cytoskeleton will exert forces to orient the matrix molecules in a similar configuration. Thus the interaction between matrix molecules and cells and then cells and matrix can drive development and propagate order from cell to cell. The cell– matrix junctions permit communications within the embryo just as gap junctions permit communications between cells. However, the cell–matrix mechanisms allow messages to be left in the matrix which may indicate migration routes or halt migrating cells and suggest a differentiation pathway. The matrix information system can thus control the temporal pattern of development. 

Transformation from Epithelium to Mesenchyme The two embryonic states of epithelial tissue and mesenchyme are not necessarily immutable, and transition from epithelia to mesenchyme and vice versa occurs during development. However, such a change requires a temporal or external inductive agent and causes dramatic upregulation and synthesis of a whole variety of special intra- and extracellular molecules as previously described. Generally, in an embryo, epithelial cells seem to derive from existing epithelial populations, but mesenchymal cells are produced initially from proliferative (germinal) epithelia and then later by amplifying mitoses within the mesenchymal population. Changes from one cell state to another are considered important in development. The early migrating cells derived from the primitive streak have a mesenchymal morphology yet become epithelial when they reach their destinations forming the somites, the somatopleuric and splanchnopleuric epithelia lining the intraembryonic coelom, including the lining of the pericardial cavity. All of these epithelia become germinal centres which produce further mesenchymal populations. However, an additional group of mesenchymal cells derived from the neural crest never reverts to epithelia. Angiogenic mesenchyme, which is believed to arise from extraembryonic sources initially, is especially proliferative and capable of great migration within the embryo; it differentiates into endothelium throughout early development. A small subset of endothelial cells in the heart are induced to transform back to mesenchyme at the atrioventricular canal and the proximal outflow tract. This may be the only example of a mesenchymal population derived from an endothelial lineage14; the cells retain expression of an endothelial marker. 

Embryonic Induction and Cell Division The earliest cells of the embryo are described as ‘totipotent’, indicating that they have the capacity to differentiate into any cell type in the body. The pathways cells take to differentiation depend on the regulation of the genes within the cell and the interaction of the cell with its environment and neighbours. Information from these other sources causes shifts in the differentiative fate of the cell’s progeny. All embryonic cell populations are initially receptive to inductive signals. They respond by becoming committed to a particular pathway of development which thus restricts their ability to respond to further inductive influences (i.e., they become restricted). After a series of restrictions, cells are said to become determined. Determined cells are programmed to complete a process of development which will lead to differentiation. The determined state is a heritable characteristic of cells and can be passed on to progeny; it is stable and not dependent on environmental factors. The differentiated state is usually not heritable; it is often dependent on environmental conditions, and it may prevent further cell division. The state of determination or differentiation may be assessed by studying cells in culture. For example, melanocytes display the black pigment melanin. If melanocytes are cultured without the tyrosine needed to synthesise melanin, the cells will become pale and no longer appear differentiated. When the tyrosine is replaced into the culture medium, the cells will again synthesise melanin and return to their differentiated state, illustrating that they maintained their determination despite not being able to display their differentiation. Similar cultures of cells taken from embryos at

CHAPTER 2  Cellular Mechanisms and Embryonic Tissues

Proliferative and Quantal Mitoses Within a mesenchymal population, cell divisions will both increase the total numbers of cells but also provide the foci for changes in determination. In normal cell division, sometimes described as proliferative, transient amplifying or multiplicative mitoses, progeny similar to the parents are produced. In some situations (e.g., as a response to a local inductive influence), the cells will enter a quantal cell cycle in which the outcome is a quantal mitosis which increases the restriction of their progeny. The progeny then continues in amplifying mitoses at the progressive level of determination. It is at these quantal mitoses that binary choices are made in the embryo. 

Determinate tissues (number fixed near birth)

Neurons Skeletal muscle fibres Seminiferous tubules Renal nephrons Heart muscle fibres Pulmonary alveoli Intestinal villi Ovarian follicles

Indeterminate tissues (number not fixed near birth)

different stages of development will reveal which types of protein the cells are capable of synthesising and thus their level of determination or differentiation. Cell proteins have been classified as ‘basic’, or ‘housekeeping proteins’, if they are considered essential for cellular metabolism and are termed ‘primary’, as are the genes which regulate them. As cells become determined, they synthesise proteins appropriate to their cell group (e.g., liver and kidney cells, but not myocytes, produce arginase); this class of protein is termed ‘secondary’. Finally, at the most differentiated state, cells produce ‘tertiary’ proteins (also called ‘luxury proteins’) specific to their needs (e.g., haemoglobin in erythrocytes). This range of proteins – primary, secondary and tertiary – is an expression of stages of determination and differentiation, and they are coded by a range of genes.

13

Thyroid follicles Hepatic chords Exocrine acini Endocrine cells Blood cells

Maturity

Birth 1

3

6

9

1

3

6 9 10

30 6090

Prenatal months Postnatal years Duration of hyperplasia

• Fig. 2.7  The duration of multiplicative growth for various human tissues, (After Gilbert SF. Developmental Biology, Sinauer Associates, MA, 1992. By permission of Oxford University Press, USA.)

Stem Cells and Progenitor Cells Within a proliferating cell population, instead of a division producing two progenies with increased determination, mitosis may produce one determined progeny and another cell with the same state of determination as the parent. This latter cell can reproduce again, passing on an increased state of determination to only one offspring. This type of division has been termed ‘asymmetric’ in contrast to the ‘symmetrical’ proliferating mitoses. Stem cells are seen in development and in adult life (e.g., in the bone marrow or in the gut epithelium). Progenitor cells are those which are already determined to some extent. They may individually follow their differentiation pathway or may proliferate producing more similarly determined progenitor cells. In this case, no stem cell can be identified; all cells seem capable of either differentiation of continued mitosis. An example is populations of migrating myoblasts in the embryonic limb bud. It should be noted that the ability of stem cells to continue cell division is intricately linked to the metabolic activity of these cells. Stem cells use glycolytic pathways to generate energy, a method related to the low oxygen tension prevalent during early development and organogenesis and a necessary requirement of human stem cells grown in culture.15 Consideration of mitochondrial structure and metabolism in early embryos suggests a dynamic interplay between developmental and differentiation processes informed by cell signalling and epigenetic regulation.16 

Terminal Differentiation The differentiative fates of cells within embryos may be to become long-living lymphoblasts or neuroblasts but may just as well be to undergo ‘apoptosis’, also called ‘programmed cell death’. Apoptosis is a mechanism whereby cells bequeath their organelles to

neighbouring cells without releasing any cellular fragments which might stimulate an inflammatory response. Apoptosis occurs in the adult state as well as in embryos. In organogenesis, apoptosis allows for some slack in the system. More cells than may be needed are produced by amplifying mitosis; later the cells are supported by the local ECM or by innervation or blood supply. Those cells which are in excess and cannot be supported by these means undergo apoptosis.17 As different tissues might be expected to produce different sets of survival factors, a cell in an abnormal location deprived of its specific signals required for survival would also die. The times at which cells cease proliferative mitoses and become differentiated is different for different tissues (Fig. 2.7). Although some tissues will all enter determined pathways and differentiation before birth, other cells retain the ability to divide (e.g., as stem cells) or if environmental conditions changes, as in a wound, are able to revert temporarily to the determined state, affect a repair and then differentiate once again 

The Cell Cycle Determination, differentiation and development, in general, all result from a series of interactions in which information from one cell is presented to another and as a consequence the behaviour of one or both cells are changed. Earlier embryonic studies described the changes in shape in individual cells and cell populations which were seen in development and then how these might be modified by experimentation. Studies of genes and proteins within embryonic cells and tissues have elucidated some of the drivers and checks of development, including when they appear within the cell cycle.

14

SE C T I O N 1     Early Fetal Development

An increase in cell restriction begins with a quantal cell division; thus the mechanisms of mitosis need to be examined. Cells which are undergoing amplifying mitoses pass through a cell cycle which lasts for 12 hours or more. The cell cycle is traditionally divided into four phases, of which the most dramatic is mitosis and cell division (Fig. 2.8). The phases of the cell cycle are noted by the letters M for mitosis and G1, S and G2 for the interphase. During mitosis, the cell packs up most of its organelles, the centrioles duplicate and move to each end of the cell and begin synthesis of microtubules which are arranged as the mitotic spindle, and the chromatin in the nucleus condenses into the chromosomes. As the cell has already replicated its DNA, the chromosomes which become visible at metaphase are duplicated but held together at the centromere. The nuclear membrane breaks down, and the chromosomes gather at the equator of the cell and are drawn apart towards the poles of the spindle, where they decondense and reform intact nuclei. The cell cytoplasm divides by cytokinesis, an event which is viewed as the end of the M phase. G1 is the time period when the cell grows until it is large enough to begin DNA synthesis. It may move along to S phase (synthesis) when it has reached an appropriate size and if the environmental conditions are favourable. If the cell is not yet committed to DNA replication or is going to follow a differentiation pathway, the cell can step out of the cycle into G0 for days, weeks or longer before resuming proliferation. The majority of cells in adults are in G0. The S phase marks replication of the nuclear DNA and is followed by G2, which is the time taken for further growth. At the end of G2, the cell must be of sufficient size, have replicated all its DNA and be in a suitable environment before it can continue into mitosis. Signals from the cells or from the environment can prevent the cell moving from one phase to another. The cycle itself is driven by complexes of cyclins, so called because they undergo synthesis and degradation in each division cycle of the cell. The cyclins bind to cyclin-dependent protein kinases to trigger mitosis and to trigger DNA replication. They thus provide a checkpoint for exit from G1 and entry into S phase and exit from G2 and entry into M phase. In early embryonic cell divisions, the zygote and blastomeres are very large. Growth is not required at this time, and the cleavage divisions operate to restore the nuclear-to-cytoplasm ratio,

is

G2

Sy nt

s he

Mitosis

+

G1

• Fig. 2.8  The four stages of the cell cycle. After cell division (mitosis), the cell grows continuously until the next mitosis. The phases G1, G2 and S are parts of interphase. (If the cell is not dividing, it enters G0.)

producing cells of typical size. Under these conditions, cells pass through M phase and S phase in quick succession. After each division, the cell progeny is half the size of the parent. Later, the cycle lengthens, and various control systems come into operation. It is important that the rate of passage through the S phase allows completion of DNA replication. If a cell enters M phase before replication is complete, it will die. The cell cycle control system receives a feedback signal from incompletely replicated DNA, which will prevent movement to the G2 phase. Similar protective mechanisms ensure that DNA is only replicated once during S phase. 

Tissue Interactions For the information carried in the genome of an embryo to be expressed, the cells produced by mitotic division must be able to contact and respond to each other; this process occurs locally by the construction of gap junctions. However, as an embryo grows, it is composed of more and more cell populations, which become differentiated into tissues and separated by the matrix molecules they produce. In these more complex situations, the tissues have a repertoire of communication methods which require both epithelial and mesenchymal arrangements and their matrices working in concert. From a starting point of an early embryo at the body plan stage, all of the basic organs arise through interactions between close epithelial populations (plus their basal laminae), epithelia and mesenchyme (with the ECM) and between differentiating mesenchymal populations. The basic interactions are the same. Such sequential spatial and temporal reciprocal interactions have been termed epigenetic cascades.18

Permissive Interactions It has been shown by experimental study that neither epithelia nor mesenchyme will grow alone; each needs the other for DNA synthesis and mitosis to occur. However, in many cases, it is not the cell bodies that are required. Epithelial cells will grow in culture as long as there is some sort of mesenchymal extract in the medium, and mesenchymal cells will grow in culture as long as they can contact a basal lamina. Thus in both cases, factors in the matrix which have arisen from the cell population are enough to support growth. These basic requirements of development are termed permissive interactions. The supporting tissue may not be that usually present in development, and indeed much research time has been spent in investigating which mesenchymal tissues would support growth of specific epithelia and vice versa. However, in many cases, although growth was maintained in these experiments, development would not proceed at all or in the normal manner. For such development, more information is needed from the reciprocating tissues, and such information could enable both tissues to change in a manner that neither of them could do without the information. This is the basis of instructive interactions. 

Instructive Interactions Wessells19 proposed four general principles which can be seen in most instructive interactions: • In the presence of tissue A, responding tissue B develops in a certain way. • In the absence of tissue A, responding tissue B does not develop in that way. • In the absence of tissue A but in the presence of tissue C, tissue B does not develop in that way.

CHAPTER 2  Cellular Mechanisms and Embryonic Tissues

Narrow cleft

Hyaluronidase produced by mesenchyme cells

15

Collagen

Collagen fibrils Mesenchyme cells Hyaluronidase Epithelial cells Collagen fibrils

A

B • Fig. 2.9  Branching of a tubular duct may occur as a result of an interaction between the proliferating epithelium of the duct and its surrounding mesenchyme and extracellular matrix. A, Mesenchymal cells initiate cleft formation by producing collagen III fibrils locally within the development clefts and hyaluronidase over other parts of the epithelium. Collagen III prevents local degradation of the epithelial basal lamina by hyaluronidase and slows the rate of mitosis of the overlying epithelial cells. B, In regions where no collagen III is produced, hyaluronidase breaks down the epithelial basal lamina and locally increases epithelial mitoses, forming an expanded acinus (arrows). (After Gilbert SF. Developmental Biology, Sinauer Associates, MA, 1992. By permission of Oxford University Press, USA.)

• I n the presence of tissue A, a tissue D, which would normally develop differently, is changed to develop like B. Thus in an instructive interaction, one tissue induces another to respond in a specific way. If the target tissue can respond to the inductive signal, it is called competent. If the target tissue does not respond, it is described as non-competent. Non-competence may be because the tissue has previously responded to an earlier inductive signal which has restricted its repertoire of possible responses. As development proceeds, more and more cell populations will become non-competent as they differentiate. Inductive interactions may be more or less complicated: only the induced tissue may change or both tissues may change and participate, as in morphogenesis, or, more commonly, several reciprocal inductive interactions may be required over a prolonged period of developmental time before a specific organ or tissue will form. 

Epithelial–Mesenchymal Interactions The instructive interactions between epithelia and mesenchyme produce the general morphological changes which are seen in every system throughout embryogenesis. They are a subset of embryonic tissue interactions occurring between epithelial tissue and specific subdivisions of mesenchymal tissue which arise during differentiation. These interactions provide a mechanism for coordinating and fine tuning the mitotic rates and differentiative abilities of the two tissues. A range of interactions occurring in different systems of the embryo is described.

Branching Morphogenesis Most organs, from the lungs to the kidneys, initially develop a main duct from which branches arise often dichotomously; later these mature into typical patterns seen in the fully formed organ. The mechanism of branching morphogenesis is therefore similar in a variety of systems. Although the early descriptions of this process described only the morphogenesis, now the specific matrix molecules involved during the interaction have been identified (Fig. 2.9). At the tip of a proliferating duct, the epithelium and its basal lamina is in contact with the underlying mesenchymal cells and their ECM molecules. Local production of hyaluronidase by the

mesenchyme breaks down the basal lamina and promotes proliferation of the epithelium. Cleft production is initiated by the mesenchyme, which produces type III collagen fibrils within putative clefts. The collagen acts to protect the basal lamina from the effects of the hyaluronidase and the overlying epithelia have a locally reduced mitotic rate. The region of rapid mitoses at the tip of the acinus is thus split into two, and two branches develop from this point. If the type III collagen is removed from the cleft, branching does not occur; if excess collagen is not removed, supernumerary clefts appear. Note that this interaction may occur with any epithelial type and any subpopulation of mesenchyme.20 

Neural Ectoderm and Surface Ectoderm Interactions The interactions described are clearly seen in formation of the lens of the eye. The optic cup is an outgrowth of the diencephalon of the brain. As the cup approaches the overlying surface ectoderm, it induces a local change. The ectoderm cells become narrower at the apical region, causing the sheet of cells to curve and move towards the optic cup. Ultimately, a small lens vesicle invaginates, and the unaffected surface ectoderm becomes confluent over the structure. If the optic cup is removed, no lens vesicle develops. If the optic cup is removed and placed beneath a different portion of surface ectoderm, a similar lens vesicle is induced (Fig. 2.10). 

Neural Ectoderm and Neural Crest Mesenchyme Interactions: The ‘Fly-Paper Model’ of Skull Development The subtle interplay between the mesenchyme and the epithelial basal lamina is demonstrated in the ‘fly-paper model’ of skull development.21 Here, an interaction occurs between the neuroectoderm of the neural tube and the surrounding neural crest mesenchyme. The neuroepithelium displays fibronectin amongst other fibrous proteins in the basal lamina. This ensures the migration of the neural crest over the neural tube and into the developing face. As development proceeds, the neuroepithelium transiently expresses type II collagen in the basal lamina on the basal aspect of the neural tube, around the olfactory regions, around the optic

16

SE C T I O N 1     Early Fetal Development

(iv) Isolated optic cup can still induce lens vesicle

(iii) Tissue other than optic vesicle implanted—no induction

(ii) No optic vesicle No lens induced

(i) Lens vesicle induced by underlying optic cup

• Fig. 2.10  Induction of the lens vesicle by the optic cup. This illustrates clearly the four general principles of an instructive interaction. (Wessells,Tissue Interactions and Development, 1st ed., ©1977. Reprinted by permission of Pearson Education, Inc., New York.)

Chondrocranial elements contributing to base of the skull

Ventrolateral surfaces

Transient collagen type II distribution seen in neuroectoderm of:

– Diencephalon

Olfactory capsule

Olfactory region

Trabecula

– Mesencephalon – Rhombencephalon

Transient collagen type II distribution seen in neuroectoderm of:

Optic capsule

Optic vesicles

Otic capsule

Otic vesicles

Parachordal

Notochord First vertebra

• Fig. 2.11  Transient expression of type II collagen in the basal lamina of the neuroepithelium causes mesenchyme cells which touch it to upregulate their own synthesis of type II collagen and differentiate along a chondrogenic pathway. The pattern of expression in the neuroectoderm determines the form of the chondrocranium. (From Thorogood, P., Bee, J. & Von Der Mark, K. (1986). Transient expression of collagen type II at epitheliomesenchymal interfaces during morphogenesis of the cartilaginous neurocranium. Devi Biol. 116, 497-509.)

cups before and during lens invagination, around the otic vesicles which will form the inner ear, and on the basal and lateral surfaces of the diencephalon, mesencephalon and rhombencephalon. The notochord also expresses type II collagen in its basal lamina. Some time after the type II collagen has been removed from the basal laminae, neural crest mesenchyme adjacent to the regions

listed commences synthesis of type II collagen and ultimately differentiates along a chondrogenic pathway. It seems as if the type II collagen in the basal lamina affects the cells which contact it and causes their upregulation of type II collagen. The pattern of the expression of type II collagen in the basal laminae determines the form of the chondrocranium (Fig. 2.11). Slight alterations in

CHAPTER 2  Cellular Mechanisms and Embryonic Tissues

Epithelial cell–tissue transformations

ORAL ECTODERM

Dental epithelium

Enamel organ

17

Pre-ameloblasts Ameloblasts ENAMEL

Epithelium Epithelium causes mesenchyme to condense

Mesenchyme

Epithelial– mesenchymal interactions

Dental papilla induces epithelium to form an enamel organ Dental mesenchyme induces oral epithelium to become dental epithelium

Mesenchymal cell–tissue transformations

JAW MESENCHYME

Enamel organ epithelium induces dental papilla mesenchyme to become pre-odontoblasts and odontoblasts

Odontoblastic mesenchyme induces epithelial pre-ameloblasts to become ameloblasts

Forming dental papilla

Dental mesenchyme

Dental papilla

Pre-odontoblasts

Odontoblasts Predentine DENTINE

•

Fig. 2.12 A summary of the epithelial–mesenchymal interactions during tooth development. (From Gray’s Anatomy, 41st ed. St. Louis: Elsevier, 2015.)

the pattern of expression could have profound effects on the shape and form of the chondrocranium, perhaps producing the diversity of skull shapes seen in vertebrates. 

Surface Ectoderm and Neural Crest Interactions A further example of a reciprocal tissue interactions can be seen in mammalian tooth development summarised in Fig. 2.12.22 Tooth development begins along the dental lamina of the premaxilla, maxillae and mandible. The epithelium proliferates to form an enamel organ under the influence of the neural crest mesenchyme, which forms a dental papilla; together this unit is a tooth bud or germ. The enamel organ induces the dental papilla mesenchyme to become odontoblasts; these cells then induce the epithelial cells to differentiate into ameloblasts. The tooth is formed by matrix deposition each side of the epithelial basal lamina, enamel on one side and dentine on the other. The neural crest mesenchyme is responsible for patterning development of the pharyngeal arches, including tooth formation. Dental papilla mesenchyme is able to induce the formation of teeth in nonoral epithelium and can specify the type of tooth produced (e.g., incisor or molar). If cranial neural crest is cultured alone, it will differentiate into cartilage. When it is recombined with limb epithelium, then cartilage and bone will form. However, if cranial neural crest is recombined with mandibular epithelium, salivary islands, hair and teeth form as well as cartilage and bone, indicating that the mandibular epithelium is essential and specific for the development of teeth. Early recombination (9–11.5 days of development) of mouse mandibular epithelium (first arch) and hyoid mesenchyme (second arch) results in teeth in 90% of cases, showing that the dental lamina epithelium can induce tooth development in the head neural crest mesenchyme. The reverse recombination – early second arch epithelium and mandibular arch mesenchyme – does not produce teeth, indicating that it is only the first arch epithelium which has this property. However, later recombination

experiments (11.5–12 days of development), with second arch epithelium and first arch mesenchyme, will produce teeth. In this case, the neural crest mesenchyme has already been induced along a dental lineage.23 The local specification of particular teeth can be changed experimentally. If presumptive incisor region of the mandibular epithelium is recombined with predetermined molar papillae from post-day 12 tooth germs, the shape of the teeth can be redefined by the epithelium and incisiform teeth develop. 

Surface Ectoderm and Somatopleuric Mesenchymal Interactions in the Limb The tissues involved in limb development arise from a ridge along the flank of the embryo. Interaction of specialised regions of the somatopleuric mesenchyme with the overlying ectoderm gives rise to local, thickened regions of surface ectoderm and proliferation of the underlying mesenchyme. At these sites, the ectoderm forms a longitudinal ridge of high columnar cells, the apical ectodermal ridge (AER) and the specialised somatopleuric mesenchyme maintains its growth. The AER and underlying mesenchyme together are termed the progress zone, and the limb grows meristematically from this point (i.e., proliferation produces the next distal portion of the limb). These two populations require each other. Only the apical ectodermal ridge can promote limb outgrowth, and only limb mesenchyme will result in limb development. Positional values are assigned to the proliferating epithelial and mesenchymal populations by the progress zone; thus first the humerus is developed, then the radius and ulna, then the carpals and so on (Fig. 2.13). Within the portion of the limb which has received its positional value, the mesenchyme instructs the overlying ectoderm about the appropriate epidermal structure to develop. Parts of a chick hind limb develop different characteristics with feathers on the thigh and scales on the leg. If mesenchyme from the thigh is transplanted beneath the leg, feathers will develop instead of the normal scales. This type of experiment has been repeated by recombining neck

18

SE C T I O N 1     Early Fetal Development

Specification of axes of the limb Proximal Pr

e-

l

Ventral

st

ia

ax

ia

l 2. Craniocaudalyaxis

1. Proximodistalyaxis

Dorsal

Po

Distal

ax

3. Dorsoventralyaxis

1. Proximodistalyaxis

Progress zone Extra AER

Duplicated distal limb

Apical ectodermal ridge (AER)

Somatopleuric limb mesenchyme Leg features grow distally on wing

Leg mesenchyme 2. Craniocaudalyaxis

III

Extra ZPA IV

II III IV

Zone of polarising activity (ZPA) specifies the post axial border

Extra ZPA grafted on other side

Postaxial border with digit IV duplicated

3. Dorsoventral axis

ZPA dispersed in mesenchyme – replaced into ectoderm sleeve

No pre-axial or postaxial specification Direction of joints still indicates dorsoventral axis

•

Fig. 2.13  The three axes of the limb are specified by different interactions. The proximodistal axis is specified by the progress zone, the craniocaudal axis by the zone of polarising activity, and the dorsoventral axis by the ectoderm of the limb. The pattern of development within the limb and the ectodermal specialisations are controlled by the limb mesenchyme.

ectoderm, which will not normally develop feathers, with thigh mesenchyme; in this case, the epidermis will develop feathers. If thigh mesenchyme is inserted beneath the apical ectodermal ridge of a developing wing which is at the stage to assign radius and ulna, two things occur. First, information from the

apical ectodermal ridge about the age of the limb will override the ‘thighness’ of the mesenchyme, and it will express leg characteristics appropriate to the developmental time of the wing. Thus the proximal limb characteristics are replaced by distal ones. Second, the wing epithelium is reassigned to develop leg characteristics by

CHAPTER 2  Cellular Mechanisms and Embryonic Tissues

the mesenchyme. Thus the limb develops a fibula and tibia and the epidermis displays scales. This latter experiment shows the reciprocal nature of inductive interactions with both tissues giving and responding to information. Positional information which causes the development of the axes of the limb is controlled by both mesenchyme and epithelium. A subset of mesenchyme situated at the postaxial border of the limb bud termed the zone of polarising activity (ZPA) controls the cranio-caudal axis of the limb (i.e., where the thumb develops). Active substances released at this region cause a number five digit to form, the little finger (Fig. 2.13). The reducing influence of this substance allows development of digits four, three, two and then the thumb to develop. However, even if this system is disrupted by mixing the mesenchyme up within the limb producing five equally structured digits, the limb still displays dorsal and ventral surfaces. The inductive influence for dorsal and ventral patterning is specified by the ectodermal epithelium. 

Endoderm and Splanchnopleuric Mesenchyme Interactions in the Lung The respiratory tree derives from interactions between endodermal epithelium and surrounding splanchnopleuric mesenchyme. The trachea arises from the pharynx as a midline, ventral diverticulum which grows causally then bifurcates into the primary bronchi, which expand dorsally on each side of the oesophagus. Originally, splanchnopleuric mesenchyme surrounding the pharynx envelopes both the oesophagus and the trachea; however, the proximity of the lung buds to the pericardio-peritoneal canals, which will later give rise to the pleural cavities provides a different mesenchymal population. Proliferation of the adjacent splanchnopleuric coelomic epithelium (of the primary pleural cavities) provides investing mesenchyme around the developing trachea and lung buds from stage 13 of development. The proliferative activity decreases in stage 14, and the mesenchyme becomes arranged in zones around the developing endoderm. This investing mesenchyme contains a mixed population of cells, that which will pattern the endodermal epithelium, a subpopulation of angiogenic mesenchyme which may migrate in to form the endothelial networks surrounding the air sacs, and splanchnopleuric mesenchyme which will give rise to the smooth muscle cells which surround both the respiratory tubes and the blood vessels. After its proliferative phase, the splanchnopleuric coelomic epithelium gives rise to the visceral pleura. The control of the branching pattern of the respiratory tree resides with the splanchnopleuric mesenchyme and particularly Fgf10 expression.24 Whereas recombination of tracheal mesenchyme with bronchial respiratory endoderm causes an inhibition of bronchial branching, recombination of bronchial mesenchyme with tracheal epithelium induces bronchial outgrowths from the trachea.19,25 Initially, the tracheal mesenchyme is continuous with that surrounding the ventral wall of the oesophagus, but with lengthening and division of the tracheal bud and deviation of the lung buds dorsally, each bud becomes surrounded by its own specific mesenchyme, thus permitting regional differences between the lungs (i.e., the number of lobes or the degree of growth and maturity of a particular lung). Each lung develops by a process of dichotomous branching as described in branching morphogenesis. Tenascin, an ECM molecule (also known as hexabrachion or cytotactin), is present in the budding and distal tip regions but absent in the clefts. Conversely, fibronectin is found in the clefts and along the sides of the developing bronchi but not on the budding and distal tips.26 

19

Endoderm and Splanchnopleuric Mesenchyme Interactions in the Gastrointestinal Tract Liver. The liver is a very precocious embryonic organ, functioning as the main centre for haemopoiesis in fetuses. It develops from an endodermal evagination of the foregut and from the septum transversum mesenchyme, a region of unsplit lateral plate mesenchyme at the very rostral edge of the disc before head folding. The development of the liver is intimately related to the development of the heart as the vitelline, followed by the umbilical veins, are disrupted by the septum transversum to form a hepatic plexus the forerunner of the hepatic sinusoids. Endodermal epithelial cells from the foregut proliferate and extend as lines of epithelial cells into the septum transversum mesenchyme. Contact of endodermal epithelium with the mesenchymal cells induces them to form blood islands and endothelium. The advance of the endodermal epithelial cells promotes the conversion of more and more septum transversum mesenchyme into endothelium and blood cells with only a little remaining to form the scanty (human) liver capsule and interlobular connective tissue. The morphogenesis of the liver lobes is patterned by the septum transversum mesenchyme, and both endoderm and septum transversum mesenchyme are required for normal liver development.27 If a mechanical barrier is inserted across the mesenchymal hepatic area just caudal to the endodermal outgrowth, liver tissue will develop normally cranial to the barrier where it is in contact with the endoderm. However, caudal to the barrier, the mesenchyme will form endothelial cells and hepatic lobes, but there will be no hepatocytes present. Experiments in which either the epithelium or the mesenchyme is changed will not result in liver development (e.g., cephalic and somitic mesenchyme do not promote the differentiation of hepatic endoderm, and intestinal endoderm cells combined with hepatic mesenchyme will not differentiate into hepatocytes). However, all derivatives of the lateral plate mesenchyme, both somatopleuric and splanchnopleuric mesenchyme, can promote the differentiation of hepatic endoderm, although not so strongly as hepatic mesenchyme, and lateral plate mesenchyme will form blood sinusoids under these conditions. It is inferred that matrix or cell surface properties are common throughout the lateral plate mesenchyme but are different from axial mesenchymal cells.28  Gastric mucosa. Within the gastrointestinal tract, the local organisation of the mucosa and smooth muscle layers is under the control of the local splanchnopleuric mesenchyme. Recombination experiments of chick gut epithelium and mouse mesenchyme and vice versa show that the patterning of the intestinal villi is determined by the underlying mesenchyme. However, the epithelial cells produce enzymes associated with the relevant species (e.g., mouse epithelium produces lactase and chick epithelium sucrase, regardless of the origin of the underlying mesenchyme).29 Indeed, culture of human pluripotent stem cells has resulted in the development of human small intestinal organoids, which can be transplanted into mice to mature.30,31 

Intraembryonic Mesoderm and Intermediate Mesenchyme Interactions The metanephric kidney develops from three sources, an evagination of the mesonephric duct, the ureteric bud; a local condensation of mesenchyme termed the metanephric blastema; and

20

SE C T I O N 1     Early Fetal Development

angiogenic mesenchyme, which migrates into the metanephric blastema slightly later to produce the glomeruli and vasa recta.32-34 It may also be the case that innervation is necessary for metanephric kidney induction. During embryonic development, functional mesonephric kidneys develop but are remodelled in male embryos as parts of the reproductive tracts. The mesonephric kidneys develop on the posterior abdominal wall and extend ultimately from the pleural region to the lumbar, with both development and regression proceeding in a craniocaudal progression. However, in the metanephric kidney, a proportion of the mesenchyme remains as stem cells, which continue to divide and enter the nephrogenic pathway later as individual collecting ducts lengthen. Thus the temporal development of the metanephric kidney is patterned radially with the outer cortex being the last part to be formed. In each kidney, a ureteric bud arises as a diverticulum from the mesonephric duct and grows dorsally to enter the metanephric mesenchymal population. The bud bifurcates when it comes into contact with the metanephric blastema as a result of local ECM molecule synthesis by the mesenchyme.35 Both chondroitin sulphate proteoglycan synthesis and chondroitin sulphate GAG processing are necessary for this and consequent branching of the ureteric bud.36 Subsequent divisions of the ureteric bud and the mesenchyme form the gross structure of the kidney with major and minor calyces; the distal branches of the ureteric ducts form the collecting ducts of the kidney. As the collecting ducts elongate, the metanephric mesenchyme condenses around them. The ureteric bud undergoes a further series of bifurcations within the surrounding metanephric mesenchyme, forming smaller ureteric ducts. The metanephric mesenchyme condenses around the dividing ducts into smaller condensations, which then undergo a mesenchyme to epithelium transformation forming vesicles. To initiate this transformation, the cells cease production of mesenchymal matrix molecules and commence production of epithelial ones. This has been demonstrated in tissue culture. Initially, syndecan can be detected between the mesenchymal cells in the condensate. The cells cease expression of the cadherin N-CAM, fibronectin and collagen I and commence production of the cadherin L-CAM (also called ‘uvomorulin’) and the basal laminal constituents laminin and collagen IV. The mesenchymal clusters thus convert to small groups of epithelial cells which undergo complex morphogenetic changes (Fig. 2.14). Each epithelial group elongates and forms a comma-shaped and then an S-shaped body, which elongates further. It then fuses to a branch of the ureteric duct at its distal end while expanding as a dilated sac at the proximal end. The sac involutes with local cellular differentiation such that the outer cells become the parietal glomerular cells, while the inner ones become visceral epithelial podocytes. The podocytes develop in close proximity to invading capillaries, which derive from local angiogenic mesenchyme37; this third source of mesenchyme produces the endothelial and mesangial cells within the glomerulus. Both the metanephric-derived podocytes and the angiogenic mesenchyme produce fibronectin and other components of the glomerular basal lamina. The isoforms of type IV collagen within this layer follow a specific programme of maturation, which occurs as the filtration of macromolecules from the plasma becomes restricted.38 Interestingly, although the interactions in kidney development were usually focused on the induction of metanephric mesenchyme by the ureteric bud epithelium, it had been noted that when

cultured across a filter, the inductive stimulus was quite weak. In contrast, fragments of spinal cord were found to be very potent inducers of metanephric mesenchyme, initiating epithelialisation. This suggested that perhaps nerves arriving at the interaction site during development were of some importance. Indeed, it has been shown that blocking nerve growth factor receptor mRNA in the developing kidney not only prevents receptor synthesis but also that nephrogenesis is also completely halted.39 The use of human pluripotent stem cells provide additional study routes of embryonic kidney cell populations; however, it is not yet clear how well these in vitro populations reflect normal human kidney development.40 

Other Cells Types Affecting or Affected by Local Interactions It seems that the basic epithelial–mesenchymal interaction may not be so basic or simple after all. Whereas initial observations were made on cultured embryos, later studies were made on cultured embryonic explants which could be perturbed in some manner. The range of inductive tissues was ascertained using other mesenchymes or epithelia from the same embryo from different embryos of the same species and even from different species. Now it seems that the homogeneity of the mesenchymes under examination may have been assumed. The presence of angiogenic mesenchyme may be fundamental for some interactions. Early in development, it arises extraembryonically from the parietal hypoblast. Later, angiogenic mesenchyme is seen close to endodermal epithelia but not ectodermal. It is not clear whether the majority of angiogenic mesenchyme arises close to endodermal populations as it has now been shown to arise from somite derived mesenchyme.41 Early embryonic angioblasts are highly invasive, moving in every direction throughout embryonic mesenchymal tissue. It is likely that these cells contribute to interactive processes, especially in those organs where close proximity of endothelia to specialised cells is a feature. Similarly, the innervation of blood vessels, glandular cells and myoepithelial cells may need to be achieved early in development, at the time of the early interactions. Indeed, the condition Hirschsprung disease, which results in a failure of neural crest cells to colonise the gut appropriately and form local constituents of the enteric nervous system, is thought to occur because of disordered basal laminal molecules. The enteric nervous system arises from neural crest cells at somite levels 1 to 7 and from 28 onwards. Normally, the crest cells invade the splanchnopleuric mesenchyme around the endoderm and site themselves in the putative submucosal and myogenic layers. The splanchnopleuric mesenchyme will develop into both the lamina propria connective tissue lineages and the smooth muscle cells of the muscularis mucosa and the muscularis propria. Hirschsprung disease is characterised by a dilated segment of colon proximally and lack of peristalsis in the segment distal to the dilation. Infants with this condition show delay in the passage of meconium, constipation, vomiting and abdominal distension. A mouse model has been investigated which demonstrates the same pathology and symptoms.42 In normal mice, laminin and type IV collagen are found beneath the mucosal and serosal epithelia and around the blood vessels. In mutant mice, there is a broad zone of these basal laminal proteins around the entire outer gut mesenchyme with an increase in the amount of laminin, type IV collagen and heparan sulphate,

CHAPTER 2  Cellular Mechanisms and Embryonic Tissues

Epithelial ureteric bud and metanephric mesenchyme

21

Metanephric mesenchyme transforms to form comma-shaped epithelial vesicle

Comma-shaped vesicles become S-shaped

Distal Renal corpuscle Proximal

Tubule simple cuboidal

S-shaped vesicles elongate, forming parts of nephron The proximal end invaginates and, with blood vessels, forms the renal corpuscle

Capsule (simple squamous)

Podocyte

Loop

• Fig. 2.14  In the developing kidney there is a mesenchyme-to-epithelial transformation. The epithelial ureteric bud forms the collecting ducts in the kidney. It induces the metanephric mesenchyme to transform into local epithelial vesicles, which develop into the nephrons.

specifically in the aganglionic portion of bowel. It is suggested that the overabundance of basal laminal components prevents the neural crest cells from penetrating the gut wall; their new position outside the gut does not confer on them the environmental stimuli for enteric nerve differentiation. Consequently,

these crest cells differentiate into autonomic ganglia and nerves similar to the parasympathetic nerves which normally modulate enteric nervous system activity. This system demonstrates the importance of local mesenchymal and ECM activity for the normal induction of neurons as well as epithelial cells. 

22

SE C T I O N 1     Early Fetal Development

Cytokines and Growth Factors As a result of experiments to find the most appropriate media for maintaining the proliferation of cells in culture, two families of ‘factors’ were identified, arising initially from lymphocytes and from platelets. Culture of lymphocytes and macrophages revealed a family of factors within the supernatant which was able to facilitate cell–cell communication and proliferation. These soluble factors were isolated and collectively termed cytokines. The cytokines include subtypes of interleukin (IL), which cause the main T- and B-cell proliferation; types of interferon (IFN), which are mainly antiviral in nature; tumour necrosis factor (TNF) α and β, which are cytotoxic; transforming growth factor β (TGFβ), which inhibits T- and B-cell proliferation; and granulocyte-macrophage colonystimulating factor (GM-CSF) with factors for granulocytes and macrophages individually, which promote growth. The proliferative actions of cytokines are mediated by specific cell receptors on the surface of target cells. Culture of mammalian cells was found to be more successful with blood serum added to the medium rather than plasma. Whereas serum is the fluid which remains after blood has clotted, plasma is obtained by removing the cells without permitting clotting to occur. The difference between these two fluids is the presence of factors released from platelets as the blood clotted. Experiment showed that an extract of platelets alone added to the medium would support cell-culture proliferation, and the extract was termed a growth factor. The particular growth factor from the platelets was shown to be a protein and named platelet-derived growth factor (PDGF). For PDGF to have an effect on a target cell, the cell must display an appropriate receptor protein on its surface as in the action of cytokines. Receptor proteins for cytokines and growth factors are part of the cell glycocalyx along with inter alia CAMs and integrins. Large families of growth factors have now been identified, not all of them proteins; steroid hormones which act on intracellular receptor protein are an example. Growth factors have been divided into broad- and narrow-specificity classes or families. PDGF is a broad-specificity factor as is epidermal growth factor (EGF). PDGF acts on fibroblasts, smooth-muscle cells and neuroglial cells, and EGF acts on epidermal cells and on many embryonic epithelia. Other broad-specificity growth factors are the insulin-like growth factors (IGFI, IGFII) (previously termed ‘somatomedins’), stimulating cell metabolism and with other growth factors stimulating cell proliferation; fibroblast growth factor (FGF) with subgroups, again being inductive in embryos; and transforming growth factor β (TGFβ), having been identified in lymphocytes and also grouped as a cytokine yet being produced widely by many cell types. The TGFβ family also includes activins and bone morphogenetic proteins (BMPs), which similar to TGFβ, may suppress growth as well as stimulate it. Of the narrow-specificity factors, nerve growth factor (NGF) and related brain-derived neurotrophic factor (BDNF), and neurotrophins 3 and 4 promote survival of neurons; erythropoietin promotes proliferation and differentiation of erythrocytes; and

interleukin-3 (IL-3) and related haemopoietic colony-stimulating factors (CSFs), which are also classed as cytokines, stimulate proliferation of blood cell precursors. Cytokines and growth factors can signal a wide variety of cellular effects, including stimulation or inhibition of growth, differentiation, migration and so on.43 Because each family is so large and because there are a similarly extensive number of receptors, the possible signalling combinations are considerable. Often, a single growth factor can bind with varying affinities to individual receptor family members. Whereas some of these receptors are monogamous, recognising only one isoform of the growth factor, others are polygamous, recognising all isoforms. The effects of any individual growth factor may therefore depend on which receptor isoform, or ratio of isoform receptors, is displayed on the cell surface. It is now clear that developmental information resides not in any single molecule but rather in the combination of molecules, to which a cell is exposed as development progresses. Thus varying combinations of growth factors, in varying concentrations, can elicit quite different effects on similar cells.44 As well as an expansion in the range of growth factors identified within development, knowledge of the genes which code for them and ways of demonstrating them has also contributed to the complexity of understanding embryological processes. Many genes have been shown to be ubiquitous throughout embryos and conserved through evolution, making viable transfer of tissues between animal groups possible. Responses to deletion of specific gene action, or application of cytokines and growth factors at inappropriate times and places in the embryo add to the body of knowledge, but because interactions are driven by complex cascades of many growth factors, each study can only add a small piece to the jigsaw. Stern45 noted that embryos generate complexity with only a few extracellular signals, each of which has multiple roles at different developmental times. Thus each time we appear to understand a developmental process and declare a ‘default’ pathway of development that is understood, more complexity is uncovered. 

Conclusion This chapter gives an overview of the morphology of the cells within an embryo and how they join to form tissues. The cell membrane and the intercellular organelles which affect it are considered along with the cytoskeletal elements which support apical specialisations and cell membrane movements and give the three-dimensional shape to a range of body cells. Epithelia and mesenchyme are presented, and the importance of the transition of one cell phenotype to the other is considered. The main epithelial–mesenchymal interactions, including examples of branching morphogenesis, are outlined for a range of developing systems. The terminology for cell–cell interactions and the cell cycle is presented. An overview of the main families of cytokines and growth factors is given. Access the complete reference list online at ExpertConsult.com. Self-assessment questions available at ExpertConsult.com.

References 1. Katz-Jaffe MG, McReynolds S, Gardner DK, Schoolcraft WB. The role of proteomics in defining the human embryonic secretome. Mol Hum Repro. 2009;15:271–277. 2. Syggelou A, Iacovidou N, Atzori L, et  al. Metabolomics in the developing human being. Pediatr Clin North Am. 2012;59:1039–1058. 3. Macklon NS, Bronsens JJ. The human endometrium as a sensor of embryo quality. Biol Reprod. 2014;91(98):1–8. 4. Takasato M, Little MH. The origin of the mammalian kidney: implications for recreating the kidney in  vitro. Development. 2015;142:1937–1947. 5. Alberts B, Johnson A, Lewis J, et al. Molecular Biology of the Cell. 6th ed. New York: Garland Science; 2014. 6.  St Jonston D, Sanson B. Epithelial polarity and morphogenesis. Curr Opin Cell Biol. 2011;23:540–546. 7. Gorfinkiel N, Blanchard GB. Dynamics of actomyosin contractile activity during epithelial morphogenesis. Curr Opin Cell Biol. 2011;23:531–539. 8. Rajasekaran SA, Beyenbach KW, Rajasekaran AK. Interactions of tight junctions with membrane channels and transporters. Biochimica et Biophysica Acta. 2008;1778:757–769. 9. Garrod D, Chidgey M. Desmosome structure, composition and function. Biochimica Biophysica Acta. 2008;1778:572–587. 10. Walko G, Castañón MJ, Wiche G. Molecular architecture and function of hemidesmosomes. Cell Tissue Res. 2015;360:529–544. 11. Doyle AD, Yamada KM. Mechanosensing via cell-matrix adhesions in 3D microenvironments. Exp Cell Res. 2016;343(1):60–66. 12. Zamir EA, Rongish BJ, Little CD. The ECM moves during primitive streak formation— computation of ECM versus cellular motion. PLoS Biol. 2008;6(10):e247. 13. Atherton P, Stutchbury B, Jethwa D, Ballestrem C. Mechanosensitive components of integrin adhesions: role of vinculin. Ext Cell Res. 2016;343(1):21–27. 14. Snarr BS, Kern CB, Wessels A. Origin and fate of cardiac mesenchyme. Dev Dyn. 2008;237: 2804–2819. 15. Rafalski VA, Mancini E, Brunet A. Energy metabolism and energy-sensing pathways in mammalian embryonic and adult stem cell fate. J Cell Sci. 2012;125:5597–5608.

16. Folmes CDL, Terzic A. Metabolic determinants of embryonic development and stem cell fate. Reprod Fertil Dev. 2013;27:82–88. 17. Zakeri Z, Penaloza CG, Smith K, et al. What cell death does in development. Int J Dev Boil. 2015;59:11–22. 18. Hall BK. Evolutionary Developmental Biology. London: Chapman and Hall; 1992. 19. Wessells NK. Tissue Interactions and Development. Menlo Park, CA: Benjamin/Cummings; 1977. 20. Harunaga JS, Doyle AD, Yamada KM. Local and global dynamics of the basement membrane during branching morphogenesis require protease activity and actomyosin contractility. Dev Biol. 2014;394:197–205. 21. Thorogood P. The developmental specification of the vertebrate skull. Development. 1988;103(suppl):141–153. 22. Lumsden AGS. Neural crest contribution to tooth development in the mammalian embryo. In: Maderson PFA, ed. Developmental and Evolutionary Aspects of the Neural Crest. New York: Wiley; 1987:261–300. 23. Jernvall J, Thesleff I. Reiterative signaling and patterning during mammalian tooth morphogenesis. Mech Dev. 2000;92:19–29. 24. Agha EE, Bellusci S. Walking along the fibroblast growth factor 10 route: a key pathway to understand the control and regulation of epithelial and mesenchymal cell-lineage formation during lung development and repair after injury. Scientifica (Cairo). 2014;2014:538379. 25. Hilfer SR, Rayner RM, Brown JW. Mesenchymal control of branching pattern in the fetal mouse lung. Tissue Cell. 1985;127:523–538. 26. Abbott LA, Lester SM, Erickson CA. Changes in mesenchymal cell-shape, matrix collagen and tenascin accompany bud formation in the early chick embryo. Anat Embryol. 1992;183:299–311. 27. Asahina K, Zhou B, Pu WT, Tsukamoto H. Septum transversum-derived mesothelium gives rise to hepatic stellate cells and perivascular mesenchymal cell in developing mouse liver. Hepatology. 2011;53:983–995. 28. Le Douarin N. An experimental analysis of liver development. Med Biol. 1975;53:427–455. 29. Haffen K, Kedinger M, Simon-Assmann P. Cell contact dependent regulation of enterocyte differentiation. In: Lebenthal E, ed. Human Gastrointestinal Development. New York: Raven Press; 1989. 30. Watson CL, Mahe MM, Múnera J, et  al. An in vivo model of human small intestine using pluripotent stem cells. Nat Med. 2014;20:1310–1314.

31. Finkbeiner SR, Freeman JJ, Wieck MM, et  al. Generation of tissue-engineered small intestine using embryonic stem cell-derived human intestinal organoids. Biol Open. 2015;12:1462–1472. 32. Grobstein C. Inductive interaction in the development of the mouse metanephros. J Exp Zool. 1955;130:319–340. 33. Saxen L, Koskimies O, Lahti A, et  al. Differentiation of kidney mesenchyme in an experimental model system. J Adv Morphogen. 1968;7:251–293. 34. Saxen L. Failure to demonstrate tubule induction in a heterologous mesenchyme. Dev Biol. 1970;23:511–523. 35. O’Brien LL, McMahon AP. Induction and patterning of the metanephric nephron. Semin Cell Dev Biol. 2014;36:31–38. 36. Fouser L, Avner ED. Normal and abnormal nephrogenesis. Am J Kidney Dis. 1993;21:64– 70. 37. Ekblom P, Sariola H, Karkinen-Jaaskelainen M, Saxen L. The origin of the glomerular endothelium. Cell Differentiation. 1982;11:35–39. 38. Bard JBL, Woolf AS. Nephrogenesis and the development of renal disease. Neurol Dial Transplant. 1992;7:563–572. 39. Sariola H, Saarma M, Sainio K, et  al. Dependence of kidney morphogenesis on the expression of nerve growth factor receptor. Science. 1991;254:571–573. 40. Takasato M, Little MH. The origin of the mammalian kidney: implications for recreating the kidney in  vitro. Development. 2015;142:1937–1947. 41. Christ B, Huang R, Scaal M. Formation and differentiation of the avian sclerotome. Anat Embryol. 2004;208:333–350. 42. Gershon MD. Phenotypic expression by neural crest-derived precursors of enteric neurons and glia. In: Maderson PFA, ed. Developmental and Evolutionary Aspects of the Neural Crest. New York: John Wiley; 1987. 43. Sporn MB, Roberts AS. Peptide growth factors and their receptors. Vols. 1 and 2. Berlin: Springer-Verlag; 1990. 44. Jessell TM, Melton DA. Diffusable factors in vertebrate embryonic induction. Cell. 1992;68:257–270. 45. Stern C. Neural induction: old problem, new findings, yet more questions. Development. 2005;132:2007–2021.

22.e1

3

Staging Embryos in Development and the Embryonic Body Plan PATRICIA COLLINS

KEY POINTS • T his chapter presents the concepts and timing of embryonic development. • The problems of using staging systems to describe a continuous process are discussed. • The method behind staging of animal development is presented. • A revision of the timing of early human development is presented. • Embryonic and obstetric stages of development are presented. • The embryonic body plan, main embryological stages and their approximate times are presented.

A variety of staging systems for human embryos were devised in the early years of the past century. To enhance this information, studies on other animals were undertaken and externally similar embryos compared. However, devising a staging system is very different from describing a day-by-day alteration of external characteristics.

Chick and Mouse Embryo Staging Series In recent years, the external characteristics of laboratory animal species have been available and shown within staging schemes. Computing power now permits the manipulation of external images, sectional information and three-dimensional (3D) representations of internal structures in embryos. Databases of developmental information of laboratory animals have been collated in collaborative projects by those involved in experimental embryology. Chick development was described by Hamburger and Hamilton as a series of 46 stages over the 20-day incubation period.1ab-3 Photographs and movies of all stages of chick development are available on an online database, e-Chick Atlas. For the mouse similar staging systems have been developed by Theiler4 based on the Streeter staging of human embryos (see later) and continued by Kaufman.5 Kaufman noted the care with which specimens needed to be prepared and sectioned and the level of experience necessary for the interpretation of serial sections in order to understand the complexity of spatial and temporal development of body systems and internal organs. The

use of magnetic resonance imaging (MRI) to obtain images of both external features and sectional anatomy is available online (emouseatlas.org) based on Kaufman’s original images. MRI studies of mouse embryos are also providing a further way of seeing digital images through any plane.6 Both chick and mouse databases now display the expression of a range of genes within the developing organs and tissues linked to sections and 3D reconstructions of embryos. 

Human Stage Series Staging of human embryos has always started from a different standpoint to those used in animal series; stages are not a serialisation of external features. Human embryos were first placed in a stage series by Mall,7 founder of the Department of Embryology of the Carnegie Institution of Washington. His work was continued by George Streeter8-11 in the 1940s and O’Rahilly and Müller since that time.12-15 The monograph Developmental Stages in Human Embryos12 has been a mainstay of embryonic developmental stages. Human embryos are assigned to a stage based on the developmental status of many body systems in concert, not on any one parameter alone. Development from fertilisation averages 266 days, or 9.5 months; the embryonic period, extending from fertilisation to about 58 days, is divided into 23 stages. The embryonic period ends when bone marrow is seen replacing cartilage in the humerus, a time defined by Streeter. The original studies on human staging were based on 600 embryos within the Carnegie Collection obtained from hysterectomies, and specimens were formalin fixed. It is not clear if all of these embryos were normal. The time at which an embryo enters and leaves a particular stage varies because of a number of factors, including placental health, genetic factors and individual growth rate. In vivo imaging techniques of very early development prompted the revision of some of the ages previously assigned to early embryonic stages. O’Rahilly and Müller15 note that greatest length, the length of an embryo, exclusive of the lower limbs, is a valuable parameter which can be measured antenatally. By adding the number 29 to the greatest length, within the range 3 to 33 mm, an age in days can be broadly estimated. The revision of stage, the age and the specific measurement of greatest length are taking time to percolate through newer embryological studies. It would be of benefit to all if the references for the staging system used were given and terms used were specified. The main features 23

SE C T I O N 1     Early Fetal Development

of the stages of human development are given before a consideration of obstetric staging of the first trimester of pregnancy. 

are becoming defined and result in neurulation and the beginning of somite formation, more clearly seen in stage 10, in which embryos typically have 7 to 12 pairs of somites. The formation of the neural plate and the beginnings of its rostral fusion contribute to the morphological movements of head folding, when the cardiac area, which was rostral to the neural epithelium, becomes ventral and forms a boundary of the cranial intestinal portal. Stages 6b to 10 are concerned with embryogenesis when morphogenetic movements affecting the whole embryo move widely dispersed cell populations closer and into their relevant positions for local interactive processes to commence. A stage 11 embryo is at the gateway of organogenesis, and all body systems can be seen to originate from this point. 

Main Stages in the Embryonic Period

Trophoblast

Oocyte

Syncytiotrophoblast

Lacunae

Cytotrophoblast

Chorion

Ootid

Zygote

Neural crest

11

10 After folding

Neural tube

9 Somites

Intraemb. coelom

Neural groove

8 Before folding

7 Notochord

Intervillous spaces Villi

Primary yolk sac

Parietal hypoblast

Neurenteric canal

6b

Secondary yolk sac

5c

6a

The criterion for stage 11 is the presence of 13 to 20 pairs of somites (Fig. 3.2); during this stage, the rostral neuropore closes.

Extraemb. mesenchyme

Visceral hypoblast Hypoblast

The Stage 11 Embryo, Body Plan Stage

Implanted previllous

5b

5a

4 Implantation

3 Free blastocyst hatched from zona

2 Compaction

Preimplantation

Fertilisation 1 First cleavage

Description of stage

Embryonic development is not apparent before stage 6. Stages 1 to 5 are concerned with setting up the cell populations for implantation; most of the cell lines generated are extraembryonic and involved in establishing the placenta and fetal membranes. In stage 6a, the primordial germ cells are sequestered into the extraembryonic mesoblast, and in stage 6b, the primitive streak appears. From this time, intraembryonic cell populations are generated, and the morphological movements of these populations produce a recognisable embryo. Fig. 3.1 shows the formation of cell populations during stages 1 to 11 matched to estimated age. The proliferation of cells at the primitive streak occurs through stages 6b, 7 and 8, when the notochord is first evident, and provides cell populations which pass within the embryo. By stage 9, the neural populations

Primitive streak

24

Haemopoiesis

Secondary yolk sac

Inner cell mass

Allantois

Extraemb. mesoblast

Connecting stalk

Epiblast Amnion Primordial germ cells Primitive streak Notochordal process/plate Embryonic endoderm Mesoblast

Mesenchyme Somatopleuric and splanchnopleuric coelomic epithelium Epithelial somites

Caudal eminence Neural ectoderm

Neural crest Ectodermal placodes

Surface ectoderm Approx. age in days

1

2–3

4–5

6

16–18 7–12

•

18–21

24–27 21–25

Fig. 3.1 Developmental processes occurring during the first 10 stages of development. In the early stages, a series of binary choices determines the cell lineages. Generally, the earliest stages are concerned with formation of the extraembryonic tissues, and the later stages are concerned with the formation of embryonic tissues. (From Gray’s Anatomy, 41st ed. St. Louis: Elsevier, 2015.)

28–30 26–29

CHAPTER 3  Staging Embryos in Development and the Embryonic Body Plan

Fusion occurs from the rhombencephalon rostrally towards the mesencephalon and from the region of the future optic chiasma towards the mesencephalic roof. The optic primordia have begun evagination towards the surface ectoderm. The otic vesicle which will invaginate and give rise to the inner ear (cochlea and semicircular ducts) has not yet formed but the surface epithelium has thickened and begun to invaginate. Around the developing pharynx, the mandibular processes are present, but the maxillary processes have yet to arise. The second pharyngeal arch, the hyoid, is present but not the third. Ventral to the foregut is the heart, this develops very precociously and is seen in stage 9 embryos as tube-like with a united ventricular portion but still separate atrial components. The specialised cells of the coelom, which will give rise to the myocardium, can be identified as can the matrix produced locally between the endocardium and the myocardium. Cardiac contractions commence at the beginning of stage 10 when the heart has a recognisable ventricle, bulbus cordis and arterial trunk, and a cardiac loop can be distinguished; the organ is already asymmetrical. By stage 11, the sinus venosus, atria, left and right ventricles, truncus arteriosus and substantial dorsal aortae can be identified. The heart is connected to a range of endothelial vessels and plexuses which are most mature cranially and still forming caudally. The fluid in the vascular system contains relatively few cells. It ebbs and flows because of the pulsations of the myocardium, which also cause movement of fluid in the intraembryonic coelom. These

25

combined circulations are sufficient to provide nutrient supply to the embryonic tissues. The intraembryonic coelom is especially important at this stage of development. The coelom arises from confluent spaces which appear in the embryo from stage 9. As head folding occurs, these spaces coalesce to form a horseshoe-shaped cavity within the embryonic body, which passes between the endoderm of the gut and the ectoderm of the body wall on each side of the developing fore- and midgut, and meets in the midline beneath the rostral neuropore as the future pericardial cavity. The ends of the horseshoe are in wide communication with the extraembryonic coelom around the embryo and within the chorion. The walls of the coelom are composed of germinal epithelia, which provide mesenchymal cell populations for the connective tissues and smooth muscle of the respiratory and gastrointestinal tracts, the body wall and especially the heart. The myocardium arises directly from the dorsal pericardial wall in the folded embryo. The ventral pericardial wall will give rise to the serous, parietal pericardial layer. The foregut develops as the head fold elevates and the pericardial cavity swings ventrally. It is flattened dorsoventrally, extending laterally to form the pharyngeal pouches. The buccopharyngeal membrane is present at this stage and may begin to rupture. There is only small indication of the future respiratory primordium. The liver is represented by the septum transversum mesenchyme and underlying endoderm, but the latter is still widely connected to the yolk sac. The nephric system consists of a solid nephrogenic

Neural tube

Pharynx Pericardial cavity

Pericardial cavity

Amnion

Pericardioperitoneal canal Yolk sac wall

Foregut

Entrance to pericardioperitoneal canal Neural tube

Somite Midgut

Yolk sac wall

Left umbilical vein

Peritoneal cavity Hindgut

Umbilical cord

A

B • Fig. 3.2  A, The embryo at stage 11, showing the position of the intraembryonic coelom (contained by the walls coloured blue). B, The three major epithelial populations within a stage 11 embryo, viewed from a ventrolateral position. The neural tube lies dorsal to the gut. Ventrally, the intraembryonic coelom crosses the midline at the level of the foregut and hindgut but is lateral to the midgut and a portion of the foregut. (From Gray’s Anatomy, 41st ed. St. Louis: Elsevier, 2015.)

26

SE C T I O N 1     Early Fetal Development

cord lateral to somites 8 to 13. The cloacal membrane is situated ventrally after tail folding just caudal to the connecting stalk which passes to the developing placenta. The primordial germ cells can be identified in the embryo at stage 11. They are initially in the mesenchyme around the yolk sac and allantoic walls. With tail folding, they are brought into the body cavity with the hindgut epithelium and surrounding mesenchyme, and then by amoeboid movement and by growth displacement, they migrate dorsocranially. They do not reach the gonads until stage 15, some 9 days later, when the local cell populations are developed sufficiently to receive them. During stage 11, the most important epithelial layers attain their position (i.e., surface epithelium, notochord, neural epithelium, somites, gut epithelium and the lining of the coelomic cavity; see Fig. 3.2). The germinal epithelia of the somites and the coelomic cavity then generate extensive mesenchymal populations at the same time that the neural crest mesenchyme cells are migrating within the head and neck. Finally, each epithelium is surrounded and supported by different mesenchymal populations (i.e., the neural tube and notochord are surrounded by neural crest mesenchyme in the head and somatopleuric mesenchyme in the trunk). The gut epithelium is surrounded by splanchnopleuric mesenchyme, which becomes specialised, particularly around the respiratory diverticulum. The surface

Mandibular process

First pharyngeal arch

Neurulation Pericardial cavity and heart

ectoderm is supported by somatopleuric mesenchyme in the trunk and by neural crest mesenchyme in the head. The coelomic epithelia themselves produce specialised epithelial populations which give rise to the gonads, adrenal medulla and the lining of the uterine tubes; these epithelia are all supported by local mesenchyme. Angiogenic mesenchyme is found throughout the embryo from stage 9. It is capable of extensive migration and differentiates into endothelium or blood cells throughout the embryo. Angiogenic mesenchyme is found within the endodermal and splanchnopleuric layers but never within ectoderm derived tissues. Stage 11 may be considered to be the body plan stage. To achieve it, genes functioning across the whole embryo are in operation. As stage 11 fades into stage 12, organogenesis is underway, and the epithelial–mesenchymal interactions which result in all development are now operating along local lines. Diversity of differentiative outcomes is now possible by upregulation of specific genes in specific regions of the embryo. Between stage 11 and stage 23, a period of about 30 days, the embryo grows in length from 3 mm to 30 mm. It passes from a general vertebrate embryo to a fully formed but immature human. Fig. 3.3 illustrates the dramatic increase in size and developmental status during this time, and Fig. 3.4 shows the relative time at which individual systems progress in development. 

Maxillary process

Pinna

Eyelids

Optic vesicle

Otic vesicle

Somites

Cerebral hemisphere Yolk sac

Connecting stalk

Hand

Upper limb bud

Stage 10

Connecting stalk

Connecting stalk

Lower limb bud

Stage 13

Umbilical cord

Stage 16

Stage 18

Reflection of amnion Epiblast population Primitive node Primitive streak Connecting stalk

Stage 6

Embryonic stage Size (mm) Approximate age (days)

6

10

13

16

18

20

23

0.4

1.5–3

4–6

8–11

13–17

21–23

28–30

16–18

26–29

30–33

35–40

41–45

46–50

53–58

• Fig. 3.3  The external appearance and size of embryos between stages 6 and 23. Early in development, external features are used to describe the stage (e.g., somites, pharyngeal arches or limb buds). (From Gray’s Anatomy, 41st ed. St. Louis: Elsevier, 2015. Adapted with permission from Rodeck CH, Whittle MJ. Fetal Medicine. London: Churchill Livingstone, 1999.)

CHAPTER 3  Staging Embryos in Development and the Embryonic Body Plan

Embryonic stages Weeks post-ovulation

External appearance

6

1

2

7

8

3

9

10 11 12 13 14 15 16

4

5

Head and tail folding Pharyngeal arches

19

20

21 22 23

7

8

9

Upper lip

Digits on hand

Palate

External ear

Anterior lobe pituitary

Otic vesicle

First neural crest cells

18

6

Neurulation

Nervous

17

27

10

11

12

Eyelids fuse

Posterior lobe pituitary

Optic cup

Membranous labyrinth

Trachea Lung buds

Respiratory

Further division of bronchi

Primary bronchi

Gastrointestinal

Fore-, mid-, Thyroid hindgut Liver

Pharyngeal pouches dorsal and ventral

Midgut loop returns to abdomen

Midgut loop rotating

Pancreas Urorectal septum

Rotation of stomach

Mesonephros

Urinary

Metanephric nephrons

Mesonephric duct

Major calyces

Ureteric bud Germ cells in allantois wall

Reproductive

Kidneys ascend

Minor calyces

Müllerian ducts

Indifferent gonad

Uterus and uterine tubes

Testis differentiating

Vagina

External genitalia indifferent

Cardiovascular

Primitive vascular system

Septum primum

Septation of ventricles

Heart beats

Spleen

Testis at inguinal canal Prostate

External genitalia differentiating

Septum secundum

Heart tube

Musculoskeletal

Somite period 20 days .................................. 30 days Forelimb bud

Forelimb digit rays Hindlimb bud

Cartilaginous

Membranous

part of skull

part of skull

• Fig. 3.4  A timetable of development of the body systems. The development of individual systems can be seen progressing from left to right. Embryonic stages and weeks of development are shown. Embryonic stages are associated with external and internal morphological features rather than embryonic length. To identify the systems and organs at risk at any time of development, follow a vertical progression from top to bottom. (From Gray’s Anatomy, 41st ed. St. Louis: Elsevier, 2015.)

Obstetric Timing and Staging of Embryos and Fetuses The obstetric timescale is involved in estimating a day of delivery and then assessing the fetus to see if it seems appropriately aged to deliver at that time. The commencement of gestation is determined clinically by counting from the date of the last menstrual period. Estimated in this manner, a pregnancy averages 280 days, or 10 lunar months (40 weeks). Fig. 3.5 shows age in weeks; the embryonic, fetal and perinatal periods; and their relationship to trimesters of pregnancy. The 2-week discrepancy between these scales can be seen. Generally, books written for obstetricians or fetal physicians use the lower, obstetric scale, and embryology books use the upper, embryological scale. Fig. 3.6 shows details of the estimated length of embryological stages and how they relate to the obstetric timescale. It is recommended that sonographically determined ages of embryos and fetuses, usually expressed in the obstetric timescale, should be specific, giving weeks and days.16 A reported age of 5 weeks and 2 days is two stages earlier than an age of 5 weeks and 6 days. As improvements in imaging of the first trimester using high resolution 3D transvaginal sonography are made, awareness of both timescales is important, and interpretation of embryonic stage should be clear. The terms ‘gestational’, ‘gestational week’, and ‘gestational age’ are considered ambiguous by O’Rahilly and Müller13; however,

the terms are used widely and colloquially in the obstetric literature. These authors also recommend the greatest length (GL) exclusive of the lower limbs as the best prenatal measurement of embryos directly or derived ultrasonically rather than the older measurement of crown–rump length (CRL).15 Consideration and caution of the shared measurements used to generate predictive data against which to correlate fetal health is very pertinent. Unless the most appropriate measurements are collected, the outcomes of any correlations or predictions will not provide meaningful information. The successful delivery and survival of preterm infants at ages equivalent to 19 or 20 embryonic weeks of development has illustrated that estimations of fetal age have become less important than estimations of fetal maturity, which depend on aspects of maternal health and placental growth. A number of biometric indices used to determine fetal age in utero have been evaluated in ultrasound studies for accuracy. Charts of first-trimester growth based on biparietal diameter, head circumference and abdominal circumference of normal singleton fetuses, correlated against CRL (from 45–84 mm) are considered to be more accurate than gestational age by menstrual dates.17 It is suggested that the femur length:head circumference ratio may be a more robust ratio to characterise fetal proportions than femur length:biparietal diameter,18,19 and combining kidney length, biparietal diameter, head circumference and femur

SE C T I O N 1     Early Fetal Development

28

Late neonatal period (7–28 days) Early neonatal period (birth–7 days)

Implantation period

Embryonic stages

1

2

3

4

5

6

7

Fetal period

8

9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39

2

1

Perinatal period

3

4

5

6

7

8

9

10

Age of embryo (weeks) Age of embryo (months)

Implantation Fertilisation 1

2

3

1

4

5

6

7

8

2

9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41

3

4

5

6

7

8

9

10

Last menstruation

Pregnancy (weeks) Pregnancy (lunar months) Estimated date of delivery

First trimester

Second trimester

Third trimester (of pregnancy)

•

Fig. 3.5 The two timescales used to depict human development. Embryonic development, in the upper scale, is counted from fertilisation (or from ovulation, i.e., in postovulatory days; see O’Rahilly and Müller12). Times given for development are based on this scale. The clinical estimation of pregnancy is counted from the last menstrual period and is shown on the lower scale; throughout this book, fetal ages relating to neonatal anatomy and growth will have been derived from the lower scale. Note that there is a 2-week discrepancy between these scales. The perinatal period is very long because it includes all preterm deliveries. (From Gray’s Anatomy, 41st ed. St. Louis: Elsevier, 2015.) Embryo Obstetric week week Days

week week

Last menstruation

1 2

Endometrial growth 5

6

1

3

11

12

13

2

4

17

18

19

20

3

5

23

24

25

26

27

4

6

30

31

32

33

34

5

7

41

6

8

48

7

9

8

10

Fertilisation, cleavage 0 stages 1–4

1

7

8

9

10

14

15

16

21

22

Implantation, placentation, stages 5–6 Primitive streak gastrulation, stages 7–8 Embryo folding, stage 9 Body plan

28

35

Stage and range shown in colours

2

3

4

Days post-fertilisation

42

10 15 18

49

56

29

11 36

12

37

38

13

39

14 40

16 43

44

17 45

46

47

19 50

57

51

21

58

52

20 53

54

55

22 11

23

• Fig. 3.6  Details of the estimated times of embryonic stages alongside first trimester weeks of pregnancy.

CHAPTER 3  Staging Embryos in Development and the Embryonic Body Plan

length also increases the precision of dating.20 Johnsen et  al18 reported that analysis of measurements of biparietal diameter and head circumference at 10 to 24 weeks’ gestation gave a gestational age assessment of 3 to 8 days greater than charts in use at that time. A multicentre study of fetal growth INTERGROWTH-21st aims to standardise the collection of anthropometric measurements of fetal and childhood growth,21 and The World Health Organization similarly is promoting consideration of ethnicity and social factors when collecting such data.22 These data will contribute to local and population specific growth charts against which normal development can be viewed. 

Conclusion This chapter outlines a commonly used staging system for human embryonic development. Human embryos in the Carnegie Collection were staged according to the system initiated by Streeter

29

and continued by O’Rahilly and Müller. It is based on internal and external criteria and is not just a sequence of size or external features. The staging system has been revised using the results of ultrasound examination of pregnancies of known commencement. The internal and external features of a stage 11 embryo are given. The duration of pregnancy in obstetric terms is based on the date of the last menstrual period; thus the obstetric ‘age’ of an early embryo is different to the Carnegie stage. Care must be taken in recording the age of an embryo or fetus so that these different timescales can be reconciled. A chart of estimated embryonic stages and the obstetric timescale is given. It is hoped that the anthropometric measurements of fetuses in a range of countries and cultures will help develop accurate biometric indices which can be used to estimate fetal age and health. Access the complete reference list online at ExpertConsult.com. Self-assessment questions available at ExpertConsult.com.

References 1a. Hamburger V, Hamilton HL. A series of normal stages in the development of the chick embryo. J Morphol. 1951;88:49–92. 1b. Hamburger V, Hamilton HL. A series of normal stages in the development of the chick embryo. Dev Dyn. 1992;195:231–272. 2. Sanes JR. On the republication of the Hamburger–Hamilton stage series. Dev Dyn. 1992; 195:229–230. 3. Hamilton V. Afterword: the stage series in the chick embryo. Dev Dyn. 1992;195:273–275. 4. Theiler K. The House Mouse. Development and Normal Stages From Fertilization to 4 Weeks of Age. 2nd ed. Berlin: Springer-Verlag; 1989. 5. Kaufman MH. The Atlas of Mouse Development. London: Elsevier; 1992. 6. Petiet AE, Kaufman MH, Goddeeris MM, et al. High-resolution magnetic resonance histology of the embryonic and neonatal mouse: a 4D atlas and morphologic database. Proc Natl Acad Sci USA. 2008;105:12331–12336. 7. Mall FP. On stages in the development of human embryos from 2 to 25 mm long. Anat Anz. 1914;46:78–84. 8. Streeter GL. Developmental horizons in human embryos. Description of age group XIII, embryos about 4 or 5 millimeters long, and age group XIV, period of indentation of the lens vesicle. Carnegie Institution of Washington Publication 557. Contrib Embryol. 1945;31:27–63.

9. Streeter GL. Developmental horizons in human embryos. Description of age groups XV, XVI, XVII, and XVIII, being the third issue of a survey of the Carnegie Collection. Carnegie Institution of Washington Publication 575. Contrib Embryol. 1948;32:133–203. 10. Streeter GL. Developmental horizons in human embryos. Description of age groups XIX, XX, XXI, XXII, and XXIII, being the fifth issue of a survey of the Carnegie Collection (prepared for publication by CH. Heuser & GW Corner). Carnegie Institution of Washington Publication 592. Contrib Embryol. 1951;34:165–196. 11. Streeter GL. Developmental horizons in human embryos. Description of age group XI, 13 to 20 somites, and age group XII, 21 to 29 somites. Carnegie Institution of Washington. Publication 541. Contrib Embryol. 1942;30:211–245. 12. O’Rahilly R, Müller F. Developmental Stages in Human Embryos. Publication 637. Carnegie Institution of Washington; 1987. 13. O’Rahilly R, Müller F. Mini-review: prenatal ages and stages—measures and errors. Teratology. 2000;61:382–384. 14. O’Rahilly R, Müller F. The Embryonic Human Brain. An Atlas of Developmental Stages. 2nd ed. Wiley-Liss; 1999. 15. O’Rahilly R, Müller F. Developmental stages in human embryos: revised and new measurements. Cells Tissues Organs. 2010;192:73–84. 16. Galan HL, Pandipati S, Filly RA. Ultrasound evaluation of fetal biometry and normal and

abnormal fetal growth. In: Callan PW, ed. Ultrasonography in Obstetrics and Gynecology. St. Louis: Saunders; 2008:225–265. 17. Salomon LJ, Bernard JP, Duyme M, et  al. Revisiting first-trimester fetal biometry. Ultrasound Obstet Gynecol. 2003;22:63–66. 18. Johnsen SL, Rasmussen S, Sollien R, Kiserud T. Fetal age assessment based on ultrasound head biometry and the effect of maternal and fetal factors. Acta Obstet Gynecol Scand. 2004;83:716–723. 19. Johnsen SL, Rasmussen S, Sollien R, Kiserud T. Fetal age assessment based on femur length at 10-25 weeks of gestation, and reference ranges for femur length to head circumference ratios. Acta Obstet Gynecol Scand. 2005;84:725–733. 20. Konje JC, Abrams KR, Bell SC, Taylor DJ. Determination of gestational age after the 24th week of gestation from fetal kidney length measurements. Ultrasound Obstet Gynecol. 2002;19:592–597. 21. Uauy R, Casanello P, Krause B, et al. International Fetal and Newborn Growth Consortium for the 21st Century. 2013 Conceptual basis for prescriptive growth standards from conception to early childhood: present and future. BJOG. 2013;120(suppl 2):3–8, v. 22. Merialdi M, Widmer M, Gülmezoglu AM, et  al. WHO multicentre study for the development of growth standards from fetal life to childhood: the fetal component. BMC Pregnancy Childbirth. 2014;4:157.

29.e1

4

Teratology SARAH G. OBICAN AND ANTHONY R. SCIALLI

KEY POINTS • A  t baseline, each pregnancy has a 2% to 4% risk for a congenital anomaly diagnosed at birth. • The adverse effects of exposures on embryo-fetal development depend on the agent, dose, and timing of exposure. • Resources are available for up-to-date information on specific exposures during pregnancy.

Introduction A teratogenic exposure is one that has the ability to interfere with normal development of the fetus. Some exposures increase the risk for structural anomalies, others may interfere with fetal organ development, and others may increase the risk for other adverse pregnancy outcomes, including intrauterine growth restriction, preterm birth and intrauterine fetal demise. This chapter discusses some of the exposures that might increase the risk for abnormal development in human pregnancy. At baseline, each pregnancy has a 2% to 4% probability of a structural anomaly diagnosed at birth.1 Chemically induced anomalies, including those caused by medication exposure, are thought to occur in fewer than 1% of these cases.2 

Historical Perspective In the 19th century, experimental teratology focused on frogs and birds in which, for example, hypoxia could cause developmental aberrations. However, the mammalian uterus was thought to be impervious to external hazards, and genetic abnormalities were blamed for the occurrence of malformations. The notion that human embryos might be harmed by environmental factors was a revolutionary concept until Norman Gregg, an Australian physician, noted in his practice an increase in children with congenital cataracts after a rubella epidemic. He identified what came to be called the ‘congenital rubella syndrome’ with a combination of additional findings, including heart defects, hearing loss, thrombocytopenia and poor growth.3 In response to the increased interest in studying birth defects, the Teratology Society was formed in 1960. Shortly thereafter, the field was changed by the unexpected tragedy of thalidomide. Thalidomide, a sedative–hypnotic, given in the usual (medicinal) doses caused fetal malformations in the absence of toxicity to the mother. Since this discovery of what is now called selective 30

embryotoxicity, drug testing regulations have changed. For example, in 1962, the Food, Drug and Cosmetic Act was expanded to include the Kefauver-Harris Amendment, which requires drug manufacturers to provide proof of effectiveness and safety before approval as well as provide information regarding side effects. By 1966, the US Food and Drug Administration (FDA) instituted standard test protocols for drug testing in pregnant laboratory animals. 

Mechanisms of Teratogenicity Experience in experimental teratology led to the development of potential mechanisms for abnormal development, articulated by James Wilson4 (Table 4.1). Wilson’s mechanisms included the idea that impairment of survival and function in differentiated cells in key locations in an embryo could perturb development. This concept gave rise to the ‘all-or-nothing’ principle in which it was believed that before gastrulation (approximately postconception day 14), cells in the embryo were largely undifferentiated and could substitute for one another, decreasing the ability of an exposure to cause selective malformations without destroying the entire embryo. This all-or-nothing principle has not proved invariable in experimental teratology, but it remains true that it is more difficult to produce malformations with experimental damage to undifferentiated compared with differentiated embryonic tissues. Mechanisms of congenital malformations more recently have been understood in terms of developmental pathways that might be inhibited by a specific exposure. For example, DiGeorge syndrome, which is often associated with a deletion in the long arm of chromosome 22, includes disruption of Tbx1, a gene that plays a role in development of cells in the secondary heart field. As a consequence, conotruncal heart defects can be seen in affected children.5 It has not been identified, however, whether isotretinoin therapy, which also has been associated with conotruncal heart defects, works by way of interference with Tbx1. There are basic cell behaviours during embryo development that may serve as targets for exposures. Cell populations in the embryo migrate to targeted locations, and interference with migration may cause abnormal development. Neural crest cells migrate into the branchial arches, for example, and interference results in a group of disorders of the jaw, ears, and zygomatic arch, among other defects. Neurons migrate from their birthplace in the centre of the brain towards the periphery, and inhibition of migration can cause microcephaly. Cells also can induce behaviours in neighbouring cells. The tips of the ureteric

CHAPTER 4 Teratology

TABLE 4.1 Mechanisms of Teratogenesis

Mutation Chromosomal aberrations Mitotic interference Altered nucleic acid synthesis and function Lack of precursors, substrates and coenzymes for biosynthesis Altered energy sources Enzyme inhibition Osmolar imbalance Changed membrane characteristics From Wilson JG. Mechanisms of teratogenesis. Am J Anat 136(2):129–131, 1973.

  

buds induce the mesenchyme of the developing kidney to form functioning nephrons. Cells produce substances that act at a distance to modify cell differentiation. For example, cells in the medial (postaxial) limb bud secrete Sonic Hedgehog protein, which diffuses across the limb paddle to help determine the identity of the individual digits. The greater understanding of cellular and molecular events during embryo development has given rise to an opportunity for understanding mechanisms of abnormal development in more detail. Although the mechanistic details of malformations in human beings associated with exposures are incompletely understood, we expect that developments in the genetic basis of malformations and in cell biology will lead to our improved understanding of exposure-associated malformations. 

Underlying Principles The mechanisms of abnormal development were the basis of one of James Wilson’s principles. The full set of principles is listed in Table 4.2 as developed in the 1950s and 1960s.1 Of note is Wilson’s fourth principle, which tells us that malformations are not the only kind of developmental toxicity of importance. An exposure that causes an organ, say the brain, not to function correctly can be devastating even if there are no recognisable structural malformations in the child. We commonly call these adverse effects developmental toxicity, which is a more helpful term than teratogenicity in not requiring a definition of what exactly counts as a malformation. An exposure producing nonmalforming developmental effects does not necessarily also produce malformations. Indeed, an exposure that produces one kind of malformation does not of necessity produce any other kind of malformation. Thalidomide, for example, produces only certain kinds of limb defects, not all kinds of limb defects.6 Effects of developmentally toxic exposures are specific, producing a finite grouping of adverse effects. Dr Wilson captured this idea of specificity in his third principle, and the Public Affairs Committee of the Teratology Society made it explicit in 2005.7 Wilson’s sixth principle is among the most important for practitioners because it reminds us that for all medications, other chemicals, and physical agents, there are an exposure level that produces no harm, an exposure level that produces death and a range of exposures in between that produces a gradation of effects. It is not useful to talk about agents (drugs, chemicals, radiations) as teratogenic or nonteratogenic. It is preferable to

31

TABLE 4.2 Wilson’s Principles 1. Susceptibility to teratogenesis depends on the genotype of the conceptus and the manner in which this interacts with environmental factors. 2. Susceptibility to teratogenic agents varies with the developmental stage at the time of exposure. 3. Teratogenic agents act in specific ways (mechanisms) on developing cells and tissues to initiate abnormal embryogenesis (pathogenesis). 4. The final manifestations of abnormal development are death, malformation, growth retardation and functional disorder. 5. The access of adverse environmental influences to developing tissues depends on the nature of the influences (agent). 6. Manifestations of deviant development increase in degree as dosage increases from the no-effect to the totally lethal level. From Wilson JG. Current status of teratology. General principles and mechanisms derived from animal studies. In Handbook of Teratology, vol 1. General Principles and Etiology, pp. 47–74, JG Wilson, FC Fraser (eds.), New York: Plenum Press, 1977.

  

talk about teratogenic exposures, an exposure including the identity of the agent and the dose level at which it is encountered. X-irradiation during pregnancy was associated with microcephaly and mental retardation when exposure levels were about 50 cGy from the atomic bombings of Japan.8 Flying across the United States is associated with estimated radiation exposures about 8000 times lower9 and would not be expected to have the same effects. We do not, therefore, characterise x-ray as teratogenic or nonteratogenic. It depends, among other things, on dose. How much evidence is needed before it is worthwhile warning patients and health care providers about possible adverse effects of exposures in reproducing men and women? There is some controversy in this area because there are potential adverse effects of excessive warning, namely anxiety, the interruption of otherwise wanted pregnancies, and the discontinuation of important, even essential, medical therapy. It has been fashionable from time to time to claim that most human teratogenic exposures were first identified by ‘astute clinicians.’ Thalidomide is often cited as evidence of this claim because the first publications on thalidomide birth defects came from one paediatrician in Germany and another in Australia who described clusters of children with phocomelia and wondered whether there was a pregnancy exposure as the cause. The astute clinician model has been described in mathematical terms,10 but the astute clinician model produces only a hypothesis that must be confirmed by other evidence. The association between rubella and congenital cataracts and between thalidomide and phocomelia were, in fact, doubted by some teratologists until additional evidence was forthcoming. Perhaps clinicians whose hypotheses are confirmed may be as lucky as they are astute. For a determination of causation in teratology, methods have been advanced that are based on the Hill criteria.11 These criteria (Table 4.3) were most famously applied in the consideration by the US Surgeon General of the evidence for a causal relationship between cigarette smoking and lung cancer.12 To entertain a conclusion of causation, you need not satisfy each of the Hill criteria, but the more you satisfy, the more confident you can be that an association is causal. In this chapter, we discuss some exposures that have been associated with developmental toxicity in human pregnancy. In some

SE C T I O N 1     Early Fetal Development

32

TABLE 4.3 Bradford Hill Criteria 1. Strength of the association (the likelihood that the association is not due to chance, bias, or confounding); strength of the association refers to findings in human epidemiological studies 2. Consistency of the association (the association is reproduced in different populations); consistency of the association refers to findings in human epidemiological studies 3. Specificity (uniqueness of the association both with respect to the exposure and with respect to outcome). In teratology, specificity results in a distinctive pattern of birth defects that appear repeatedly and consistently. 4. Temporal relationship (the putative cause comes before the effect) 5. Coherence (the association is compatible with related knowledge) 6. Biologic gradient (there is a dose–response effect) 7. Biologic plausibility (the association does not violate known principles) 8. Experiment (reducing the putative cause reduces the effect) 9. Analogy (evidence is similar to that for similar cause-effect relationships) From Hill AB. The environment and disease: association or causation? Proc R Soc Med 58:295–300, 1965.

  

instances, there is evidence that the association is causal, but in other cases, a causal association cannot be concluded. We recognise, however, that giving patients advice about exposures during pregnancy does not require conviction that causation criteria have been satisfied. For example, we recommend that women who have taken lithium during pregnancy consider fetal echocardiography, even though causation criteria are not satisfied that lithium causes Ebstein anomaly or any other heart defect. In the end, counselling about exposures relies on the same kinds of judgments about adverse outcomes that we use every day as clinicians considering possible risks and benefits of medication therapy. 

Personalised Risk Assessment and Resources Several excellent books are available as resources. However, soon after publication, books in this ever-changing field have the potential to be outdated. Online databases offer summaries of pregnancy exposures written and frequently updated by teratology experts include TERIS (http://depts.washington.edu/terisdb/teris web/index.html), REPROTOX (Reprotox.org), and Briggs (available through http://wolterskluwer.com). Lactmed (https://toxnet. nlm.nih.gov/newtoxnet/lactmed.htm) is a free online resource for medication exposure and lactation. There are two networks of teratology information services, one in North America called the Organization of Teratology Information Specialists (OTIS; http://www.mothertobaby.org) and one serving Europe, the European Network of Teratology Information Services (ENTIS; http://www.entis-org.eu). Both networks are staffed with physicians, genetic counsellors and teratology experts who offer individualised risk assessments and up-to-date information for any drug or environmental exposure during pregnancy and lactation. These complimentary services are available to both health care providers and patients. These networks also conduct prospective studies and patient follow-up after pregnancy exposures. Pregnancy registries open to enrolment may be located from the FDA’s Office of Women’s Health (www.fda.gov/ScienceRese arch/SpecialTopics/WomensHealthResearch/ucm251314.htm). 

Selected Human Exposures Medication A survey of 1000 pregnancies in southwest France found that 99% of the women received a prescription for at least one drug during pregnancy with a mean of 13.6 medications per woman.13 In other studies, approximately two thirds of pregnant women took at least one medication during pregnancy, and about 60% of patients used a prescription medication.14 In the past few decades, the overall use of medication and the number of women using 4+ prescription medications anytime in pregnancy more than doubled, and for the first trimester, it more than tripled.15 Thalidomide. No other therapeutic agent has had a greater impact on how we think about teratogenic potential as thalidomide. In 1957, thalidomide was marketed as a sedative and antiemetic. Thalidomide did not cause acute toxicity in adults, making it one of the few agents that are selectively embryotoxic. In the late 1950s and 1960, there were few case reports of phocomelia, an unusual limb reduction defect in which the hand and foot arise from the shoulder or hip. By 1961, Dr Lenz in Germany and Dr McBride in Australia independently noted an association between phocomelia and exposure to thalidomide. Thalidomide exposure was associated with specific limb reduction defects, oesophageal and duodenal atresia, congenital heart defects (tetralogy of Fallot), external ear and cranial nerve abnormalities, and renal agenesis. The sensitive time period for the limb defects was 21 to 36 days postconception. Although thalidomide was removed from the market, it was found later to be effective for erythema nodosum leprosum. In 1998, the US FDA approved Thalomid for this use. In 2006, the medication was approved for multiple myeloma. Prescription and dispensing of thalidomide is strictly controlled in the US through a Risk Evaluation and Mitigation Strategy (REMS)16 to prevent exposure during pregnancy.  Isotretinoin. Isotretinoin (13-cis-retinoic acid), a derivative of vitamin A (retinol), is an effective treatment of cystic acne vulgaris. Use of the medication increases the risk for spontaneous abortion and the development of a specific set of anomalies, including neural crest-related facial and palate defects, micrognathia, external and internal ear anomalies (microtia or anotia), conotruncal heart defects, thymic abnormalities and deficits in intelligence.17-20 Effects on cognition can occur in the absence of structural anomalies.20 There is no associated risk for poor fetal outcome in cases in which isotretinoin was discontinued before pregnancy. The current recommendation is to discontinue isotretinoin 1 month before conception, but considering the half-life of 29 hours, after 1 week off therapy, maternal blood concentrations should be negligible. Use of topical retinoids does not increase the risk for structural malformations or developmental delay due to decreased availability of the medication and its metabolites in maternal plasma.21  Warfarin. In 1948, warfarin was marketed as a potent rodenticide because of its capability of inducing internal haemorrhage.16 Warfarin prevents vitamin K from acting as a cofactor in hepatic synthesis of factors II, VII, IX and X and was adapted for human clinical use because of its oral bioavailability and reversibility by vitamin K. Warfarin was associated with embryo-fetal growth restriction, nasal hypoplasia, fibula hypoplasia and stippled epiphysis.22,23 Embryotoxicity is associated with exposure

CHAPTER 4 Teratology

between 6 and 9 weeks of gestation,23 although one report did not note a warfarin embryopathy in those exposed before 8 weeks of gestation.24 Other poor perinatal outcomes associated with warfarin exposure include an increased risk for stillbirth, spontaneous abortion, preterm birth and low birth weight.24-26 The underlying maternal disease may contribute to the increased risk for poor pregnancy outcomes. Warfarin is still used by some practitioners in pregnancy, especially in women with mechanical heart valves. Heparin and low-molecular-weight heparin (LMWH) are the mainstays of anticoagulant therapy during pregnancy, but they may not be effective enough, especially in women with mechanical heart valves. More favourable maternal and fetal outcomes may be associated with the use of low-dose warfarin (60% of individuals

• Fig. 6.3  Representative killer immunoglobulin-like receptor (KIR) A and B haplotypes of the KIR gene family with known binding of human leukocyte antigen (HLA)-C epitopes (C1 or C2) depicted above their cognate receptors. KIR2DS4 binds a few C1 and C2 allotypes, but more than 60% of individuals have a truncated form of KIR2DS4. KIR2DS4 only recognises some HLA allotypes carrying that epitope. Framework KIR genes that are present in all haplotypes are shown as black boxes. Activating KIRs are shown as blue boxes and inhibitory KIR as red boxes.

Uterine Natural Killer Cell Recognition of Trophoblast

Maternal KIR–Fetal HLA-C Combinations Influence Reproductive Success

Uterine NK cells express an array of receptors, some of which are known to bind to the HLA class I molecules expressed by extravillous trophoblast.4 Unlike blood NK cells, all uNK cells express high levels of the C-type lectin family member CD94/NKG2A, which binds to HLA-E, resulting in inhibition of NK-cell cytotoxicity. Neither ligand or receptor shows significant polymorphism, and this is likely to be the signal that stops uNK cells from killing trophoblast and the maternal cells in the decidua. Uterine NK cells also express members of the killer immunoglobulin-like receptors (KIRs) family of receptors. These are carried together as a haplotype on chromosome 19 and have different binding specificities.11 The KIR also differ in the length of their cytoplasmic tail that either results in an inhibitory or an activating signal to the NK cell. Those that have a short tail (S) are activating (KIR2DS), and those that are long (L) are inhibitory (KIR2DL) receptors. In all populations, there are two main KIR haplotypes, A and B; these differ in the presence of additional activating receptors in the B haplotype. HLA-C, which is the only polymorphic MHC class I molecule expressed by extravillous trophoblast, is the dominant ligand for several KIR receptors. These HLA-C allotypes all fall into two groups, C1 and C2, based on a dimorphism at amino acid 80 of the α1 domain. KIR that bind HLA-C distinguish between C1 and C2 as mutually exclusive epitopes as shown in Fig. 6.3. The maternal–fetal immunologic interaction that occurs at the site of implantation between uNK and trophoblast therefore involves two gene systems, maternal KIR and fetal HLA-C molecules. Because these are both polymorphic and both maternal and paternal HLA-C allotypes are expressed on trophoblasts, the exact KIR–HLA-C interaction differs in each pregnancy. Some KIR–HLA-C combinations appear to be more favourable to trophoblast invasion than others, thus affecting reproductive outcome. 

Genetic studies of large pregnancy cohorts have now shown that mothers with two KIR A haplotypes (KIR AA genotype) are at increased risk for disorders of pregnancy, including preeclampsia and other GOS if the fetus carries an HLA-C allele with a C2 epitope inherited from the father.12 Conversely, mothers with a KIR B haplotype (containing activating KIR2DS1 that can also bind C2 epitopes) are at low risk, but instead these mothers have an increased risk for delivering a large baby.13 When the fetus is C1/C1 homozygous, the mother’s KIR genotype has no effect, so a C2 epitope is the crucial fetal ligand (Fig. 6.4). Three major conditions of pregnancy–recurrent miscarriage, FGR and pre-eclampsia–all show the same association. Overall our results suggest that receptor–ligand interactions leading to strong uNK inhibition result in decreased trophoblast invasion and compromised fetal development. Findings in mice support this. Binding of the inhibitory receptor Ly49A on murine uNK cells to a single extra MHC molecule on trophoblast results in decreased uterine vascular remodelling and reduced fetal growth.14 A key question remains: how does the presence of the KIR B haplotype reduce this risk? In Europeans, the protective genes on the KIR B haplotype map to the region where the activating KIR for C2 (KIR2DS1) is located. Protection from pre-eclampsia is likely to be due to counterbalancing uNK activation when KIR2DS1 binds C2. Indeed, when KIR2DS1 on uNK cells binds to C2, this increases secretion of cytokines that enhance trophoblast invasion in vitro.15 If trophoblast invasion in vivo is correspondingly enhanced, this could lead to improved placental perfusion and better fetal growth. In support of this model, we find that highbirth-weight pregnancies are associated with mothers who have inherited the activating receptor KIR2DS1 on the KIR B haplotype and a fetus with an HLA-C allele bearing a C2 epitope. The

CHAPTER 6  The Immunology of Implantation

Baby’s HLA-C Mother’s KIR haplotype C1 C1

KIR2DL1 ↓ A A ↑ A B

B B

C1 C2

C2 C2

↓ ↑

↑ KIR2DS1

↓

↓

↑

↑ KIR2DS1

•

Fig. 6.4 Certain combinations of maternal killer immunoglobulin-like receptor (KIR) and fetal human leukocyte antigen (HLA)-C genotypes increase susceptibility to pre-eclampsia, recurrent miscarriage or fetal growth restriction. A cross (x) indicates the increased risk for a poor clinical outcome. Maternal KIR A haplotype carries the inhibitory KIR2DL1 that can bind the C2 epitope carried by some fetal HLA-C alleles. KIR B haplotypes can also include the activating KIR2DS1 that binds C2.

effect is significant resulting in an estimated average increase in birth weight of some 200 g.13 Conversely, KIR AA mothers who have two copies of KIR2DL1 the inhibitory receptor for C2 and have a fetus with a C2 epitope show reduced birth weight compared with fetuses that lack C2. These effects on both large and small babies are most significant when the fetal C2-bearing allele is paternally derived (Fig. 6.5). Pregnancies at both extremes of birth weight more likely to experience serious obstetric complications. Large babies are at risk for fetal obstruction, which can result in prolonged labour, fetal death from asphyxia and postpartum haemorrhage.16 On the other hand, when spiral artery modification is inadequate, poor placental perfusion can manifest as increased risk for preeclampsia, recurrent miscarriage or FGR, with increased maternal and fetal morbidity. Because humans have a large brain and narrow pelvis relative to other primates, the obstetric dilemma is particularly acute, and there is strong selective pressure to maintain human birth weight between these two extremes. Although many genes and environmental influences have an impact on fetal growth, the genetic studies suggest that KIR–HLA-C interactions play a role in maintaining an optimal birth weight in human populations.

0.4

Birth weight frequency

Population frequency

Special care transfer frequency 0.3

0.2

0.1

0.0

1000

2000

3000

53

4000

5000

6000

Birth weight (g)

Low Increased frequency of KIR AA+ paternal C2

Normal

High Increased frequency of KIR2DS1+ paternal C2

Poor spiral artery transformation

Normal transformation

Exceptional spiral artery transformation?

• Fig. 6.5  The presence of maternal KIR2DS1 is associated with increased birth weight if fetus has inherited a human leukocyte antigen (HLA)-C allele with a C2 epitope. Distribution of birth weights in Norwegian MoBa cohort is shown together with percentage of babies transferred to special care baby unit. The cohort was divided into high- (>90th percentile), normal- (6th–89th percentile) and low- (2 multiples of the median value for gestation) with no associated fetal malformation had an increased risk for adverse outcomes such as preeclampsia, IUGR and antepartum fetal death.84 The implication is that increased placental permeability may lead to impaired pregnancy outcome. Abnormal Doppler values and abnormal placental shape identify those at greatest risk for perinatal death and preterm delivery from placental damage. Villous repair after loss of trophoblast involves deposition of fibrin, and in vitro studies using horseradish peroxidase confirm these as sites of increased permeability and so are responsible for the increased release of AFP into the maternal circulation.85 In a normal term placenta, fibrin deposits that may be responsible for the passage of macromolecules cover approximately 7% of the villous surface. Another paratrophoblastic transfer route for smaller molecules is provided by the transtrophoblastic channels, approximately 20 nm in diameter and seen only by electron microscopy. These exist to allow transfer of water-soluble, lipophobic molecules with an effective molecular diameter smaller than 1.5 nm and may be important for the regulation of fluid balance. Under certain circumstances such as fetal hydrops, increased fetal venous pressure or reduced fetal oncotic pressure, these channels dilate such that not only water but also fetal proteins may pass into the maternal circulation.83 

Physiology of Fetoplacental Blood Flow Fetal size and thus oxygen and nutritional demands rapidly outstrip growth of the placenta such that by term, 1 g of placenta supports 6 g of fetus. To meet these demands, the peripheral villous placenta differentiates such that by term, the proportion of descending aortic blood flow entering the umbilical arteries is increased to 40%, and the diffusive capacity is increased 10-fold. These alterations are almost wholly dependent on the exponential elaboration of terminal villi in the second half of pregnancy. As in the uteroplacental circulation, which becomes ‘denervated’ by trophoblast, the villous tree remains free of fetal autonomic influences and is dependent upon fetal cardiac output because it is devoid of nerves. The fetoplacental circulation competes with the lower fetal body for aortic blood. The umbilical arteries receive this large proportion of descending aortic blood flow because of low impedance.7 Doppler studies of the umbilical arteries indicate a progressive fall in fetoplacental vascular impedance, reflected by increasing end-diastolic flow velocity. During the first trimester, end-diastolic velocities are absent, becoming consistently present by 14 weeks of gestation. Thereafter the steady rise in end-diastolic velocity parallels differentiation of the villous tree into its mature form. The dramatic changes in peripheral capillarisation of villi throughout pregnancy (see Fig. 7.11) contribute to changes in umbilical artery blood flow in addition to the local vasomotor regulatory process in muscularised stem villous arterioles.75 Systemic vascular beds have a relatively short distance between arterioles and venules and these are bridged by many parallel capillaries, so that impedance or flow is regulated by autonomically

68

SE C T I O N 2     The Placenta

innervated precapillary sphincters; these structures regulate blood flow across a wide range, for example, in muscle that may exercise or rest. By contrast, fetoplacental blood flow must be constant and ever increasing; the capillary bed of the peripheral villi is much longer than in muscle (2000–4000 μm), less richly branched, and is focally dilated into sinusoids within terminal villi (see Figs. 7.8 and 7.11).7 

Conclusions This chapter has aimed to discuss the clinical relevance of normal human placental development for obstetrics. Furthermore, a wide range of important clinical problems, such as adult cardiac disease, have their origins in placental maldevelopment and pathology.

Increasing interest in placental research and important contributions by clinicians, especially collaboration to obtain Doppler and real-time ultrasound information of the placenta just before delivery, has led to important advances in our understanding of the pathological basis of placental insufficiency syndromes that cause stillbirth and premature death. In the near future, more widespread acceptance amongst maternal-fetal medicine clinicians of the value of making a prenatal diagnosis of placental pathology may lead to advances in the therapeutic options for at-risk women. Access the complete reference list online at ExpertConsult.com Self-assessment questions available at ExpertConsult.com

References

1. Toal M, Chan C, Fallah S, et  al. Usefulness of a placental profile in high-risk pregnancies. Am J Obstet Gynecol. 2007;196(4):363. e1–e7. 2. Zhong Y, Zhu F, Ding Y. Serum screening in first trimester to predict pre-eclampsia, small for gestational age and preterm delivery: systematic review and meta-analysis. BMC Pregnancy Childbirth. 2015;15:191. 3. Dunk C, Huppertz B, Kingdom J. Fetal medicine. In: Rodeck CH, Whittle MJ, eds. Development of the Placenta and Its Circulation. 2nd ed. St. Louis: Elsevier; 2007. 4. Torpin R. The Human Placenta. Springfield, IL: Charles C Thomas; 1969. 5. Liu CC, Pretorius DH, Scioscia AL, et  al. Sonographic prenatal diagnosis of marginal placental cord insertion: clinical importance. J Ultrasound Med. 2002;21(6):627–632. 6. Silver RM. Abnormal placentation: placenta previa, vasa previa, and placenta accreta. Obstet Gynecol. 2015;126(3):654–668. 7. Benirschke K, Kaufmann P. Pathology of the Human Placenta. 4th ed. New York: SpringerVerlag; 2006. 8. Plazenton Schuhmann. Begriff, entstehung, funktionelle anatomie. In: Becker V, Schiebler TH, Kubli F, eds. Die Plazenta des Menschen. Stuttgart, Germany: Thieme Verlag; 1981:199–207. 9. Zimmermann P, Eirio V, Koskinen J, et  al. Doppler assessment of the uterine and uteroplacental circulation in the second trimester in pregnancies at high risk for pre-eclampsia and/ or intrauterine growth retardation: comparison and correlation between different Doppler parameters. Ultrasound Obstet Gynecol. 1997;9(5):330–338. 10. Mayhew TM. Stereology and the placenta: where’s the point? a review. Placenta. 2006;27(suppl A):S17–S25. 11. Egbor M, Ansari T, Morris N, et al. Morphometric placental villous and vascular abnormalities in early- and late-onset pre-eclampsia with and without fetal growth restriction. BJOG. 2006;113(5):580–589. 12. Ball E, Bulmer JN, Ayis S, et al. Late sporadic miscarriage is associated with abnormalities in spiral artery transformation and trophoblast invasion. J Pathol. 2006;208(4):535–542. 13.  Wigglesworth JS. Vascular organization of the human placenta. Nature. 1967;216(5120): 1120–1121. 14. Borell U, Fernstrom I, Westman A. Eine arteriographische studie des plazentarkreilaufs. Geburtshilfe Frauenheilhd. 1958;18:1–9. 15. Konje JC, Huppertz B, Bell SC, et  al. 3-dimensional colour power angiography for staging human placental development. Lancet. 2003;362(9391):1199–1201. 16.  Moll W. Physiologie der maternen plazentaren durchblutung. In: Becker V, Schiebler TH, Kubli F, eds. Die Plazenta des Menschen. Stuttgart, Germany: Thieme Verlag; 1981:172–194. 17. McDermott M, Gillan JE. Chronic reduction in fetal blood flow is associated with placental infarction. Placenta. 1995;16(2):165–170. 18. Knott JG, Paul S. Transcriptional regulators of the trophoblast lineage in mammals with hemochorial placentation. Reproduction. 2014;148(6):R121–R136. 19. Jauniaux E, Englert Y, Vanesse M, et  al. Pathologic features of placentas from

singleton pregnancies obtained by in vitro fertilization and embryo transfer. Obstet Gynecol. 1990;76(1):61–64. 20a. Kaufmann P, Castellucci M. Extravillous trophoblast in the human placenta. Trophoblast Res. 1997;10:21–65; 20b. Moser G, Windsperger KPollheimer J, de Sousa Lopes SC, Huppertz B. Human trophoblast invasion: new and unexpected routes and functions. Histochem Cell Biol. 2018;150(4):361–370. 21. Moser G, Gauster M, Orendi K, et al. Endoglandular trophoblast, an alternative route of trophoblast invasion? analysis with novel confrontation co-culture models. Hum Reprod. 2010;25(5):1127–1136. 22. Frank HG, Malekzadeh F, Kertschanska S, et al. Immunohistochemistry of two different types of placental fibrinoid. Acta Anat (Basel). 1994;150(1):55–68. 23. Simmons DG, Fortier AL, Cross JC. Diverse subtypes and developmental origins of trophoblast giant cells in the mouse placenta. Dev Biol. 2007;304(2):567–578. 24. Kemp B, Kertschanska S, Kadyrov M, et  al. Invasive depth of extravillous trophoblast correlates with cellular phenotype: a comparison of intra- and extrauterine implantation sites. Histochem Cell Biol. 2002;117(5):401–414. 25. Huppertz B, Kertschanska S, Frank HG, et al. Extracellular matrix components of the placental extravillous trophoblast: immunocytochemistry and ultrastructural distribution. Histochem Cell Biol. 1996;106(3):291–301. 26. Frank HG, Huppertz B, Kertschanska S, et  al. Anti-adhesive glycosylation of fibronectin-like molecules in human placental matrix-type fibrinoid. Histochem Cell Biol. 1995;104(4):317–329. 27. Harris LK, Jones CJ, Aplin JD. Adhesion molecules in human trophoblast—a review. II. extravillous trophoblast. Placenta. 2009;30(4):299–304. 28. Feinberg RF, Kliman HJ, Lockwood CJ. Is oncofetal fibronectin a trophoblast glue for human implantation? Am J Pathol. 1991;138(3):537–543. 29.  Tseng JJ, Chou MM. Differential expression of growth-, angiogenesis- and invasion-related factors in the development of placenta accreta. Taiwan J Obstet Gynecol. 2006;45(2):100–106. 30. Kadyrov M, Kingdom JC, Huppertz B. Divergent trophoblast invasion and apoptosis in placental bed spiral arteries from pregnancies complicated by maternal anemia and early-onset preeclampsia/intrauterine growth restriction. Am J Obstet Gynecol. 2006;194(2):557–563. 31. Kadyrov M, Schmitz C, Black S, et  al. Preeclampsia and maternal anaemia display reduced apoptosis and opposite invasive phenotypes of extravillous trophoblast. Placenta. 2003;24(5):540–548. 32. Zybina TG, Frank HG, Biesterfeld S, Kaufmann P. Genome multiplication of extravillous trophoblast cells in human placenta in the course of differentiation and invasion into endometrium and myometrium. II. Mechanisms of polyploidization. Tsitologiia. 2004;46(7):640–648. 33. Dunk CE, Gellhaus A, Drewlo S, et  al. The molecular role of connexin 43 in human trophoblast cell fusion. Biol Reprod. 2012;86(4):115.

34. Silver RM, Landon MB, Rouse DJ, et  al. Maternal morbidity associated with multiple repeat caesarean deliveries. Obstet Gynecol. 2006;107(6):1226–1232. 35. Fernandez H, Al-Najjar F, ChauveaudLambling A, et al. Fertility after treatment of Asherman’s syndrome stage 3 and 4. J Minim Invasive Gynecol. 2006;13(5):398–402. 36. Burton GJ, Jauniaux E, Watson AL. Maternal arterial connections to the placental intervillous space during the first trimester of human pregnancy: the Boyd collection revisited. Am J Obstet Gynecol. 1999;181(3):718–724. 37. Schapps JP, Hustin J. In  vivo aspect of the maternal-trophoblastic border during the first trimester of gestation. Trophoblast Res. 1988;3:39–48. 38. Jauniaux E, Jurkovic D, Campbell S, et  al. Investigation of placental circulations by color Doppler ultrasonography. Am J Obstet Gynecol. 1991;164(2): 486–468. 39. Jauniaux E, Gulbis B, Burton GJ. The human first trimester gestational sac limits rather than facilitates oxygen transfer to the foetus—a review. Placenta. 2003;24(suppl A):S86–S93. 40.  Burton G.J., Jaunaiux E.. Maternal vascularisation of the human placenta: does the embryo develop in a hypoxic environment? Gynecol Obstet Fertil. 2001;29(78):503–508. 41. Huppertz B, Gauster M, Orendi K, et  al. Oxygen as modulator of trophoblast invasion. J Anat. 2009;215(1):14–20. 42.  Hazan AD, Smith SD, Jones RL, et al. Vascularleukocyte interactions: mechanisms of human decidual spiral artery remodeling in vitro. Am J Pathol. 2010;177(2):1017–1030. 43. Lima PD, Zhang J, Dunk C, et  al. Leukocyte driven-decidual angiogenesis in early pregnancy. Cell Mol Immunol. 2014;11(6): 522–537. 44. Smith SD, Choudhury RH, Matos P, et  al. Changes in vascular extracellular matrix composition during decidual spiral arteriole remodeling in early human pregnancy. Histol Histopathol. 2016;31(5):557–571. 45. Smith SD, Dunk CE, Aplin JD, et al. Evidence for immune cell involvement in decidual spiral arteriole remodeling in early human pregnancy. Am J Pathol. 2009;174(5):1959–1971. 46. Smith SD, Choudhury RH, Matos P, et  al. Changes in vascular extracellular matrix composition during decidual spiral arteriole remodeling in early human pregnancy. Histol Histopathol. 2016;31(5):557–571. 47. Kaufmann P, Black S, Huppertz B. Endovascular trophoblast invasion: implications for the pathogenesis of intrauterine growth retardation and preeclampsia. Biol Reprod. 2003;69(1): 1–7. 48. Zhou Y, Fisher SJ, Janatpour M, et al. Human cytotrophoblasts adopt a vascular phenotype as they differentiate. A strategy for successful endovascular invasion? J Clin Invest. 1997;99(9):2139–2151. 49. Boyd JD, Hamilton WJ. The Human Placenta. Cambridge, United Kingdom: Heffer; 1970. 50. Brosens I, Robertson WB, Dixon HG. The physiological response of the vessels of the placental bed to normal pregnancy. J Pathol Bacteriol. 1967;93(2):569–579. 51. Jauniaux E, Greenwold N, Hempstock J, et al. Comparison of ultrasonographic and Doppler mapping of the intervillous circulation in normal and abnormal early pregnancies. Fertil Steril. 2003;79(1):100–106.

68.e1

68.e2

References

52. Tong S, Marjono B, Mulvey S, et al. Low levels of pregnancy-associated plasma protein-A in asymptomatic women destined for miscarriage. Fertil Steril. 2004;82(5):1468–1470. 53. Ball E, Robson SC, Ayis S, et al. Early embryonic demise: no evidence of abnormal spiral artery transformation or trophoblast invasion. J Pathol. 2006;208(4):528–534. 54. Brosens I. The uteroplacental vessels at term— the distribution and extent of physiological changes. Trophoblast Res. 1988;3:61–67. 55. Burton GJ, Hempstock J, Jauniaux E. Oxygen, early embryonic metabolism and free radicalmediated embryopathies. Reprod Biomed Online. 2003;6(1):84–96. 56. Roberts VH, Lo JO, Salati JA, et  al. Quantitative assessment of placental perfusion by contrast-enhanced ultrasound in macaques and human subjects. Am J Obstet Gynecol. 2016;214(3):369. e1–e8. 57. Burton GJ, Jauniaux E. Placental oxidative stress: from miscarriage to preeclampsia. J Soc Gynecol Investig. 2004;11(6):342–352. 58. Jauniaux E, Watson A, Burton G. Evaluation of respiratory gases and acid-base gradients in human fetal fluids and uteroplacental tissue between 7 and 16 weeks’ gestation. Am J Obstet Gynecol. 2001;184(5):998–1003. 59. Burton GJ, Woods AW, Jauniaux E, et  al. Rheological and physiological consequences of conversion of the maternal spiral arteries for uteroplacental blood flow during human pregnancy. Placenta. 2009;30(6):473–482. 60. Bewley S, Cooper D, Campbell S. Doppler investigation of uteroplacental blood flow resistance in the second trimester: a screening study for pre-eclampsia and intrauterine growth retardation. Br J Obstet Gynaecol. 1991;98(9):871–879. 61. Viero S, Chaddha V, Alkazaleh F, et al. Prognostic value of placental ultrasound in pregnancies complicated by absent end-diastolic flow velocity in the umbilical arteries. Placenta. 2004;25(8-9):735–741. 62. Demers S, Girard M, Roberge S, et  al. Firsttrimester placental and myometrial blood perfusion measured by three-dimensional power Doppler in preeclampsia. Am J Perinatol. 2015;32(10):920–926. 63. Brosens IA, Robertson WB, Dixon HG. The role of the spiral arteries in the pathogenesis of pre-eclampsia. J Pathol. 1970;101(4): Pvi. 64. Khong TY, De Wolf F, Robertson WB, Brosens I. Inadequate maternal vascular response to placentation in pregnancies complicated by pre-eclampsia and by small-forgestational age infants. Br J Obstet Gynaecol. 1986;93(10):1049–1059.

65. Demir R, Kaufmann P, Castellucci M, et  al. Fetal vasculogenesis and angiogenesis in human placental villi. Acta Anat (Basel). 1989;136(3):190–203. 66. Aplin JD, Whittaker H, Jana Lim YT, et  al. Hemangioblastic foci in human first trimester placenta: distribution and gestational profile. Placenta. 2015;36(10):1069–1077. 67. Mayhew TM. A stereological perspective on placental morphology in normal and complicated pregnancies. J Anat. 2009;215(1):77–90. 68. Wilkins-Haug L, Roberts DJ, Morton CC. Confined placental mosaicism and intrauterine growth retardation: a case-control analysis of placentas at delivery. Am J Obstet Gynecol. 1995;172(1 Pt 1):44–50. 69. Fogarty NM, Mayhew TM, Ferguson-Smith AC, Burton GJ. A quantitative analysis of transcriptionally active syncytiotrophoblast nuclei across human gestation. J Anat. 2011;219(5): 601–610. 70. Fogarty NM, Ferguson-Smith AC, Burton GJ. Syncytial knots (Tenney-Parker changes) in the human placenta: evidence of loss of transcriptional activity and oxidative damage. Am J Pathol. 2013;183(1):144–152. 71. Lee W, Ginsburg KA, Cotton DB, Kaufman RH. Squamous and trophoblastic cells in the maternal pulmonary circulation identified by invasive hemodynamic monitoring during the peripartum period. Am J Obstet Gynecol. 1986;155(5):999–1001. 72. Johansen M, Redman CW, Wilkins T, Sargent IL. Trophoblast deportation in human pregnancy—its relevance for pre-eclampsia. Placenta. 1999;20(7):531–539. 73. Sibley CP, Bauman KF, Firth JA. Molecular charge as a determinant of macromolecule permeability across the fetal capillary endothelium of the guinea-pig placenta. Cell Tissue Res. 1983;229(2):365–377. 74. Alfirevic Z, Neilson JP. Doppler ultrasonography in high-risk pregnancies: systematic review with meta-analysis. Am J Obstet Gynecol. 1995;172(5):1379–1387. 75. Kingdom JC, Burrell SJ, Kaufmann P. Pathology and clinical implications of abnormal umbilical artery Doppler waveforms. Ultrasound Obstet Gynecol. 1997;9(4):271–286. 76. Su EJ. Role of the fetoplacental endothelium in fetal growth restriction with abnormal umbilical artery Doppler velocimetry. Am J Obstet Gynecol. 2015;213(suppl 4):S123–S130. 77. Castellucci M, Muhlhauser J, Zaccheo D. The Hofbauer cell: the macrophage of the human placenta. In: Andreani D, Bompiani GD, Di Mario U, et al., eds. Immunobiology of Normal and Diabetic Pregnancy. New York: John Wiley; 1990:135–144.

78. Kaufmann P, Mayhew TM, Charnock-Jones DS. Aspects of human fetoplacental vasculogenesis and angiogenesis. II. Changes during normal pregnancy. Placenta. 2004;25(2-3): 114–126. 79. Bracero LA, Beneck D, Kirshenbaum N, et al. Doppler velocimetry and placental disease. Am J Obstet Gynecol. 1989;161(2):388–393. 80. Giles WB, Trudinger BJ, Baird PJ. Fetal umbilical artery flow velocity waveforms and placental resistance: pathological correlation. Br J Obstet Gynaecol. 1985;92(1):31–38. 81. McCowan LM, Mullen BM, Ritchie K. Umbilical artery flow velocity waveforms and the placental vascular bed. Am J Obstet Gynecol. 1987;157(4 Pt 1):900–902. 82. Kingdom JC, Kaufmann P. Oxygen and placental villous development: origins of fetal hypoxia. Placenta. 1997;18(8):613–621; discussion 623–626. 83. Kaufmann P, Schroder H, Leichtweiss HP. Fluid shift across the placenta: II. Fetomaternal transfer of horseradish peroxidase in the guinea pig. Placenta. 1982;3(4):339–348. 84. Alkazaleh F, Chaddha V, Viero S, et  al. Second-trimester prediction of severe placental complications in women with combined elevations in alpha-fetoprotein and human chorionic gonadotrophin. Am J Obstet Gynecol. 2006;194(3):821–827. 85. Brownbill P, Edwards D, Jones C, et al. Mechanisms of alphafetoprotein transfer in the perfused human placental cotyledon from uncomplicated pregnancy. J Clin Invest. 1995;96(5): 2220–2226. 86. Kaufmann P, Scheffen I. Placental development. In: Pollin R, Fox W, eds. Neonatal and Fetal Medicine—Physiology and Pathophysiology. Orlando: WB Saunders; 1992. 87. Kaufmann P. Basic morphology of the fetal and maternal circuits in the human placenta. Contrib Gynecol Obstet. 1985;13:5–17. 88. Kaufmann P, Luckhardt M, Leiser R. Threedimensional representation of the fetal vessel system in the human placenta. Trophoblast Res. 1988;3:113–137. 89. Becker V, Schiebler TH, Kubli F. Die Plazenta Des Menschen. Stuttgart, Germany: Thieme Verlag; 1981. 90. Castellucci M, Scheper M, Scheffen I, et al. The development of the human placental villous tree. Anat Embryol (Berl). 1990;181(2):117–128.

8

Placental Function in Maternofetal Exchange COLIN SIBLEY AND MARK DILWOR TH

KEY POINTS • M  aternofetal exchange across the placenta provides the solutes and water needed for fetal development and growth and enables the waste products of fetal metabolism to be transferred to the maternal circulation. • The placental exchange barrier consists of the syncytiotrophoblast epithelial cell layer, basement membrane and connective tissue, and the fetal capillary endothelium. All contribute to the barrier, but the syncytiotrophoblast is probably the most important locus of regulation of maternofetal exchange. • Driving forces for maternofetal exchange are, depending on the molecule in question, electrochemical gradients or hydrostatic gradients (or both) between maternal and fetal circulations. • The placenta is highly permeable to small lipophilic molecules such as oxygen. These therefore rapidly cross the exchange barrier with the rate of transfer being mainly dependent on uterine and umbilical blood flow. • The placenta has a low permeability to larger hydrophilic molecules. These therefore only diffuse slowly across the placenta with the rate of transfer being more dependent on the properties of the placental barrier rather than blood flow. • Transfer of a hydrophilic molecule is likely to require selective transporter proteins in the plasma membranes of the syncytiotrophoblast (channels or carriers) or, for larger molecules, vesicles enabling endocytosis at one membrane and exocytosis at the other. • Placental dysfunction includes abnormalities in maternofetal exchange and can lead to pathologies, including fetal growth restriction (FGR). Such dysfunction may be stratified into vascular defects with abnormal blood flow or nonvascular defects with abnormalities of the syncytiotrophoblast. Development of new treatments for FGR will need to target these different phenotypes of placental dysfunction.

Introduction This chapter summarises current understanding of the mechanisms of maternofetal exchange across the placenta. A full description of these mechanisms would combine details of the physiological

processes involved with information about the identities, properties and genetic control of the relevant molecular species. However, there remains much to be learnt before such a comprehensive review is possible. Therefore we describe the key principles required for a full understanding of the maternofetal exchange of any solute and then provide examples of how these apply to selected substances. Finally, we consider the clinical relevance of maternofetal exchange in relation to fetal growth restriction. Additional detailed coverage of aspects of placental transfer which are beyond the scope of this chapter may be found elsewhere.1-5 A variety of species have been used to study placental transport, but considerable care must be taken in extrapolating from these to the human because of the great diversity of placental morphology and function.4 Animal studies do provide an important foundation for understanding placental function,6 but here we focus on work on human placenta. A variety of in vitro and in vivo techniques have been used to study the human placenta, and these are considered in detail elsewhere.4,5,7,8 Complete characterisation of a transport mechanism should broadly include four sets of information: (i) the amount of substance transferred per unit time and per unit surface area, the flux, in both maternofetal and fetomaternal directions, the difference between the two giving the magnitude and direction of the net flux; (ii) the magnitude and factors controlling the driving force for the transfer of a substance (e.g., plasma concentrations along the length of the exchange surface and blood flow); (iii) the route of transfer, for example, whether across the plasma membranes and through the cytosol (transcellular route) or via extracellular water-filled channels (paracellular route); and (iv) the role and contribution of the placenta’s own metabolic processes. Historically, measurement of flux was the focus of attention, but more recently, the focus has shifted to the cellular and molecular aspects of transport with the use of in vitro and molecular techniques. However, there is an ongoing requirement for the physiological relevance of mechanistic components deduced from in  vitro techniques to be reconciled with overall flux and accretion. It must also be borne in mind that transport by components of the placenta does not always lead to transfer across the placenta because some of the transport will satisfy the metabolic needs of the placenta itself. 

The Placental Exchange Barrier The human placenta is of the haemochorial type so that blood delivered into the intervillous space via the spiral arteries (Fig. 8.1) 69

70

SE C T I O N 2     The Placenta

directly bathes the syncytiotrophoblast lining of the villi (i.e., there is no endothelium separating blood from this epithelium). The syncytiotrophoblast is also unusual in that it a true multinucleated syncytium with no lateral intercellular spaces akin to those found in other epithelia (but see later discussion on paracellular routes) and is usually considered to be the main barrier to exchange. It has two plasma membranes: the microvillous, maternal-facing plasma membrane (MVM) and the fetal-facing basal plasma membrane (BM). Underlying the syncytiotrophoblast, there is an extracellular matrix (ECM) of connective tissue and finally the capillary

endothelium bathed in fetal blood. Although ECM will not present a major barrier to most solutes, it is likely that, as in other epithelia, it will create a slow-moving pool of fluid (an ‘unstrirred layer’) that will affect the nature of electrochemical gradients across the syncytiotrophoblast. The fetal capillary endothelium has lateral intercellular spaces through which small molecules can diffuse. However, the diffusion of large proteins, known to cross the placenta, such as immunoglobulin G and alpha-fetoprotein, is restricted through these spaces so that the endothelium is a significant barrier to such molecules.9 

Types of Exchange Mechanisms The different types of mechanisms of exchange across the placenta are summarised in Fig 8.2. The placental exchange barrier limits solute transfer to varying degrees. Lipophilic substances (e.g., oxygen) that dissolve readily in the plasma membrane will rapidly diffuse across the barrier.6 On the other hand, for hydrophilic substances (e.g., sodium ions, amino acids), the placenta is a significant barrier to transfer. However, this barrier is not absolute because there are multiple pathways available for hydrophilic solutes to pass across the placenta, including pores, channels, carriers (including cotransporters and exchangers), pumps and vesicles. These can be matched to mechanisms such as filtration (pores), diffusion (pores and channels), facilitated diffusion and secondary active transport (carriers), primary active transport (pumps) and endocytosis and exocytosis (vesicles). Pores allow for the movement of solute and solvent through a paracellular, extracellular water-filled pathway such that the transferred substances do not have to cross any plasma membranes to traverse a layer of tissue. Pores may allow transfer by diffusion of solute alone or, by bulk flow, of solute and solvent together. The extent of diffusion through pores can be altered by changes in electrochemical gradients and, in the case of bulk flow, by changes in plasma hydrostatic and osmotic pressures.10 There is clear physiological evidence, both in vivo and in vitro, that transfer through pores does occur in human placentas11-13 as well as in the placentas of several other species.14 Because the syncytiotrophoblast is a true syncytium as described earlier, the morphological correlates of the pores are unclear. However, there is evidence that naturally

VT MVM

BM

UC FC N

SA IVS

•

Fig. 8.1  The placental barrier. This primarily consists of the syncytiotrophoblast and the fetal capillary (FC) endothelium. Of these structures, it is primarily the two polarised plasma membranes, the microvillous (MVM) and the basal plasma membrane (BM) of the syncytiotrophoblast, that restrict the transfer of molecules like glucose and amino acids. IVS, intervillous space; N, nucleus of syncytiotrophoblast; SA, spiral artery; UC; umbilical cord; VT, villous tree.

O2

Mannitol

Glucose

Amino acid Na+

K+ Ca+

ATP

MVM

Na+ Ca+ H+

Ca+

Na+

ATP

ATP

a

b

c

d

Na+ e

K+ f

IgG Cl−, K+ g

h

BM

i

• Fig. 8.2  Schematic of the major transfer mechanisms across the microvillous membrane (MVM) and basal membrane (BM) of the syncytiotrophoblast with examples of the solutes transferred. (a) Diffusion of relatively lipophilic substances; (b) paracellular route for hydrophilic substances; (c) facilitated diffusion; (d) cotransport; (e) exchange; (f and h) active transport; (g) ion channels and (i) endocytosis-exocytosis. ATP, Adenosine triphosphate; IgG, immunoglobulin G. (Reproduced and adapted from Desforges M, Sibley CP. Placental nutrient supply and fetal growth. Int J Dev Biol. 2010;54(2-3):377–390.)

CHAPTER 8  Placental Function in Maternofetal Exchange

occurring areas of syncytial denudation, found in all normal placentas, could provide a large pore with contributions from other extracellular fluid-containing routes.15 This paracellular route of transfer is quantitatively of major importance for the transfer of small hydrophilic solutes such as calcium ions and chloride ions.16,17 Of course, the transcellular route through the syncytiotrophoblast, using channels and carriers, is likely to be qualitatively of greater importance, allowing fine tuning of net flux. Channels are integral membrane proteins through which ions may diffuse down electrochemical gradients either into or out of cells. Although passive, the diffusion of solute through channels is selective, gated and saturable and may be functionally asymmetric. These properties allow cells to modify the extent of inward and outward solute movements caused by diffusion in response to homeostatic signals mediated by intracellular, autocrine, paracrine and endocrine agents and by effects at the genome. This probably allows a range of normal processes and a broad repertoire of reactions to abnormal processes. Carriers are integral membrane proteins that selectively combine with a solute and can carry it from one side of the membrane to the other. The combining site is only exposed to one side of the membrane at a time. Channels and carriers show different behaviours. In general terms, if a solute is added to the far side (the trans side) of the membrane, then the combining site will be able to return to the near side (the cis side) more quickly than it would otherwise. The combining site will be on the near side more often and will remove solute more frequently. A carrier will thus carry more solute if it is ‘transstimulated’, but a channel will not respond in this way. Some carriers can transport more than one solute at a time. A cotransporter carries two solutes in the same direction; an exchanger swaps solutes. This allows cells to coordinate the movement of disparate solutes. Pumps carry solutes against concentration gradients. This is called ‘active transport’ because energy (as adenosine triphosphate (ATP)) is used up in the process. A good example is the active extrusion of sodium by cells in exchange for potassium on the Na+/K+-ATPase (sodium pump). This is primary active transport. Carriers can harness the gradients generated by pumps by linking solute movements to sodium movements. This is secondary active transport, which allows cells to move solutes against concentration gradients and thus to control their surroundings more subtly than if diffusion gradients were the only forces present. Vesicles are formed on one side of an epithelium such as the syncytiotrophoblast by invagination of the plasma membrane and, on the opposite side of the cell, fuse with the plasma membrane and open onto the extracellular space. Solute and water may be taken up into the forming vesicle by simple entrapment (‘fluidphase’ endocytosis), or solute may be taken up specifically by binding to receptors on the surface of the area of membrane about to vesiculate. Vesicles may move around the cytoplasm randomly by Brownian motion or may be directed by the cytoskeleton. 

Factors Affecting Maternofetal Exchange Most placental exchange is driven by diffusion or modifications of this process. Factors affecting diffusion will thus affect the magnitude of net flux. The rate of diffusion is determined by the concentration gradient across the barrier and the permeability of the barrier and its components. Different substances have different concentration gradients.3 Permeability varies among solutes according to their size, shape and lipophilicity (permeability to hydrophobic molecules being much greater than that

71

of hydrophilic ones as mentioned earlier). The permeability of the placenta to hydrophilic solutes increases towards term in various animals,18 although this has not been studied in humans. A potential difference across the exchange barrier will affect the transfer of charged solutes. Potential differences between mother and fetus have been measured in some species,19 but it is unclear whether these are generated by the placenta. In humans, a potential difference has been measured in vitro both across the MVM and across the entire exchange barrier of isolated mature intermediate villi derived from term placentas (magnitude ∼4 mV; fetal side, negative.)20 In vivo, a small maternofetal potential difference was measured in women in the third trimester21 of similar magnitude to that found in vitro. However, at term, there is reported to be no significant maternofetal potential difference.22 This question of the magnitude and polarity of potential difference across the placental exchange barrier is difficult to address experimentally in humans but is of fundamental importance in understanding the driving forces for ions and other charged solutes. The pattern and magnitude of blood flow affects exchange.23 A hydrophobic substance (e.g., oxygen) crosses the membrane so quickly that it has effectively gone from the maternal side of the placenta as soon as it arrives; the rate-limiting steps of transfer will be the rate at which it arrives and the rate at which it is taken away. The transfer of such substances is said to be ‘flow limited’; if placental blood flow is deranged, oxygen delivery, for example, will be impaired. Furthermore, the pattern of blood flow will affect the efficiency of exchange. If the blood flows are in opposite directions (countercurrent flows), the exchange will be more efficient than if they are in the same direction (concurrent flows). The human placenta is thought to have an intermediate arrangement in efficiency called ‘multivillous pool flow’.23 A hydrophilic substance, on the other hand (e.g., an amino acid) will have much lower permeability across the placenta, transfer is slow and its concentration in the maternal circulation hardly changes across the exchange barrier. The transfer of such substances is therefore relatively unaffected by blood flow, and their transfer is said to be ‘membrane’ or ‘diffusion’ limited. It is important to understand the distinction between flow- and membrane-limited diffusion when considering the phenotypes of placental dysfunction as related to FGR (see final section of this chapter). The placenta is a metabolically active organ, which significantly affects the traffic of solutes such as oxygen, amino acids and carbohydrates. Control of placental metabolism, as well as of transport, by hormonal, genetic or intrinsic means, by the mother or fetus, is likely to be of considerable importance. Finally, it should be noted that although the placenta shares many characteristics with other tissues (e.g., sodium pumps and intracellular signalling apparatus), we can identify features which are less prominent in other organs, and these need to be mentioned as a background to any discussion of function or pathology. First, the supply of some substances is vastly in excess of fetal accretion,24 but other fluxes are more obviously related to fetal requirements. Second, transfer represents diverse phenomena; some substances (e.g., glucose) are transferred by one or two mechanisms only, and alterations in these mechanisms are relatively easy to detect. Other substances (e.g., sodium) are transferred by many specific mechanisms, none of which is dominant, and alterations in these systems are more difficult to detect. Water flux, which is greater than for any other molecule,25 appears to be particularly complex. There is evidence in rats26,27 that net water flux may be the balance of osmotic flow in the maternofetal direction following active transport of ions and bulk flow in the fetomaternal direction down

72

SE C T I O N 2     The Placenta

a hydrostatic pressure gradient. Alteration in water transfer may thus reflect a wide range of changes and is therefore not simple to understand and will not be easy to manipulate. 

Specific Examples of Transferred Substances Respiratory Gas Exchange Gas exchange at the placenta is determined by the following factors: (i) the concentration gradient of the gas across the placenta; (ii) the gas-carrying capacities of fetal and maternal blood; (iii) the rates of fetal and maternal placental blood flow; (iv) the relative spatial orientation of the two blood flows; and (v) the permeability properties and surface area of the membranes involved. Membrane permeability and total surface area change over gestation but cannot respond to short-term perturbations, and the relative orientation of blood flows is also fixed. Blood flow in the intervillous space is thought not to occur before 10 to 12 weeks of gestation.28 After intervillous blood flow is established, transfer processes seem to fit a multivillous pool flow model best.23 Blood flows and concentration gradients can change, in the short term, in normal physiological states and, in the long term, in abnormal states such as preeclampsia and FGR. Oxygen supply. Throughout pregnancy, fetal blood has a higher haemoglobin (Hb) concentration than maternal blood and a higher affinity for oxygen (O2) because of a lower affinity for 2,3-diphosphoglycerate.29 Whereas fetal blood at term has an O2-carrying capacity of 25 mL/dL maternal blood can carry only 15 mL/dL. There are many ways of expressing O2 exchange, but the crucial physiological variable is fetal O2 uptake because it determines the capacity for oxidative metabolism. In sheep, for example, homeostatic mechanisms keep this variable remarkably constant in the face of wide variations in other variables (see earlier discussion). It is not until fetal O2 uptake drops below a critical level (0.6 mmol O2/min/kg) that metabolic acidosis ensues. A small drop in umbilical artery O2 content results in a large increase in the amount of O2 transferred from mother to fetus. This is due to the relative characteristics of HbA and HbF O2 dissociation curves for the mother and fetus, respectively. The result of this relationship is that the single most important determinant of O2 transfer from maternal to fetal blood is the O2 content of blood perfusing the placenta from the umbilical arteries. This mechanism keeps fetal O2 uptake relatively constant during shortterm changes in umbilical and uterine blood flow via compensatory changes in fractional O2 extraction.30 Although responses to short-term hypoxia are seen in clinical situations such as cord compression, maternal exercise, fetal activity or uterine contractions, no effect on long-term outcome has been demonstrated. However, long-term hypoxia is important to fetal well-being as demonstrated by studies on women living at high altitudes.31  Carbon dioxide removal. Most carbon dioxide (CO2) in the blood is hydrated to hydrogen ions (H+) and bicarbonate (HCO3− ) ions; this conversion is catalysed by carbonic anhydrase (CA) inside red blood cells. Some CO2 is associated with deoxygenated Hb, as carbamino-Hb, and only a small amount is in solution. In the guinea pig, transfer of HCO3− relies on the presence of CA on both MVM and BM,32 suggesting that HCO3− must be converted back to CO2 before transfer across the placenta. Carbon dioxide is lipid soluble, so it is readily transferred across the placenta by diffusion. In the same study, a small amount

of transfer of the HCO3− ion, in exchange for lactate or chloride ions, was seen when CA activity was inhibited with acetazolamide. Fetal plasma (umbilical vein) Pco2 at term is between 38 and 45 mm Hg,33 but maternal arterial Pco2 is between 26 and 34 mm Hg34 (lower than prepregnancy values because of hyperventilation). This concentration gradient therefore drives CO2 transfer. However, because diffusion of the small, lipophilic CO2 molecule is rapid, relative fetal and maternal placental blood flows are the critical determinants of its rate of transfer. Consequently, reduced uteroplacental or umbilical blood flow leads to respiratory acidosis in the fetus; this is rapidly corrected if normal blood flow is reestablished. 

Acid–Base Balance The role of the placenta in fetal acid–base balance is poorly understood. Mechanisms for regulating acid–base status are a dynamic element of fetal metabolism. For example, as normal gestation proceeds, the pH of blood in the umbilical vessels falls.35 These changes do not occur in isolation. Po2 also declines during normal gestation, and fetal Hb rises.35 Acid equivalents produced by the fetus during metabolism cannot be eliminated by CO2 transfer across the placenta and require transport of protons from fetal to maternal circulations or of bicarbonate in the reverse direction. Although there is good evidence of a Cl−/HCO3− exchanger in the synctriotrophoblast,17,36,37 its role in HCO3− transport has not been elucidated. Better studied is the sodium-proton exchanger (NHE) of which several isoforms have been identified in the human placenta.38,39 NHE is highly active in the MVM, where it exchanges Na+, moving down its concentration gradient into the syncytiotrophoblast, for H+ which is therefore eliminated into the maternal circulation. NHE expression and activity on the MVM is lower in FGR pregnancies than those with normally grown babies,40 and this could contribute to the acidosis that some FGR babies develop. Lactate is traditionally viewed as a byproduct of anaerobic respiration. However, in a fetus, lactate may be an important source of energy for the fetus and the placenta even when oxygen and glucose supplies are adequate.41 Umbilical venous lactate concentrations are higher than the umbilical arterial levels, and both are higher than the levels in the maternal circulation.41 This suggests that lactate is secreted into both circulations by the placenta.42 Small-for-gestational age (SGA) fetuses have lower umbilical arterial and venous Po2 and pH values with higher Pco2 and lactate values than in normally grown fetuses.35,42 This elevated lactate concentration might suggest a reduced oxidative capacity in SGA fetuses; the lower Po2 and raised Pco2 are likely to be indicative of reduced placental blood flow. There is good evidence that lactate is taken up by both MVM and BM of the syncytiotrophoblast via isoforms 1 and 4 of the lactate/H+ co-transporter (also known as the monocarboxylate transporter).43 The activity of this transporter in the BM, but not MVM, is reduced in pregnancies with FGR,44 and this may contribute to the Lacticacidaemia associated with this condition. 

Ions Because the permeability of the human placenta to small hydrophilic substances is so high, it seems likely that the major component of both maternofetal and fetomaternal fluxes of ions are by diffusion through a paracellular route. As already noted, understanding the exact contribution of such diffusion to ion exchange

CHAPTER 8  Placental Function in Maternofetal Exchange

is hampered by the lack of a clear understanding of the maternofetal potential difference Furthermore, the presence of specific ion transporters in the MVM and BM of the syncytiotrophoblast suggests that there is at least a small transcellular component of exchange which, being regulatable, might be qualitatively most important. In rats, there is good evidence that sodium is actively transported to the fetus.45 Analysis of the classical data of Flexner and colleagues, obtained by in  vivo measurements of Na transfer,24 suggests that although the bulk of sodium transfer to the fetus might be by passive means, the human placenta also does not act solely as a simple filter of sodium. Several routes for transport of sodium have been demonstrated in human placental preparations, and these include sodium channels in the MVM,46 the NHE as described earlier, Na+/amino acid cotransport (see later section on amino acids) and the Na+/K+-ATPase.47 However, all of these may contribute to placental homeostasis as well as to fetal growth. Certainly Na+/K+-ATPase in the syncytiotrophoblast has a key role in cellular homeostasis, as in all cells. Interestingly, the activity of this transporter on the MVM, like that of NHE, is reduced in FGR,47 and this could impair the functioning of all Na+-coupled solute transporters in this condition. Chloride transfer across the placenta has been poorly explored. There is evidence that the bulk of maternofetal chloride transfer is by passive diffusion but that transcellular routes do contribute a quantitatively small fraction.17 Several such routes have been identified, including channels48 and the Cl−/HCO3− exchanger described earlier, but the relative contribution of each of these to maternofetal exchange rather than syncytiotrophoblast homeostasis is unknown.

Placental transfer of the divalent cations calcium, magnesium and phosphorus involves common features. First, transport from a mother to her fetus is against a concentration gradient, the concentration of each of these solutes being higher in fetal plasma than maternal49 this suggests that active transport mechanisms underlie the placental transfer of all three ions. Second, the net placental transport of each of the divalent ions increases over the last third of gestation, coincident with the mineralisation of the fetal skeleton.49 This coordinated gestational increase in placental transfer, despite different transport mechanisms being involved for the individual ions, is remarkable. Unfortunately, the means by which this coordination is brought about is not yet known. Although phosphorous and magnesium transfer are poorly understood, the mechanism of calcium transfer across the human placenta has been reasonably well described. Fetal plasma concentrations of total and ionised calcium are higher than maternal, and there is strong evidence that there is active, transcellular transport of this cation across the placenta.4 This transcellular transport of calcium most likely involves three steps (Fig. 8.3). The first of these is being transfer from the maternal blood to the trophoblastic cytosol across the MVM of the syncytiotrophoblast. The electrochemical gradient for calcium movement across this plasma membrane is favourable: the median potential difference across the MVM, when measured in  vitro, was –22 mV (trophoblast negative),20 and the intracellular ‘free’ (ionised) calcium concentration is likely to be of the order of 10–7 M,50 four orders of magnitude lower than the extracellular ‘free’ calcium concentration of 10–3 M in plasma. This favourable inwardly directed electrochemical gradient makes channels [Ca2+] = 1.2 mmol/L

Ca2+ Ca2+

Calbindin-D9K or D28K Ca2+

ADP + Pi

PMCA

Maternofetal

Maternofetal

Fetomaternal

TRPV6

Maternal side Microvillous membrane

ATP

[Ca2+] = 0.1 µmol/L

Basal membrane Fetal side

Ca2+ Paracellular

73

Transcellular

[Ca2+] = 1.4 mmol/L

• Fig. 8.3  Schematic showing the paracellular and transcellular routes of Ca2+ transfer across the syncytiotrophoblast. Paracellularly, there are maternofetal and fetomaternal fluxes, though the fetomaternal flux is lower because of the prevailing electrochemical gradient. Transcellularly, Ca2+ enters the syncytiotrophoblast across the microvillous plasma membrane (MVM) down an electrochemical gradient from maternal blood (∼1.2 mmol/L) into the trophoblastic cytosol (0.1 μmol/L). This diffusion is likely to involve channels such as the transient potential vanilloid type 6 (TRPV6). When inside the cytosol of the trophoblast, Ca2+ binds to a calcium binding protein, with calbindin-D9K (and calbindin-D28K) thought to be important for intracellular buffering of Ca2+. At the basal membrane (BM), Ca2+ is effluxed across the BM against an electrochemical gradient and into fetal blood through the actions of the plasma membrane calcium ATPase (PMCA). This process maintains a relatively hypercalcaemic (∼1.4 mmol/L) environment in fetal versus maternal blood. ADP, Adenosine diphosphate; ATP, adenosine triphosphate.

74

SE C T I O N 2     The Placenta

the likely route of calcium diffusion into the syncytiotrophoblast across the MVM, and evidence from mice shows that the Ca2+ selective channel, transient receptor potential, vanilloid 6 (TRPV6) plays such a role in this species.51 Furthermore, this channel is expressed in human syncytiotrophoblasts.52 Although it seems that TRPV6 is an excellent candidate for the Ca2+ entry route, it should be noted that other Ca2+ selective channels are expressed in human syncytiotrophoblasts, suggesting multiple routes of entry.52 The second step in the transcellular transfer of calcium across the placenta involves translocation across the trophoblast cytosol, without generating marked fluctuations in intracellular calcium concentration. This is likely to be achieved mainly by the presence of various calcium-binding proteins. In particular, the role of a 9-kDa calcium-binding protein (calbindin-D9k) has been the focus of several studies. In rat placentas, the mRNA expression of calbindin-D9k increases markedly over the last third of gestation, and this induction is temporally associated with the gestational increase in the unidirectional maternofetal clearance of calcium across this tissue.53 This suggests that the protein is stoichiometrically involved in transplacental calcium transport. However, calbindin-D9k knockout mice do not have defective placental calcium transport,54 perhaps because of compensation by other proteins. This protein is present in human trophoblast cells55 as are other calcium-binding proteins such as calbindin-D28k,56 which may also be involved in transcellular calcium transfer across the syncytiotrophoblast. The third step in the transcellular transfer of calcium across the placenta involves efflux of calcium from the trophoblast cytosol across the BM and hence across the fetal capillary endothelium into fetal plasma. Because this step involves transport against a chemical gradient, an active mechanism is implicated. Indeed, the transfer of calcium across the BM in the human placenta is now known to occur via the plasma membrane calcium ATPase (PMCA or Ca2+-ATPase).57,58 As already noted, there is a marked increase in calcium flux across the placenta to the fetus towards the end of gestation, presumably to enable the rapid skeletal mineralisation that happens at this time. In rats, this increase in calcium transport appears to be mediated by a sharp increase in calbindin-D9k expression,53 but in human placentas, calcium pump activity on the BM increases significantly from 32 to 37 weeks.59 The latter might be regulated by parathyroid hormone-related peptide (PTHrP): the midmolecule fragment 38 to 94 of this hormone does acutely stimulate the activity of human placental calcium pump activity in vitro.60 

Glucose Glucose is the primary metabolic substrate for energy in both fetuses and placentas. In human placentas, uptake of radiolabelled d-glucose or methyl d-glucose, a nonmetabolisable analogue, across the MVM and BM of the syncytiotrophoblast, was found to be rapid, stereospecific, Na+-independent and inhibitable by cytochalasin B, phloretin and phlorizin.61,62 These are characteristics of the GLUT family of Na+-independent sugar transporters which undoubtedly play the major role in transplacental glucose transfer. At least four different GLUT isoforms are expressed in the syncytiotrophoblast of a first trimester placenta (GLUT1, 3, 4 and 12).63,64 GLUT1 is the predominant isoform in the syncytiotrophoblast at term.63,65 GLUT3 is also expressed at term in the MVM but at much lower levels than in the first trimester,66

suggesting it is of greater importance in early pregnancy. Both first trimester and term human placentas show greater GLUT1 expression on the MVM compared with the BM.65,67 This asymmetry in GLUT1 distribution together with the much greater surface area of the MVM may well result in syncytial glucose concentrations approaching maternal, providing a maximal gradient for transfer to the fetus. The driving force for facilitated glucose transfer across the placenta is the maternofetal concentration gradient with fetal whole blood or plasma concentrations being lower than maternal.68 From the Km values for the glucose transporters in the MVM and BM of the human syncytiotrophoblast, 31 mM61 and 23 mM,62 respectively, it can be concluded that not only is transport likely to be linearly related to maternal concentration in the physiological range but also that transplacental glucose transport will not be saturated under physiological conditions. A consequence of this is that maternal hyperglycaemia in diabetes with suboptimal metabolic control will result in increased placental glucose transfer with potentially increased fetal insulin secretion and fetal overgrowth. GLUT4 and GLUT12 are sensitive to regulation by insulin, and there is evidence that glucose uptake is stimulated by insulin in the first trimester.63 However, GLUT1 and GLUT 3 are not insulin sensitive, and to date, there is no good evidence for hormonal control of glucose transfer across late gestation placentas.69 

Amino Acids Amino acids are required for fetal protein synthesis, as energy substrates and for other functions such as cell volume regulation. The concentration of almost all amino acids is higher in umbilical venous blood than in maternal uterine arterial blood from the midtrimester onwards, implying active transport from the mother to the fetus across the syncytiotrophoblast.70 In support of this, a wide range of amino acid transporters have now been characterised in the MVM and BM of the syncytiotrophoblast5; each transporter mediates the uptake of several different amino acids, and each specific amino acid can be transported by several different transporters. Concentrations within placental tissue itself are higher than in either maternal or fetal serum,71 so the interesting question is how net flux from mother to the fetus occurs. There appear to be three different classes of transporter present in the syncytiotrophoblast which enable net flux – accumulative, exchanger and facilitative72 (Fig. 8.4). The active step is at the MVM, where amino acids have to transported against their concentration gradient. This can be achieved by accumulative transporters, which in many cases use the inwardly directed sodium gradient created by Na+/K+ATPase in an energy-dependent process. An example of such a secondary active process is the system A amino acid transporter which co-transports alanine, serine or glycine with sodium into the syncytiotrophoblast. Alternatively, other transport systems such as system y+ for cationic amino acids can use the electrical gradient across the MVM (inside negative) to drive amino acid accumulation into the syncytiotrophoblast. Exchangers can swap one amino acid for another across the plasma membrane; for example, the steep outwardly directed gradient for glycine across the MVM (arising from accumulative transport) can enable accumulation of leucine by exchange on the system L transporter. These exchange systems therefore cannot change the total quantity of amino acid in the syncytiotrophoblast, but they can change the composition. For net flux to occur, amino acids have to leave the syncytiotrophoblast across the BM. Because of the high syncytiotrophoblast

CHAPTER 8  Placental Function in Maternofetal Exchange

Microvillous membrane

Basal membrane

Fetal capillary endothelium

Syncytiotrophoblast

Maternal blood arterial

75

Fetal blood arterial

Paracellular routes

Accumulative transporters

Exchange transporters

Exchange transporters

Flow

Flow and mixing

Accumulative transporters

Facilitative transporters

Venous

Arrows indicate movement of amino acids

Venous

• Fig. 8.4  Schematic of amino acid transfer routes across the syncytiotrophoblast. Amino acid transfer is reliant upon a range of transport proteins/systems with different specificities and modes of action. The complex interplay between these transporters is summarised above. (Replicated with permission from Lewis RM, Brooks S, Crocker IP, et al. Review: modelling placental amino acid transfer—from transporters to placental function. Placenta 2013;34(suppl):S46–S51.)

cytosol amino acid concentration, this is down a concentration gradient, and recent evidence suggests that this uses both exchangers and facilitated diffusion through specific efflux pathways.72,73 Interestingly accumulative transporters such as system A are also expressed on the BM5; presumably, they have a role here in maintaining the amino acid gradients required for the exchangers to operate. Although the fundamental steps involved in amino acid transfer as described earlier now seem fairly clear, the overall mechanism of net flux of the 20 amino acids required by a growing fetus is complex. As noted, although the transporters are selective, they have overlapping affinities for different amino acids, which therefore compete; there has to be integration between accumulative, exchange and facilitative transporters as they each alter the quantity and composition of amino acid in the syncytiotrophoblast; finally, amino acids are used and metabolised by the syncytiotrophoblast and other placental cell types, again modulating transfer to the fetus. In silico modelling of placental amino acid transfer is now being used to resolve this complexity.74,75 This is particularly important because abnormal placental amino acid transfer appears to be an important component in the aetiology of FGR, as described further later. A number of hormones, growth factors and cytokines affect placental amino acid transfer. For example, insulin, insulinlike growth factor 1 (IGF-1), leptin, interleukin-6 and tumour necrosis factor alpha all upregulate system A transporter activity; corticosteroids appear to downregulate activity of this transporter, and adiponectin inhibits insulin-stimulated amino acid transfer (recently reviewed by Dimasuay and colleagues3). Such circulating signals and other factors affecting nutrient transfer, including blood flow, oxygen and maternal nutrition, appear to be sensed by the mammalian (or mechanistic) target of rapamycin (mTOR) pathway.3 It is likely that mTOR has a key role in modulating placental amino acid transfer both by modulating the activity of

amino acid transporters and by affecting the trafficking of these transporters to the syncytiotrophoblast plasma membrane. In the placental nutrient-sensing model, it is proposed3 that the syncytiotrophoblast integrates signals from the maternal and fetal compartment that modulate placental function and match fetal growth to maternal nutrient availability. 

Immunoglobulin G In women, passive immunity is conferred on the fetus by the selective transfer of immunoglobulin G (IgG) by the placenta. Selective IgG transfer has been considered as involving an endocytosis–exocytosis model with three steps: (i) IgG destined for transfer to the fetus is taken up from maternal plasma by endocytosis via specific Fc receptors in coated pits (specialised areas of plasma membrane which have a fuzzy electron-dense outline, under the electron microscope, because of a protein called ‘clathrin’) on the microvillous plasma membrane of the syncytiotrophoblast; (ii) Fc receptor-­ bound IgG within coated vesicles, formed by invagination of the coated pits, is protected from digestion by lysosomes within the syncytiotrophoblast; and (iii) vesicles containing undigested IgG fuse with the basal plasma membrane of the syncytiotrophoblast, exocytose and release the IgG into the interstitial space. Studies in which IgG in the syncytiotrophoblast has been localised under the electron microscope, as well as biochemical experiments, mainly support the first part of the model described,76-80 although not every study has found IgG in coated pits and vesicles.81 Evidence suggests that this uptake involves the human neonatal Fcϒ receptor (hFcRn).82 Unlike other Fcϒ receptors, hFcRn has a much higher affinity for IgG at pH 6.0 than it does at neutral pH and is therefore unable to bind IgG at the extracellular face of the MVM. It is therefore most likely that IgG in relatively high concentrations in maternal plasma is taken up by fluid-phase endocytosis and then binds to hFcRn in the acidic environment

76

SE C T I O N 2     The Placenta

of endosomes inside the syncytiotrophoblast. It is in this form that IgG is transferred across the syncytiotrophoblast (step ii). Experimental evidence for exocytosis in step (iii) of the model is equivocal; direct evidence for exocytosis is weak.78-81 When IgG reaches the interstitial space on the fetal side of the syncytiotrophoblast, it still has to traverse the basement membrane and fetal capillary endothelium. Although the former does not appear to present a significant barrier,78,81 the latter does9; the available data suggest that transcytosis in vesicles is a more likely route of transfer across the endothelium than is diffusion through the lateral intercellular spaces between endothelial cells.78-80 

Clinical Considerations: Maternofetal Exchange and Fetal Growth Restriction More than 95% of the solutes and water that constitute a term baby is attained by net flux across the placenta over the course of gestation (the remainder largely via yolk sac in early pregnancy and a transamniotic route). Therefore, by definition, net flux across the placenta over gestation in a pregnancy with a small baby will have been lower compared with that in a pregnancy with a larger baby. A key question is whether, in a pregnancy affected by FGR (or in the opposite condition of fetal overgrowth), the altered net flux across the placenta is a cause or consequence of the abnormal fetal growth. There is in fact now considerable evidence to support a causal role of dysfunctional maternofetal exchange across the placenta in FGR; indeed, evidence of placental dysfunction is becoming the gold standard in separating cases of small but normal babies from those with FGR.83 However, it is also becoming increasingly clear that abnormal placental transfer in FGR may be stratified into at least two separate pathologies relating to the concepts of blood flow limited and membrane limited transfer as described earlier in the chapter. Placental dysfunction in preeclampsia and FGR primarily arises from inadequate trophoblast invasion and incomplete maternal uterine spiral artery remodelling, resulting in impaired development and function of the uteroplacental vasculature and abnormal formation and renewal of the syncytiotrophoblast.84,85 Abnormal spiral artery transformation leads to ischaemia/reperfusion injury in the placenta, inducing oxidative and nitrative stress.86 The free radicals generated may also impair syncytiotrophoblast development and function. This aetiology could lead to a number of different placental abnormalities resulting in different disease phenotypes of FGR. These can be broadly categorised as (i) vascular FGR (caused by abnormal uterine and myometrial artery function, with consequent inadequate blood flow to the placenta) and (ii) nonvascular FGR (caused by defective syncytiotrophoblast function). Raised vascular resistance in FGR is detected clinically by abnormal Doppler ultrasound flow-velocity waveforms in the uterine and umbilical arteries.84 Abnormal vascular anatomy87 and impaired myometrial and chorionic plate vascular tone regulation88-91 probably underlie this raised vascular resistance. As described previously, reduced blood flow through the placenta particularly alters the diffusion gradient for transfer of small lipophilic solutes such as O2 and CO2 across the placenta and so directly restricts fetal growth. However, FGR is found in the absence of abnormal uterine or umbilical artery Doppler waveforms,83,85 suggesting that reduced blood flow is not the direct cause of reduced maternofetal

exchange in such pregnancies. Furthermore, some features of FGR cannot be ascribed to abnormal blood flow (e.g., umbilical vein plasma amino acid concentrations in FGR are significantly lower than those in normally grown babies)70; such relatively large hydrophilic molecules would have too low a permeability across the placenta for their transfer to be significantly affected by flow. It therefore seems that there is a set of FGR pregnancies in which defective syncytiotrophoblast function is the major cause of reduced maternofetal transfer. This is supported by data showing that the activity of several syncytiotrophoblast plasma membrane amino acid transporters and other nutrient transporters are decreased in FGR (reviewed by Hayward5). It is difficult to show a direct cause-and-effect relationship between decreased placental transporter activity and reduced fetal growth. However, in rats fed a low-protein diet, decreased placental amino acid transporter activity precedes FGR,92 and in mice, genetically altering the activity of the placental system A amino acid transporter is followed by FGR.93 Changes in transporter activity may directly arise from free radical damage to the syncytiotrophoblast in early pregnancy after abnormal transformation of spiral arteries. Alternatively, placental transporter activity is regulated by growth factors and hormones such as IGF-1 and leptin, and it is clear that there are changes in maternal plasma concentrations of these and of placental receptor number and activity in FGR which would be compatible with (e.g., reduced system A amino acid transporter activity3). A further causative mechanism could be via nutrient sensing proteins in the syncytiotrophoblast such as mTOR. In vitro experiments with human placental fragments or trophoblast cells show that inhibition of mTOR markedly reduced amino acid transporter activity,94,95 and it has also been shown that placental mTOR signalling activity is downregulated in FGR.95 Although the activity of some transporters is reduced in placentas from FGR pregnancies, that of others is unchanged,5 and indeed one, the syncytiotrophoblast BM Ca2+ATPase, is actually increased.96 This suggests that the change in syncytiotrophoblast transporter activity in FGR is discrete rather than being a result of generalised tissue damage. There is evidence from both human and animal studies that placental transporter activity adapts in relation to fetal growth, putatively to return an abnormal growth pattern back to normal (i.e., there is increased activity per milligram of placental membrane protein when growth is reduced compared with normal and vice versa.97,98 It could be that nonvascular FGR related to syncytiotrophoblast dysfunction is at least in part because of a failure of such adaptation. There are currently no treatments for FGR other than early delivery. However, there are clinical trials in progress of two therapies designed to improve uteroplacental blood flow – sildenafil citrate99 and vascular endothelial growth factor delivered by gene therapy.100 The outcomes of these trials will be complicated by the presence or absence of patients with nonvascular syncytiotrophoblast dysfunction as well as those with uterine and myometrial vascular dysfunction. Future work needs to be directed at techniques for stratifying pregnancies with FGR into these two groups, as well as perhaps other subgroups of placental dysfunction. Placental hormone biomarkers such as placental growth factor (PIGF) might be useful for this, but specific tools for determining placental exchange function directly are needed. Magnetic resonance spectroscopy could be one such tool,101 as could measurements of placental oxygen handling using magnetic resonance imaging.102 After stratification, targeting of drugs to either vasculature

CHAPTER 8  Placental Function in Maternofetal Exchange

or syncytiotrophoblast would likely make therapies more effective and reduce side effects; there are now exciting data to suggest that this might be achieved by packaging drugs in nanoparticles coated with tissue specific homing peptides.103 

Conclusions Although understanding of placental function in maternofetal exchange has markedly increased over the past 50 years, there is still much to learn. Knowledge of the individual components and factors that determine transfer of any particular solute is now good but improved understanding of how these contribute to net flux of that solute across the placenta is needed. Because of

77

the complexity involved and the difficulty in studying the whole system in pregnant women, this will require new methods, which will undoubtedly include in silico modelling alongside more refined animal models. This will also be important in characterising and stratifying placental dysfunction in relation to FGR and fetal overgrowth and enabling targeted treatments. Regulation of maternofetal exchange is undoubtedly important, and more work is also needed in this area, although the placental nutrientsensing model provides a good foundation for further studies. Access the complete reference list online at ExpertConsult.com. Self-assessment questions available at ExpertConsult.com.

References

1. Desforges M, Sibley CP. Placental nutrient supply and fetal growth. Int J Dev Biol. 2010;54(2-3):377–390. 2. Brett KE, Ferraro ZM, Yockell-Lelievre J, et al. Maternal-fetal nutrient transport in pregnancy pathologies: the role of the placenta. Int J Mol Sci. 2014;15(9):16153–16185. 3. Dimasuay KG, Boeuf P, Powell TL, Jansson T. Placental responses to changes in the maternal environment determine fetal growth. Frontiers Physiol. 2016;7:12. 4. Atkinson DE, Boyd RDH, Sibley CP. Placental transfer. In: Neill JD, ed. Knobil and Neil’s physiology of reproduction. 2nd ed. San Diego: Elsevier; 2006:2787–2846. 5. Hayward CE, Jones RL, Sibley CP. Mechanisms of transfer across the human placenta. In: Polin RA, Abman SH, Rowitch DH, et al., eds. Fetal and neonatal physiology. 5th ed. Philadelphia: Elsevier; 2017:121–133. 6. Dilworth MR, Sibley CP. Review: transport across the placenta of mice and women. Placenta. 2013;34(suppl):S34–S39. 7. Greenwood SL, Sibley CP. In  vitro methods for studying human placental amino acid transport placental villous fragments. Methods Mol Med. 2006;122:253–264. 8. Glazier JD, Sibley CP. In  vitro methods for studying human placental amino acid transport: placental plasma membrane vesicles. Methods Mol Med. 2006;122:241–252. 9. Firth JA, Leach L. Not trophoblast alone: a review of the contribution of the fetal microvasculature to transplacental exchange. Placenta. 1996;17(2-3):89–96. 10. Faber JJ, Anderson DF. Model study of placental water transfer and causes of fetal water disease in sheep. Am J Physiol. 1990;258(5 Pt 2):R1257–R1270. 11. Willis DM, O’Grady JP, Faber JJ, Thornburg KL. Diffusion permeability of cyanocobalamin in human placenta. Am J Physiol. 1986;250(3 Pt 2):R459–R464. 12. Thornburg KL, Burry KJ, Adams AK, et  al. Permeability of placenta to inulin. Am J Obstet Gynecol. 1988;158(5):1165–1169. 13. Bain MD, Copas DK, Taylor A, et al. Permeability of the human placenta in vivo to four non-metabolized hydrophilic molecules. J Physiol. 1990;431:505–513. 14. Stulc J. Extracellular transport pathways in the haemochorial placenta. Placenta. 1989;10(1):113–119. 15. Brownbill P, Edwards D, Jones C, et  al. Mechanisms of alphafetoprotein transfer in the perfused human placental cotyledon from uncomplicated pregnancy. J Clin Invest. 1995;96(5):2220–2226. 16. Stulc J, Stulcova B, Smid M, Sach I. Parallel mechanisms of Ca++ transfer across the perfused human placental cotyledon. Am J Obstet Gynecol. 1994;170(1 Pt 1): 162–167. 17. Doughty IM, Glazier JD, Greenwood SL, et  al. Mechanisms of maternofetal chloride transfer across the human placenta perfused in  vitro. Am J Physiol. 1996;271(6 Pt 2):R1701–R1706. 18. Sibley CP, Coan PM, Ferguson-Smith AC, et al. Placental-specific insulin-like growth factor 2 (Igf2) regulates the diffusional exchange characteristics of the mouse placenta. Proc Natl Acad Sci U S A 25. 2004;101(21):8204–8208.

19. McNaughton TG, Power GG. The maternalfetal electrical potential difference: new findings and a perspective. Placenta. 1991;12(3): 185–197. 20. Greenwood SL, Boyd RD, Sibley CP. Transtrophoblast and microvillus membrane potential difference in mature intermediate human placental villi. Am J Physiol. 1991;265(2 Pt 1):C460–C466. 21. Stulc J, Svihovec J, Drabkova J, et al. Electrical potential difference across the mid-term human placenta. Acta Obstet Gynecol Scand. 1978;57(2):125–126. 22. Mellor DJ, Cockburn F, Lees MM, Blagden A. Distribution of ions and electrical potential differences between mother and fetus in the human at term. J Obstet Gynaecol Br Commonw. 1969;76(11):993–998. 23. Schroder HJ. Comparative aspects of placental exchange functions. Eur J Obstet Gynecol Reprod Biol. 1995;63(1):81–90. 24. Flexner LB, Cowie DB, et al. The permeability of the human placenta to sodium in normal and abnormal pregnancies and the supply of sodium to the human fetus as determined with radioactive sodium. Am J Obstet Gynecol. 1948;55(3):469–480. 25. Barcroft J. Researches on prenatal life. Part 1. Oxford, United Kingdom: Blackwell Scientific Publications; 1947. 26. Stulc J, Stulcova B. Asymmetrical transfer of inert hydrophilic solutes across rat. Placenta Am J Physiol. 1993;265(3 Pt 2):R670–R675. 27. Stulc J, Stulcova B. Effect of NaCl load administered to the fetus on the bidirectional movement of 51Cr-EDTA across rat placenta. Am J Physiol. 1996;270(5 Pt 2):R984– R989. 28. Jauniaux E, Jurkovic D, Campbell S. Current topic: in  vivo investigation of the placental circulations by Doppler echography. Placenta. 1995;16(4):323–331. 29. Bauer C, Ludwig I, Ludwig M. Different effects of 2.3 diphosphoglycerate and adenosine triphosphate on oxygen affinity of adult and foetal human haemoglobin. Life Sci Pt 1 Physiol. 1968;7(23P1):1339–1343. 30. Edelstone DI, Peticca BB, Goldblum LJ. Effects of maternal oxygen administration on fetal oxygenation during reductions in umbilical blood-flow in fetal lambs. Am J Obstet Gynecol. 1985;152(3):351–358. 31.  Zamudio S. High-altitude hypoxia and preeclampsia. Front Biosci. 2007;1(12):2967–2977. 32. Hatano H, Leichtweiss HP, Schroder H. Uptake of bicarbonate Co2 in the isolated guinea-pig placenta. Placenta. 1989;10(2): 213–221. 33. Economides DL. Acid-base balance in the fetus. In: Hillier SG, Kitchener HC, Neilson JP, eds. Scientific essentials of reproductive medicine. London: W.B. Saunders; 1996:376–385. 34. Robson SC. Maternal respiratory and cardiovascular changes during pregnancy. In: Hillier SG, Kitchener HC, Neilson JP, eds. Scientific Essentials of Reproductive Medicine. London: W.B. Saunders; 1996:443–454. 35. Soothill PW, Nicolaides KH, Rodeck CH, Campbell S. Effect of gestational age on fetal and intervillous blood gas and acidbase values in human pregnancy. Fetal Ther. 1986;1(4):168–175. 36. Shennan DB, Davis B, Boyd CA. Chloride transport in human placental microvillus membrane vesicles. I. Evidence for anion exchange. Pflugers Arch. 1986;406(1):60–64.

37.  Powell TL, Lundquist C, Doughty IM, et  al. Mechanisms of chloride transport across the syncytiotrophoblast basal membrane in the human placenta. Placenta. 1998;19(4):315–321. 38. Sibley CP, Glazier JD, Greenwood SL, et  al. Regulation of placental transfer: the Na(+)/H(+) exchanger—a review. Placenta. 2002;23(suppl A):S39–S46. 39. Speake PF, Mynett KJ, Glazier JD, et al. Activity and expression of Na+/H+ exchanger isoforms in the syncytiotrophoblast of the human placenta. Pflugers Arch. 2005;450(2):123–130. 40. Johansson M, Glazier JD, Sibley CP, et  al. Activity and protein expression of the Na+/ H+ exchanger is reduced in syncytiotrophoblast microvillous plasma membranes isolated from preterm intrauterine growth restriction pregnancies. J Clin Endocrinol Metab. 2002;87(12):5686–5694. 41. Burd LI, Jones Jr MD, Simmons MA, et  al. Placental production and foetal utilisation of lactate and pyruvate. Nature. 1975;254(5502):710–711. 42.  Nicolaides KH, Economides DL, Soothill PW. Blood gases, pH, and lactate in appropriate- and small-for-gestational-age fetuses. Am J Obstet Gynecol. 1989;161(4):996–1001. 43. Settle P, Mynett K, Speake P, et al. Polarized lactate transporter activity and expression in the syncytiotrophoblast of the term human placenta. Placenta. 2004;25(6):496–504. 44. Settle P, Sibley CP, Doughty IM, et  al. Placental lactate transporter activity and expression in intrauterine growth restriction. J Soc Gynecol Investig. 2006;13(5):357–363. 45. Stulc J, Stulcova B, Sibley CP. Evidence for active maternal-fetal transport of Na+ across the placenta of the anaesthetized rat. J Physiol. 1993;470:637–649. 46. Marino GI, Kotsias BA. Expression of the epithelial sodium channel sensitive to amiloride (ENaC) in normal and preeclamptic human placenta. Placenta. 2013;34(2):197–200. 47. Johansson M, Karlsson L, Wennergren M, et al. Activity and protein expression of Na+/ K+ ATPase are reduced in microvillous syncytiotrophoblast plasma membranes isolated from pregnancies complicated by intrauterine growth restriction. J Clin Endocrinol Metab. 2003;88(6):2831–2837. 48. Brown PD, Greenwood SL, Robinson J, Boyd RD. Chloride channels of high conductance in the microvillous membrane of term human placenta. Placenta. 1993;14(1):103–115. 49. Husain SM, Mughal MZ. Mineral transport across the placenta. Arch Dis Child. 1992;67(7 Spec No):874–878. 50. Juhlin C, Lundgren S, Johansson H, et  al. 500-Kilodalton calcium sensor regulating cytoplasmic Ca2+ in cytotrophoblast cells of human placenta. J Biol Chem. 1990;265(14):8275–8279. 51. Suzuki Y, Kovacs CS, Takanaga H, et al. Calcium channel TRPV6 is involved in murine maternal-fetal calcium transport. J Bone Miner Res. 2008;23(8):1249–1256. 52. Yang H, Kim TH, An BS, et  al. Differential expression of calcium transport channels in placenta primary cells and tissues derived from preeclamptic placenta. Mol Cell Endocrinol. 2013;367(1-2):21–30. 53. Glazier JD, Atkinson DE, Thornburg KL, et  al. Gestational changes in Ca2+ transport across rat placenta and mRNA for calbindin9K and Ca(2+)-ATPase. Am J Physiol. 1992;263(4 Pt 2):R930–R935.

77.e1

77.e2

References

54. Lee GS, Lee KY, Choi KC, et al. Phenotype of a calbindin-D9k gene knockout is compensated for by the induction of other calcium transporter genes in a mouse model. J Bone Miner Res. 2007;22(12):1968–1978. 55. Belkacemi L, Gariepy G, Mounier C, et  al. Calbindin-D9k (CaBP9k) localization and levels of expression in trophoblast cells from human term placenta. Cell Tissue Res. 2004;315(1):107–117. 56. Belkacemi L, Gariepy G, Mounier C, et  al. Expression of calbindin-D28k (CaBP28k) in trophoblasts from human term placenta. Biol Reprod 68(6):1943–1950. 57. Fisher GJ, Kelley LK, Smith CH. ATP-dependent calcium transport across basal plasma membranes of human placental trophoblast. Am J Physiol. 1987;252(1 Pt 1):C38–C46. 58. Borke JL, Caride A, Verma AK, et al. Calcium pump epitopes in placental trophoblast basal plasma membranes. Am J Physiol. 1989;257(2 Pt 1):c341–c346. 59. Strid H, Powell TL. ATP-dependent Ca2+ transport is up-regulated during third trimester in human syncytiotrophoblast basal membranes. Pediatr Res. 2000;48(1):58–63. 60. Strid H, Care A, Jansson T, Powell T. Parathyroid hormone-related peptide (38–94) amide stimulates ATP-dependent calcium transport in the basal plasma membrane of the human syncytiotrophoblast. J Endocrinol. 2002;175(2):517–524. 61. Johnson LW, Smith CH. Monosaccharide transport across microvillous membrane of human placenta. Am J Physiol. 1980;238(5):C160–C168. 62. Johnson LW, Smith CH. Glucose transport across the basal plasma membrane of human placental syncytiotrophoblast. Biochimica et Biophysica Acta 26. 1985;815(1):44–50. 63. Ericsson A, Hamark B, Powell TL, Jansson T. Glucose transporter isoform 4 is expressed in the syncytiotrophoblast of first trimester human placenta. Hum Reprod. 2005;20(2): 521–530. 64. Gude NM, Stevenson JL, Rogers S, et  al. GLUT12 expression in human placenta in first trimester and term. Placenta. 2003;24(5): 566–570. 65. Jansson T, Wennergren M, Illsley NP. Glucose transporter protein expression in human placenta throughout gestation and in intrauterine growth retardation. J Clin Endocrinol Metab. 1993;77(6):1554–1562. 66. Brown K, Heller DS, Zamudio S, Illsley NP. Glucose transporter 3 (GLUT3) protein expression in human placenta across gestation. Placenta. 2011;32(12):1041–1049. 67. Hahn T, Hartmann M, Blaschitz A, et  al. Localisation of the high affinity facilitative glucose transporter protein GLUT 1 in the placenta of human, marmoset monkey (Callithrix jacchus) and rat at different developmental stages. Cell Tissue Res. 1995;280(1):49–57. 68. Ashmead GG, Kalhan SC, Lazebnik N, Nuamah IF. Maternal-fetal substrate relationships in the third trimester in human pregnancy. Gynecol Obstet Investig. 1993;35(1):18–22. 69. Ericsson A, Hamark B, Jansson N, et al. Hormonal regulation of glucose and system A amino acid transport in first trimester placental villous fragments. Am J Physiol Regul Integr Comp Physiol. 2005;288(3):R656–R662. 70. Cetin I, Marconi AM, Corbetta C, et al. Fetal amino acids in normal pregnancies and in pregnancies complicated by intrauterine growth

retardation. Early Hum Dev. 1992;29(1-3): 183–186. 71. Sooranna SR, Burston D, Ramsay B, Steer PJ. Free amino acid concentrations in human first and third trimester placental villi. Placenta. 1994;15(7):747–751. 72. Lewis RM, Brooks S, Crocker IP, et al. Review: Modelling placental amino acid transfer— from transporters to placental function. Placenta. 2013;34(suppl):S46–S51. 73. Cleal JK, Glazier JD, Ntani G, et  al. Facilitated transporters mediate net efflux of amino acids to the fetus across the basal membrane of the placental syncytiotrophoblast. J Physiol. 2011;589(Pt 4):987–997. 74. Widdows KL, Panitchob N, Crocker IP, et  al. Integration of computational modeling with membrane transport studies reveals new insights into amino acid exchange transport mechanisms. FASEB J. 2015;29(6):2583– 2594. 75. Panitchob N, Widdows KL, Crocker IP, et  al. Computational modelling of amino acid exchange and facilitated transport in placental membrane vesicles. J Theoret Biol. 2015;21(365):352–364. 76. Pearse BM. Coated vesicles from human placenta carry ferritin, transferrin, and immunoglobulin G. Proc Natl Acad Sci U S A. 1982;79(2):451–455. 77. Ockleford CD, Clint JM. The uptake of IgG by human placental chorionic villi: a correlated autoradiographic and wide aperture counting study. Placenta. 1980;1(2):91–111. 78. King BF. Absorption of peroxidase-conjugated immunoglobulin G by human placenta: an in vitro study. Placenta. 1982;3(4):395–406. 79. Leach L, Eaton BM, Firth JA, Contractor SF. Immunogold localisation of endogenous immunoglobulin-G in ultrathin frozen sections of the human placenta. Cell Tissue Res. 1989;257(3):603–607. 80. Leach L, Eaton BM, Firth JA, Contractor SF. Immunocytochemical and labelled tracer approaches to uptake and intracellular routing of immunoglobulin-G (IgG) in the human placenta. Histochem J. 1991;23(10):444–449. 81. Lin CT. Immunoelectron microscopy localization of immunoglobulin G in human placenta. J Histochem Cytochem. 1980;28(4):339–346. 82. Szlauer R, Ellinger I, Haider S, et al. Functional expression of the human neonatal Fc-receptor, hFcRn, in isolated cultured human syncytiotrophoblasts. Placenta. 2009;30(6):507–515. 83. Benton S, McCowan L, Grynspan D, et  al. Placental growth factor as a marker for placental intrauterine growth restriction. Reprod Sci. 2016;23. 306A–A. 84.  Brosens I, Pijnenborg R,Vercruysse L, Romero R. The “great obstetrical syndromes” are associated with disorders of deep placentation. Am J Obstet Gynecol. 2011;204(3):193–201. 85. Sibley CP, Turner MA, Cetin I, et al. Placental phenotypes of intrauterine growth. Pediatr Res. 2005;58(5):827–832. 86. Burton GJ, Jauniaux E. Oxidative stress. Best Pract Res Clin Obstet Gynaecol. 2011;25(3): 287–299. 87. Junaid TO, Brownbill P, Chalmers N, et  al. Fetoplacental vascular alterations associated with fetal growth restriction. Placenta. 2014;35(10):808–815. 88. Sweeney M, Wareing M, Mills TA, et  al. Characterisation of tone oscillations in placental and myometrial arteries from normal pregnancies and those complicated by

pre-eclampsia and growth restriction. Placenta. 2008;29(4):356–365. 89. Wareing M, Greenwood SL, Fyfe GK, Baker PN. Reactivity of human placental chorionic plate vessels from pregnancies complicated by intrauterine growth restriction (IUGR). Biol Reprod. 2006;75(4):518–523. 90. Mills TA, Greenwood SL, Johnstone ED, et  al. Mechanical and receptor-mediated responses of placental chorionic plate arteries are altered in fetal growth restriction. Reprod Sci. 2012;19(S3):314A–A. 91. Jones S, Bischof H, Lang I, et al. Dysregulated flow-mediated vasodilatation in the human placenta in fetal growth restriction. J Physiol. 2015;593(14):3077–3092. 92. Jansson N, Pettersson J, Haafiz A, et al. Downregulation of placental transport of amino acids precedes the development of intrauterine growth restriction in rats fed a low protein diet. J Physiol. 2006;576(Pt 3):935–946. 93. Constancia M, Hemberger M, Hughes J, et al. Placental-specific IGF-II is a major modulator of placental and fetal growth. Nature. 2002;417(6892):945–948. 94. Roos S, Kanai Y, Prasad PD, et al. Regulation of placental amino acid transporter activity by mammalian target of rapamycin. Am J Physiol Cell Physiol. 2009;296(1):C142–C150. 95. Roos S, Jansson N, Palmberg I, et  al. Mammalian target of rapamycin in the human placenta regulates leucine transport and is down-regulated in restricted fetal growth. J Physiol. 2007;582(Pt 1):449–459. 96. Strid H, Bucht E, Jansson T, et al. ATP dependent Ca2+ transport across basal membrane of human syncytiotrophoblast in pregnancies complicated by intrauterine growth restriction or diabetes. Placenta. 2003;24(5):445–452. 97. Godfrey KM, Matthews N, Glazier J, et  al. Neutral amino acid uptake by the microvillous plasma membrane of the human placenta is inversely related to fetal size at birth in normal pregnancy. J Clin Endocrinol Metab. 1998;83(9):3320–3326. 98. Coan PM, Angiolini E, Sandovici I, et  al. Adaptations in placental nutrient transfer capacity to meet fetal growth demands depend on placental size in mice. J Physiol. 2008;586(18):4567–4576. 99. Ganzevoort W, Alfirevic Z, von Dadelszen P, et  al. STRIDER: Sildenafil Therapy In Dismal prognosis Early-onset intrauterine growth Restriction—a protocol for a systematic review with individual participant data and aggregate data meta-analysis and trial sequential analysis. Syst Rev. 2014;3:23. 100. Sheppard M, Spencer RN, Ashcroft R, et  al. Ethics and social acceptability of a proposed clinical trial using maternal gene therapy to treat severe early-onset fetal growth restriction. Ultrasound Obstet Gynecol. 2016;47(4): 484–491. 101. Denison FC, Semple SI, Stock SJ, et al. Novel use of proton magnetic resonance spectroscopy (1HMRS) to non-invasively assess placental metabolism. PloS One. 2012;7(8):e42926. 102. Huen I, Morris DM, Wright C, et  al. R1 and R2 * changes in the human placenta in response to maternal oxygen challenge. Magn Reson Med. 2013;70(5):1427–1433. 103. King A, Ndifon C, Lui S, et  al. Tumorhoming peptides as tools for targeted delivery of payloads to the placenta. Sci Adv. 2016;2(5):e1600349.

9

Placental Pathology and Implications for Fetal Medicine NEIL J. SEBIRE AND JOHN C. KINGDOM

KEY POINTS

Placental Pathological Assessment

• Pathological examination of the placenta may provide useful information regarding the underlying mechanisms of a range of pregnancy complications that may guide future management and improve understanding of disease pathophysiology. • Placentas should be submitted for examination by specialist pathologists in all complicated pregnancies according to national and local guidelines. • Interpretation of the clinical significance of many placental histologic changes remains difficult, and novel approaches are required for future development in addition to traditional histologic evaluation. • Paraffin-embedded tissue blocks are stable for many years at room temperature and thus may be transferred to tertiary pathology centres if required for reassessment, using additional histologic or DNA methods, and may also be used for medicolegal assessment of disease causation. • Placental evaluation should be encouraged in all cases of intrauterine death, regardless of whether formal postmortem fetal examination is requested.

The yield of significant abnormal findings from placental pathology examination is related to the underlying clinical circumstances, and there are therefore several recommendations published regarding indications for formal placental pathological evaluation.4,5 These largely include all preterm deliveries and otherwise complicated pregnancies, either associated with maternal or fetal diseases, acute compromise to fetal health or admission to the neonatal intensive care unit (NICU). This policy results in examination of around 10% of placentas from unselected low-risk pregnancies, a proportion that will obviously be much greater in tertiary referral fetal medicine centres. In addition, protocols exist describing the suggested examination approach, including macroscopic assessment, tissue sampling and subsequent histologic evaluation to form an overall diagnostic opinion.6,7 In the majority of cases examined as part of clinical practice, sampling of the umbilical cord, membranes and placental parenchyma (normal and abnormal), including any lesions, takes place after a period of fixation, with subsequent processing and histologic evaluation of haematoxylin and eosin–stained slides from each tissue sample. Large placental tissue diagnostic archives are therefore available but are limited by being composed primarily of formalinfixed paraffin-embedded (FFPE) blocks and slides rather than frozen material that is typically obtained from fresh tissue. With the introduction of novel methods of future investigation, it is likely that routine storage of fresh placenta samples taken immediately at delivery may be required for analysis (typically for protein, metabolite or RNA studies), with obvious resource implications. Placental histologic sections are evaluated and the findings interpreted in the context of details in the clinical history such as gestational age, pregnancy complications, birth details and the initial gross placental findings (Fig. 9.1). In this regard, placental pathology reporting is in many ways more challenging than other areas such as tumour pathology because there are few placental histologic changes that are pathognomonic of a specific disease; rather, interpretation is based upon constellations of features in relation to the clinical features that are statistically associated with particular clinical presentations. Placental features are therefore helpful for determining the broad mechanisms of underlying pathological processes leading to overt clinical manifestations so as to improve our understanding of the pathogenesis of a variety of complications of pregnancy (e.g., early- and late-onset preeclampsia, in which maternal factors can considerably affect the risk for disease).8

Introduction It has long been recognised that a wide range of disorders of pregnancy are related to changes affecting the placenta, and our understanding of the underlying mechanisms of many obstetric diseases, such as fetal growth restriction (FGR) and preeclampsia, is largely derived from pathological studies of the delivered placenta. With this in mind, the potential benefits of a specialised placental pathology service include improved evaluation of pathophysiological processes in specific cases, which may affect subsequent management and recurrence risk, and as a source of material for subsequent research. This chapter provides an overview of the role of placental pathological assessment in modern fetal medicine, with examples in relation to antenatal diagnosis, and suggests how this area may develop in the near future. Extensive literature is available regarding details of specific placental pathologies in specialist texts.1-3  78

CHAPTER 9  Placental Pathology and Implications for Fetal Medicine

A

B

C

D

79

• Fig. 9.1  Photomicrographs of placental histology demonstrating extensive villitis of unknown aetiology. (A and B; haematoxylin and eosin (H&E) original magnifications ×20 and ×100, respectively) and congenital toxoplasmosis. C and D; H&E original magnifications ×40 and ×200, respectively.)

However, because histologic evaluation involves subjective assessment, a relationship exists between pathologist expertise or experience and placental reporting utility; 40% of placental cases reported by nonspecialist pathologists were erroneous compared with subspecialty assessment, including omissions and false-positive findings.9 It is therefore recommended that multidisciplinary placental teams are established in specialist centres, with close interaction of the obstetric and perinatal pathology reporting staff, and regular discussion of findings in relation to both antenatal ultrasound findings and clinical outcomes. It is hoped that recent efforts regarding consensus statements for placental reporting will reduce interpathologist variability and allow improved studies of interpretation.10

Prenatal Assessment of Specific Placental Pathologies The ability to identify pregnancies with, or at risk for, a range of placental pathologies has advanced considerably over the past 30 years, with the application of both real-time and colour Doppler ultrasound to evaluate both the placenta and its maternal and fetal circulations. The definitive placenta is formed by the end of the first trimester, such that many aspects of gross anatomy, such as shape, size, cord insertion and implantation site, can be determined from the first trimester, using either simple two-dimensional methods11 or three-dimensional volume assessment.12 During the second trimester, the placenta is larger and thus easier to assess using abdominal ultrasound imaging including Doppler assessment of the uteroplacental circulation13 (Figs. 9.2 to 9.4). Because maternal vascular malperfusion (MVM) is the most common type of placental pathology associated with early-onset preeclampsia and FGR, screening programs to identify women at

CI

• Fig. 9.2  Normal anterior placenta at 19 weeks’ gestation demonstrating a central placental cord insertion and normal sonographic appearances.

most risk have focused on incorporating uterine artery Doppler studies into screening algorithms that include clinical risk scores and biomarkers, such as serum placenta growth factor (PlGF).14 This combined approach has the potential to provide improved precision in screening for both preventable stillbirth caused by placental disease15 and FGR,16 although to date, no interventions have delivered improved perinatal outcome. Many large-scale research screening programs such as those referenced lack placental pathology findings, which is understandable because of the associated cost per case. However, the

80

SE C T I O N 2     The Placenta

C

B

A

A

B

C

D

• Fig. 9.3  Normal placental appearances on routine 20-week sonographic assessment, including various methods of placental measurements.

A

C

B

D

• Fig. 9.4  Normal (A) and abnormal (B) uterine artery Doppler waveforms. Abnormal flow is associated with fetal growth restriction with placental hyperinflation (C) caused by maternal vascular malperfusion and placental infarction (D).

inherent variability in underlying placental pathophysiology associated with stillbirth and FGR17 has the potential to confound the accuracy of screening. Uterine artery Doppler may predict the placental features associated with MVM; however, in a study of severe early-onset FGR pregnancies with abnormal

umbilical artery Doppler, 10% had normal uterine artery Doppler studies; these had a greater risk for placental diseases unrelated to MVM but with significant recurrence rates (e.g., chronic histiocytic intervillositis (CHI) and massive perivillous fibrin deposition).18

CHAPTER 9  Placental Pathology and Implications for Fetal Medicine

81

• BOX 9.1   Classification of Placental Pathologies Placental vascular processes • Maternal stromal-vascular lesions • Malperfusion (including distal villous hypoplasia, accelerated villous maturation and infarct) • Loss of integrity (including abruptio placenta and marginal abruption) • Fetal stromal-vascular lesions • Developmental (including delayed villous maturation and dysmorphic villi) • Malperfusion (including global and segmental lesions) • Loss of integrity (including fetal haemorrhage and fetomaternal haemorrhage) 

Placental inflammatory-immune processes

•

Fig. 9.5 Abnormally appearing ‘hyperinflated’ placenta in association with low first trimester serum placental associated protein A (PAPP-A) and early-onset fetal growth restriction (see Video 9.1).

• Infectious inflammatory lesions • Acute (including maternal and fetal inflammatory responses) • Chronic (including villitis and intervillositis) • Immune or idiopathic inflammatory lesions • Including villitis of unknown aetiology (chronic villitis, chronic chorioamnionitis, lymphoplasmacytic deciduitis, eosinophil T-cell fetal vasculitis) and chronic histiocytic intervillositis 

Other placental processes • Massive perivillous fibrin(oid) deposition (maternal floor infarction) • Morbidly adherent placentas (accreta) • Meconium-associated changes Adapted from Redline RW. Classification of placental lesions. Am J Obstet Gynecol 213 (4 suppl):S21-S28, 2015.

8 cm

collaborative approaches to unify the use of terminology. The currently recommended classification system is being used in this chapter (Box 9.1).10 

Interpretation of Lesions

•

Fig. 9.6 Massive placentomegaly with nonimmune fetal hydrops at 32 weeks’ gestation caused by fetomaternal haemorrhage. The mother showed features of ‘mirror syndrome’.

It has further been suggested that many placentas from earlyonset FGR or preeclampsia exhibit abnormalities of size, shape, cord insertion or parenchyma, which may be detectable antenatally. For example, ‘chorion regression’ may be associated with a ‘jelly-like’ hyperinflated placenta,19 and several specific placental pathologies have sonographically identifiable features. However, the role of ‘placental sonography’ beyond individual case assessment remains uncertain. Illustrative examples of the role of placental sonography in identifying pathologies of the placenta, umbilical cord and membranes are provided (Figs. 9.5 and 9.6 and Video 9.1). 

Classification of Placental Lesions One of the historical difficulties of interpreting literature relating to placental pathology has been inconsistent use of terminologies by clinicians, scientists and pathologists and use of multiple labels for the same entity. To address this issue, there have been recent

Some placental lesions demonstrate characteristic and unique histologic features, allowing definitive diagnosis regardless of clinical circumstances or other factors. However, such entities represent only a minority of histologic changes identified in the placenta, with the majority of lesions also being encountered in clinically uncomplicated normal pregnancies, although being more or less frequent in association with specific pregnancy complications. This overlap results in consistent data describing risks or odds ratios for the strength of association among specific histologic features and specific obstetric disorders on a population basis, but accurate interpretation of the clinical significance of specific findings in an individual case is fraught with difficulties. The details provided summarise the available data but should be interpreted with these above in mind. 

Categories of Placental Pathologies In this section, entities which are relatively common or important are described, focusing particularly on their relationship to antenatal detection and management of common clinical conditions. Extensive literature is available providing details of the full spectrum of pathologies.1-3 The categories broadly map to Box 9.1 but for ease of discussion are described in terms of their mechanisms and clinical significance.

82

SE C T I O N 2     The Placenta

Placental portion anterior

Succenturiate lobe posterior

•

Fig. 9.7  Antenatal sonogram of a case of vasa previa with a posterior succenturiate lobe (LT), with colour Doppler demonstrating fetal vessels running outside of the placenta connecting the placental masses (inset) (see Video 9.2).

Abnormalities of Placental Development The details of normal placental development are described in Chapter 7. There are a range of macroscopically identifiable disorders which are believed to be a consequence of gross abnormalities of the process of initial implantation or subsequent growth of the placental disk, resulting in abnormalities in placental shape, architecture or umbilical cord insertion site. These lesions are not usually associated with specific histologic abnormalities (although it has been suggested that abnormal placental regression may also be related to villous changes) but may be associated with increased risk for certain pregnancy complications. Eccentric or velamentous insertion and vasa praevia. The umbilical cord normally inserts into the central portion of the placental disk chorionic plate. Minor degrees of peripheral insertion are usually of no clinical significance, but because of rarefaction of the process of dichotomous branching of the surface chorionic plate arteries, the opposite side of the placenta from a marginal cord may be hypovascularized, thus reducing placental efficiency. At the extremes, umbilical cord–derived vessels may leave the margins of the placenta, found in around 1% of pregnancies, termed velamentous insertion; a variant of this is when vessels from a marginal cord insertion traverse within the membranes to accessory, or succenturiate, lobes, so as to connect them in a functional sense, to the fetus. The term vasa previa describes this arrangement when the vessels run closer to, or over, the internal os of the cervix. The fetus is then at risk for hypovolemic shock during vaginal delivery because these vessels may be damaged as labour advances, especially at the time of membrane rupture. Antenatal screening using ultrasound (for variants of placental and cord anatomy) and diagnosis (using transvaginal colour Doppler ultrasound) are lifesaving for the fetus because elective caesarean section increases fetal survival to more than 95% (Figs. 9.7 and 9.8 and Video 9.2).  Bilobata, succenturiata and other shape abnormalities.

Although the normal human placenta is discoid, there are numerous variations in shape, most of which are not associated with significant or consistent clinical complications. These include

•

Fig. 9.8  Delivered placenta from a case of vasa previa with numerous large chorionic vessels running within the fetal membranes.

placenta membranacea, in which placental villous tissue persists extensively around the gestational sac; placenta fenestrate, in which there is focal deficiency of parenchyma; placenta bilobata, in which two distinct disks are present usually with central cord insertion between the two; and placenta succenturiata, in which one of more accessory lobes is present joined to the main disk by intramembranous vessels. These deviations from a spherical placenta and central cord insertion have been suggested to reduce efficiency in some studies20 but do not threaten fetal survival unless additional pathologies are present. However, because of the abnormal anatomy, either placental parenchymal tissue or chorionic vessels may be present over the cervical os, with associated risks of trauma and haemorrhage.  Circummarginate or circumvallate placenta. As part of normal placental development, the edge of the placental parenchymal disk (basal plate) corresponds to the edge of the chorionic plate and hence the smooth junction of the amniotic cavity with the placenta. If this process is defective the edge of the chorionic plate may no longer be sited over the placental parenchymal edge, resulting in either a smooth or ridged, abnormally sited junction (circummarginate and circumvallate placentation, respectively). To some degree, this affects around 1% to 5% of placentas with little functional significance but has been associated with increased rates of antepartum haemorrhage and preterm delivery. The normal marginal sinus, where intervillous blood reenters the uteroplacental veins, can be imaged by ultrasound and may on occasion be prominent. This is of no consequence unless sited close to the internal os but can be mistaken for marginal abruption. 

Abnormalities of Placental Perfusion To function normally, maternal blood must flow, at the appropriate rate and pressure, into and through the intervillous space (uteroplacental circulation) surrounding the chorionic villi; this blood supply is arranged as functional units, each centrally perfused by a spiral artery branch. These functional units may be termed placentomes, and up to 50 exist in a normalterm placenta. Effective transplacental diffusion also requires adequate perfusion from the fetus (fetoplacental circulation). It will be apparent therefore that these processes may be

CHAPTER 9  Placental Pathology and Implications for Fetal Medicine

83

defective at any level, resulting in chronic fetal hypoxia and impaired fetal growth, and it is therefore logical to discuss these according to the anatomical area predominantly affected.

Abnormalities of Uteroplacental Flow Fetal growth restriction and preeclampsia. Pathological studies

of products of conception, delivered placentas and placental bed biopsies have demonstrated that abnormalities of normal establishment of the uteroplacental circulation are associated with, and likely the underlying pathophysiological process responsible for, a range of pregnancy complications ranging from early pregnancy failure (miscarriage), preeclampsia and FGR. It is now generally accepted that in the first trimester decidual vessels become occluded by extravillous endovascular trophoblast to protect the early conceptus from pressure and oxygen-related damage, with failure of such ‘plugging’ one of the causes of miscarriage, for example, in association with antiphospholipid antibodies. After initial endovascular invasion, during the second trimester, the trophoblast masses recanalize, and both endovascular and interstitial extravillous trophoblast of the implantation site combine to convert the distal muscular spiral artery branches into poorly muscularised, low-resistance, high-flow uteroplacental vessels supplying the intervillous space. Failure of this phase of development is associated with abnormally reactive uteroplacental vessels with increased flow resistance and reduced and abnormally pulsatile intervillous flow.21 These changes have secondary effects on chorionic villus structure and function, the combination of which results in FGR, preeclampsia or both. Although in most cases, the cause of the defective implantation remains unknown, in a minority, there may be underlying conditions, such as maternal connective tissue diseases, which are associated with identical features. Pathological evaluation of the delivered placenta in such cases may demonstrate a range of histologic features, which are now recognised as ‘typical’ changes of FGR or preeclampsia described collectively as MVM. These changes include reduced placental size and surface area, presence of decidual vasculopathy (fibrinoid necrosis or macrophages and inflammatory cells within the vessel wall (atherosis)), villous infarction, fetoplacental vasoconstriction, reduced villous branching and hypovascularity, accelerated maturation and a range of functional alterations. Although many such changes are subjective and may be identified to some degree in clinically uncomplicated pregnancies at term, the constellation of all features, especially in iatrogenic preterm deliveries, is highly suggestive of underlying MVM. The alterations in maternoplacental flow or placental shape and size are detectable antenatally based upon uterine artery Doppler and placental morphology assessment, and the secondary changes in fetoplacental flow, especially maldevelopment of the gas exchanging peripheral villi, result in changes detectable using umbilical artery Doppler sonography (Fig. 9.9).  Abruption and retroplacental haemorrhage. The placenta is normally firmly adherent to the uterus at the basal plate until the third stage of labour. If abnormally premature separation occurs, either centrally or at the margin, the consequence is retroplacental haemorrhage, which most often tracks along the uteroplacental junction, resulting in vaginal bleeding, but is occasionally ‘concealed’, being retained retroplacentally. In addition, because separation has occurred, no functional uteroplacental circulation remains in these areas, with associated complete loss of functional

•

Fig. 9.9 Multifocal basal placental infarction presenting at 37 weeks’ gestation with the features of late-onset fetal growth restriction.

capacity of the supplied villous areas, which may result in ischaemic necrosis (infarction) of the overlying placenta. It should be noted that although in some cases, unequivocal abnormal retroplacental haemorrhage with secondary overlying changes may be identified in the delivered placenta, in other cases, especially with marginal separation and vaginal bleeding, the delivered placenta may not demonstrate characteristic changes of abruption even in the presence of a typical clinical history. Ultrasound may occasionally diagnose chronic abruption,22 although often abruption is such an acute event in labour and delivery that clinical management and delivery override the utility of ultrasound imaging (Fig. 9.10). 

Abnormalities of Fetoplacental Flow It has been demonstrated that after primary abnormalities of uteroplacental perfusion, secondary changes in fetoplacental perfusion develop, such as with typical FGR caused by MVM. However, in addition, morphological changes may also occur indicating reduced fetoplacental flow in the absence of any maternal abnormalities. Such changes include either the direct documentation of chorionic vascular thrombosis or the downstream villous effects of proximal fetovascular occlusion, namely clusters of chorionic villi with normal intervillous space showing intravascular karyorrhexis, syncytial knot formation and stromal sclerosis, according to chronicity. Such changes are within the spectrum of fetal vascular occlusion (FVO) or fetal thrombotic vasculopathy (FTV). When focal, they are usually of no clinical significance, even though they may be reported more commonly in certain scenarios, such as maternal diabetes mellitus, but occasionally may be associated with underlying fetal visceral thrombosis or placental functional consequences if extensive. For example, extensive entrapment of a long, hypercoiled umbilical cord in association with FVO lesions may suggest causality in stillbirth. 

Primary Abnormalities of Villous Development In some circumstances of FGR or fetal distress, there are diffuse changes affecting fetal chorionic villi which are not associated with typical features of MVM, and it has been suggested that such cases are caused by primary abnormalities of fetal development.

84

SE C T I O N 2     The Placenta

A

B

PL Maternal surface

H

C

Fetal surface

D

• Fig. 9.10  Sonographic identification of a consolidated asymptomatic central concealed abruption in a clinic setting at 30 weeks’ gestation (A and B), which progressed after 2 inpatient days, precipitating caesarean delivery after steroid administration for fetal lung maturity and a favourable outcome. C, Histopathology. Contrast with bedside ultrasound findings in acute abruption in a labour and delivery setting (D).

Most cases of distal villous hypoplasia and villous hypermaturity are now believed to be changes secondary to alterations in uteroplacental flow, although it has been suggested that some could represent primary maldevelopment. In contrast, a generalised disorder of villous development, distal villous immaturity (DVI), is now well recognised, being identified as a generalised increase in villous stroma with immature appearing villi containing centrally located small capillaries with paucity of normal vasculosyncytial membranes.23 The consequence of this histologic finding is that the diffusion distance between maternal and fetal erythrocytes is greatly increased, impairing conductance of carbon dioxide and oxygen. DVI may therefore contribute to some instances of antenatal stillbirth, especially with larger fetuses in the context of diabetes. 

Abnormalities Primarily Affecting the Intervillous Space In addition to the maternal and fetal circulations, abnormalities affecting the normal structure or function of the intervillous space are rare but may occur and have distinctive histologic features. CHI is a condition characterised by the presence of large numbers of maternal histiocytes within the intervillous space, often associated with fibrin deposition, in the absence of known infective cause. The aetiology is unknown but is presumed autoimmune, particularly in view of the findings that presentation may be throughout pregnancy, from first trimester loss through to term, with a greater than 50% recurrence risk. Massive perivillous fibrin deposition (MPVFD) is characterised by the majority of the intervillous space being involved by perivillous fibrin, into which trophoblast proliferates, which

separates chorionic villi and prevents normal intervillous blood flow. Again, the exact mechanism remains uncertain, but there is a significant recurrence risk (20%). Both of these conditions are only reliably diagnosed on histologic examination and have no typical ultrasonographic appearances.24 

Inflammatory Lesions Inflammatory lesions may be infectious or noninfectious, presumed immune mediated. Ascending genital tract infection. Inflammation affecting the fetal membranes overlying the cervical os, with subsequent spread to involve the membranes more diffusely, the amniotic cavity and finally the fetal circulation, represents an infective process, most often with normal vaginal or cervical commensal organisms, the condition representing a loss of normal balance between defence mechanisms and colonisation. An initial localised maternal inflammatory response of the fetal membranes occurs (chorioamnionitis), which may result in stimulation of the onset of labour, or the infectious process may progress if delivery does not ensue, until the fetus mounts a systemic inflammatory response (funisitis). Ascending genital tract infection is therefore a major cause of midtrimester pregnancy loss and severe preterm delivery and even at term may act synergistically with other insults, such as hypoxiaischaemia, to cause neurological damage. Although some cases may be associated with a systemic maternal response, with fever, there is poor correlation between clinical and histologic features.  Villitis or intervillositis caused by haematogenous infection.

Because maternal blood supplies the intervillous space, maternal systemic diseases may involve the placenta, leading to either collections of inflammatory cells or fibrin within the intervillous

CHAPTER 9  Placental Pathology and Implications for Fetal Medicine

85

• • Fig. 9.11  Sonographic appearance of a placental chorioangioma including colour Doppler identification of a large functional shunt within a 6-cm placental chorioangioma. The fetus demonstrated signs of high-output cardiac failure (Video 9.3).

space (e.g., malaria), or inflammation of the villi (villitis; e.g., cytomegalovirus). When there is villitis from an infective cause, which may be viral, bacterial or protozoal, the placenta usually demonstrates patchy but diffuse involvement, with florid focal villitis, which may even be associated with villous necrosis or granuloma formation. The pattern of tissue involvement may suggest a particular organism as the aetiology, but confirmation should always be based on additional ancillary investigations. In some cases of viral infection, characteristic viral cellular inclusions may be present, making the specific diagnosis more definitive.  Villitis of unknown aetiology. Villitis indicates infiltration of chorionic villi by inflammatory cells. As noted earlier, in some cases, the pattern of inflammation may be characteristic and the aetiology determined to be an infectious agent. However, in the majority of cases in which villitis is identified, there is patchy infiltration of groups of chorionic villi by mononuclear inflammatory cells, mainly lymphocytes, with some macrophages, but no other specific findings and no infectious agent is identified. Such cases are classified as villitis of unknown aetiology (VUE). VUE may be present in clinically normal deliveries at term but is reported more frequently in association with complications such as FGR and preeclampsia. It is now established that the majority of infiltrating cells in VUE are of maternal origin, and it has therefore been suggested that this may represent a maternofetal immune-mediated process, similar in concept to graft rejection25 (see Fig. 9.1). 

Tumours and Tumourlike Lesions There are few mass lesions affecting the placenta, but there are several entities which may be detected on antenatal sonographic examination, which have clear histologic correlates and effects on clinical management. Chorioangioma. By far the commonest ‘tumour’ of the placenta is chorioangioma, which represents a benign proliferation of villous blood vessels surrounded by expanded villous stromal tissue and trophoblast. These lesions are often highly vascular on imaging and when large can develop functional shunts of

Fig. 9.12 Sonographic appearances of a placenta at 21 weeks’ gestation with severe preeclampsia, growth restriction, abnormal umbilical artery Doppler and bilateral cystic ovarian enlargement. Note the increased thickness and multiple small cysts. Amniocentesis revealed triploidy (partial hydatidiform mole), and the pregnancy was terminated by induction of labour.

the fetoplacental circulation. Such lesions are most often situated beneath the chorionic plate, may be single or multiple, and can vary in size from millimetres to more than 10 cm in diameter. Small lesions appear to have no direct clinical significance, but larger or more extensive lesions may be associated with fetal cardiac failure, polyhydramnios or nonimmune hydrops.26 Chorioangiomas may infarct in utero, resulting in spontaneous resolution of high-output cardiac failure. Fetal interventional techniques can also be used to occlude the aberrant arteriovenous malformation and restore normal fetal physiology (Fig. 9.11 and Video 9.3).  Hydatidiform mole and intraplacental choriocarcinoma.

Hydatidiform moles (HMs) represent genetically abnormal conceptions with relative overexpression of the paternal genome, leading to villous hydropic change and abnormal trophoblast hyperplasia. Depending on their pathological and genetic features, HM may be complete (CHM; diploid) or partial (PHM; triploid), with most cases presenting with vaginal bleeding or early pregnancy failure. However, in some cases, such as mosaic HM and HM with a normal co-twin, the pregnancy may continue into the third trimester with coexistence of sonographically normal placental tissue and other areas demonstrating marked hydropic change. The main clinical significance of diagnosing HM is the increased subsequent risk for persistent gestational trophoblastic disease requiring chemotherapy (15% for CHM and 0.5% for PHM). It should be noted, however, that in the first trimester, the majority of HMs sonographically appear as early pregnancy failures and may not demonstrate significant sonographically detectable hydropic change27 (Fig. 9.12). Very rarely, a focus of intraplacental choriocarcinoma may develop within an otherwise unremarkable third trimester placenta, which may lead to metastatic disease of the mother, fetus or both. Such focal lesions are not detectable sonographically and even on macroscopic examination of the delivered placenta are indistinguishable from intervillous thrombi, infarct or other lesion until the correct diagnosis is made after histologic evaluation of the lesion. 

86

SE C T I O N 2     The Placenta

Placental mesenchymal dysplasia. Placental mesenchymal dysplasia (PMD) is described here because it is increasingly recognised as a specific entity with distinctive pathological features and because it may sonographically be confused with a placenta affected by hydatidiform molar change. Typically, such cases demonstrate a sonographically homogeneously enlarged placenta with diffusely scattered hydropic cystic change in association with an apparently structurally normal fetus. The placenta may appear larger than the fetus. Histologically, such placentas demonstrate characteristic hydropic change of stem villi, without trophoblast hyperplasia, often in association with marked dilation of chorionic plate vessels. It appears that PMD may represents androgenetic or biparental mosaicism and in a minority of cases may be associated with underlying disorders such as fetal Beckwith-Wiedemann syndrome.28 

Future Approaches To date, placental pathology data has been almost exclusively based on findings of subjective morphological studies describing the frequency of various histologic lesions in specific groups. Although this approach has led to important observations related to both clinical care and underlying mechanisms of disease, further developments are likely to require additional approaches which provide objective data to minimise the effects of nonblinding, unconscious bias and may identify mechanistic rather than structural alterations. Recent technological developments in -omic approaches, such as genomics, proteomics, metabolomics and microbiomics, will have profound effects on the evaluation of tissue samples in disease, and such techniques are now being applied to the placenta, and their findings are beginning to challenge our existing paradigms of disease mechanisms and pathophysiology.29

However, with the introduction of such new capabilities, the importance of a range of factors related to sample acquisition is increasing because such factors may affect the interpretation of findings. Examples are the precise geographical localisation of sampling, in relation to the periphery, basal and chorionic plates, and cord insertion; the timing of sampling in relation to delivery; the mode of delivery; the method of protein extraction; and the temperature of storage and length of storage time. All these, and likely many yet unrecognised, factors require modifications of existing placental examination protocols, but such a multidimensional approach will lead to exciting new discoveries in relation to a wide range of placental related obstetric complications. 

Conclusion Many pregnancy complications are caused by a variety of placental pathologies, some of which are detectable antenatally through ultrasound examination. Histopathological examination of the delivered placenta may allow both confirmation of specific diagnoses and identification and mechanisms of underlying disease patterns and pathophysiology. Some pathological conditions appear to be poorly detectable antenatally and, at present, are only recognised on microscopic placental examination. It is highly likely that in addition to these abnormalities described, a range of placental functional disorders may also lead to pregnancy complications, and in this context, the development of novel additional investigations may allow more accurate detection of these disorders and potentially their early antenatal detection and prevention. Access the complete reference list online at ExpertConsult.com. Self-assessment questions available at ExpertConsult.com.

References 1. Fox H, Sebire NJ. Pathology of the Placenta. St. Louis: Elsevier Saunders; 2007. 2. Benirschke K, Burton G. Pathology of the Human Placenta. Berlin: Springer; 2012. 3. Kraus FT, Redline RW, Gersell DJ, Nelson DM, Dicke JM. Placental Pathology (Atlas of Nontumor Pathology), 1st ed. Washington: American Registry of Pathology; 2005. 4. Hargitai B, Marton T, Cox PM. Best practice no 178. Examination of the human placenta. J Clin Pathol. 2004;57:785–792. 5. Langston C, Kaplan C, Macpherson T, et al. Practice guideline for examination of the placenta: developed by the Placental Pathology Practice Guideline Development Task Force of the College of American Pathologists. Arch Pathol Lab Med. 1997;121:449– 476. 6. Cox P, Evans C. Tissue Pathway for Histopathological Examination of the Placenta. London; 2011. 7. Burton GJ, Sebire NJ, Myatt L, et al. Optimising sample collection for placental research. Placenta. 2014;35:9–22. 8. Raymond D, Peterson E. A critical review of early-onset and late-onset preeclampsia. Obstet Gynecol Surv. 2011;66:497–506. 9. Khong TY, Gordijn SJ. Quality of placental pathology reports. Pediatr Dev Pathol. 2003;6:54–58. 10. Redline RW. Classification of placental lesions. Am J Obstet Gynecol. 2015;213(suppl 4):S21–S28. 11. Milligan N, Rowden M, Wright E, et  al. Two-dimensional sonographic assessment of maximum placental length and thickness in

the second trimester: a reproducibility study. J Matern Fetal Neonatal Med. 2015;28:1653– 1659. 12. Schwartz N, Coletta J, Pessel C, et  al. Novel 3-dimensional placental measurements in early pregnancy as predictors of adverse pregnancy outcomes. J Ultrasound Med. 2010;29:1203– 1212. 13. Bahlmann F, Fittschen M, Reinhard I, et al. Reference values for blood flow velocity in the uterine artery in normal pregnancies from 18 weeks to 42 weeks of gestation calculated by automatic Doppler waveform analysis. Ultraschall Med. 2012;33:258–264. 14. Ghosh SK, Raheja S, Tuli A, et  al. Serum PLGF as a potential biomarker for predicting the onset of preeclampsia. Arch Gynecol Obstet. 2012;285:417–422. 15. Bhide A, Thilaganathan B, Singh T, et  al. Role of second-trimester uterine artery Doppler in assessing stillbirth risk. Obstet Gynecol. 2012;119:256–261. 16. Crovetto F, Triunfo S, Crispi F, et al. First trimester screening with specific algorithms for early and late onset fetal growth restriction. Ultrasound Obstet Gynecol. 2016;48(3):340–348. 17. Walker MG, Fitzgerald B, Keating S, et  al. Sex-specific basis of severe placental dysfunction leading to extreme preterm delivery. Placenta. 2012;33:568–571. 18. Deter RL, Levytska K, Melamed N, et  al. Classifying neonatal growth outcomes: use of birth weight, placental evaluation and individualized growth assessment. J Matern Fetal Neonatal Med. 2016;29:3939–3949. 19. Porat S, Fitzgerald B, Wright E, et  al. Placental hyperinflation and the risk of adverse perinatal outcome. Ultrasound Obstet Gynecol. 2013;42:315–321.

20. Salafia CM, Yampolsky M, Misra DP, et  al. Placental surface shape, function, and effects of maternal and fetal vascular pathology. Placenta. 2010;31:958–962. 21. Burton GJ, Woods AW, Jauniaux E, Kingdom JCP. Rheological and physiological consequences of conversion of the maternal spiral arteries for uteroplacental blood flow during human pregnancy. Placenta. 2009;30:473–482. 22. Walker M, Whittle W, Keating S, Kingdom J. Sonographic diagnosis of chronic abruption. J Obstet Gynaecol Can. 2010;32:1056–1058. 23. Fitzgerald B, Kingdom J, Keating S. Distal villous hypoplasia. Diagnostic Histopathol. 2012;18:195–200. 24. Sebire NJ, Sepulveda W. Correlation of placental pathology with prenatal ultrasound findings. J Clin Pathol. 2008;61:1276–1284. 25. Tamblyn JA, Lissauer DM, Powell R, et al. The immunological basis of villitis of unknown etiology [review]. Placenta. 2013;34:846–855. 26. Zanardini C, Papageorghiou A, Bhide A, Thilaganathan B. Giant placental chorioangioma: natural history and pregnancy outcome. Ultrasound Obstet Gynecol. 2010;35: 332–336. 27. Fowler DJ, Lindsay I, Seckl MJ, Sebire NJ. Routine pre-evacuation ultrasound diagnosis of hydatidiform mole: experience of more than 1000 cases from a regional referral center. Ultrasound Obstet Gynecol. 2006;27:56–60. 28. Kaiser-Rogers KA, McFadden DE, Livasy CA, et al. Androgenetic/biparental mosaicism causes placental mesenchymal dysplasia. J Med Genet. 2006;43:187–192. 29. Law KP, Han T-L, Tong C, Baker PN. Mass spectrometry-based proteomics for pre-eclampsia and preterm birth. Int J Mol Sci. 2015;16: 10952–10985.

86.e1

10

Development of the Heart and Cardiovascular System in Relation to Cardiac Abnormalities MICHAEL ASHWORTH

KEY POINTS • T he heart is largely derived from mesoderm. • A single heart tube forms with venous and arterial connections. It elongates by addition of cells at either end from the surrounding mesenchyme. • The tube folds to the right and lies in the pericardial cavity. • The atria and ventricles form by ballooning from the tube; septation of the atria and ventricles is largely achieved by this method. • The endocardial cushions at the atrioventricular (AV) junction and outflow tract complete septation of the AV junction and outflow tracts and give rise to the AV and arterial valves. • The epicardium gives rise to the mesenchyme of the heart, the veins and most of wall of the coronary arteries. • The epicardial coronary arteries grow into the aortic root. • The conduction tissue is derived from primitive myocardium of the original heart tube. • A complex series of venous precursors contribute to the abdominal veins. • Aortic arches 3, 4 and 6 give rises to the vessels of the head, neck and thorax.

Introduction The heart is an organ largely derived from mesoderm, with a contribution from ectodermally derived neural crest cells. Its development is critically dependent upon a cascade of events tightly regulated in time and space.1 The first recognisable sign of heart development in the embryo is the formation of the heart tube within the lateral splanchnic mesoderm late in the third week. Invisible to the classical morphologist are a series of preceding genetic steps on which the morphological changes are critically dependent. These include the specification of precursor cells in the bilaminar embryonic disc, their migration to the heart fields and the specification of cell types within the developing heart. By the end of the seventh week, the heart, although tiny, is essentially fully formed. It does not develop in isolation but is intimately related to surrounding 88

structures such as the pharynx, and heart development is both influenced by and influences these surrounding structures. Unsurprisingly, defects in the heart are associated with defects in these associated structures. From very early in its development, the heart is a beating structure and contains a flowing liquid, so mechanical forces are also important in shaping its development.2 Heart development is contributed to by multiple genes, many of which have multiple functions in development, with considerable overlap.3 If a defect develops, it is likely to be modified by growth and subsequent alterations in haemodynamics. Because heart development is a sequential and complex process, no single gene defect leads to a single specific heart defect. There has been a proliferation of information on heart development in recent decades, with genetic experiments in mouse, chick and zebrafish embryos having determined details of cell lineage. Although much remains unknown, the genetic mechanisms regulating heart development are beginning to be understood. 

Brief Overview of Heart Development The bulk of the work on early cardiac development comes from studies in fish, chick and mouse embryos.4-6 Within the cardiac crescent (the group of mesodermal cells lying in the splanchnic mesoderm anterior to the buccopharyngeal membrane and that show patterns of gene expression committing them to cardiogenesis), paired endothelial-lined tubes develop with their long axis in the long axis of the embryonic disc (Fig. 10.1). With folding of the embryo, these paired tubes fuse medially over part of their length to form the primitive single heart tube. The surrounding mesoderm of the cardiac crescent differentiates to provide an investing sleeve of myocardium, both layers separated by extracellular matrix termed cardiac jelly. The inflow is caudal, a continuation of the primitive veins, and the outflow cranial and connected to the paired dorsal aortae. The pericardial cavity develops initially on the dorsal surface of the developing heart as part of the intraembryonic coelom. With folding of the embryo and formation of the single

CHAPTER 10  Development of the Heart and Cardiovascular System in Relation to Cardiac Abnormalities

89

•

Fig. 10.1  Schematic representation of a cross-section of the trilaminar embryo before embryonic folding. The coelomic cavity is developing within the mesoderm, localising the paired heart tubes to the splanchnic mesoderm on the ventral aspect. As the embryo folds, the lateral edges are brought into apposition, the endoderm fuses to form the gut, and the paired heart tubes fuse in the midline ventral to the gut. The coelom forms the pericardial cavity. The ectoderm enfolds the other structures, and the amniotic cavity extends completely to surround the embryo.

heart tube, the pericardial cavity comes to lie ventral to the heart tube, which is connected dorsally to the mesenchyme by the dorsal mesocardium. The septum transversum grows into the intraembryonic coelom partially to divide the pericardial cavity from the peritoneal cavity. The pericardial cavity is further divided by ingrowth of tissue from the lateral body walls, the pleuropericardial folds, that fuse with each other and the foregut mesenchyme to completely enclose the pericardial cavity and create two pleural cavities still joined to the peritoneal cavity. The dorsal mesenchyme breaks down to leave the heart connected to the body wall only at the arterial and venous poles. The space thus created is the transverse pericardial sinus. The primitive myocardium of this primary heart tube shows regular contractions by the third week (embryonic age). This heart tube elongates by addition of mesodermal cells at its two poles (and from the dorsal mesocardium until that structure involutes) and undergoes rightward looping (Fig. 10.2). The primitive heart tube is the scaffold to which the atrial and ventricular chambers are added by ballooning from its myocardium. Septa are formed, dividing the right and left sides of the atria, ventricles and great arteries. Heart formation is completed with the development of valves, the conduction system and the formation of the coronary arteries by the ingrowth of extracardiac tissues derived from the neural crest and from the proepicardium situated in the septum transversum. 

The Heart Fields Heart progenitor cells are located in two small patches, one on either side of the midline in the epiblast of the bilaminar embryonic disc, close to the cranial part of the primitive streak. The earliest known committed cardiac precursors express the T-box transcription factor Eomesodermin\Tbr2, which activates another transcription factor MESP1.7 These progenitor cells migrate together through the primitive streak and form two plates of lateral mesoderm cells positioned anteriorly. Specification of cardiogenic

• Fig. 10.2  Looped heart. A dissection of an embryo of approximately 35 days. The pericardial sac has been opened to expose the heart. The heart is looped to the right. The bulboventricular sulcus is seen as a notch on the inferior surface. The inflow is located behind the ventricular mass and the outflow is superior.

mesoderm has already started during this cellular migration. The progenitor cells migrate such that the medial-lateral arrangement of these cells will become the cranial-caudal axis of a linear heart tube. With the formation of the embryonic coelom, they occupy the splanchnic mesoderm and fuse in the midline cranially to form the cardiac crescent. The cells of this cardiac mesoderm express the cardiac specific transcription regulator genes NKX2.5 and GATA-4.8 The cells of the splanchnic mesoderm, one on each side of the body, interact with adjacent tissues (Fig. 10.3). The splanchnic mesoderm is positioned adjacent to the foregut endoderm (see Fig. 10.1), which is thought to provide inductive signals to begin myocardial differentiation.9 Endoderm also has a mechanical role in assembly of the heart tube. By its active contraction, it pulls the bilateral fields of cardiogenic mesoderm towards the midline, permitting them to fuse and form a single heart tube. The primitive, single heart tube initially functions not so much to support the embryonic circulation as to provide a scaffold into which the cells from the second heart field migrate to effect chamber formation. The second heart field cells are first located medial to the cardiac crescent and subsequently lie in mesoderm underlying the pharynx before they are added to the heart.10 The cranial part of the second heart field, the anterior heart field, which is identified by FGF10 expression, contributes to the formation of right ventricular and outflow tract myocardium. Cells in the posterior second heart field express Isl1, but not anterior heart field markers, contribute to atrial myocardium. The nomenclature of the heart fields may be confusing10 because not all authors use exactly the same terms, and the heart fields are dynamic, changing in position and with time. The first and second heart fields develop sequentially, and the anterior and posterior heart fields describe anatomical positions. It appears that the overall cellular environment is critical for this process because cells taken from a different location or at a

90

SE C T I O N 3     Fetal Physiology and Pathology

Pharynx\foregut

BMP FGF

+

–

β-catenin/WNT

+

Midline

– WNT inhibitors

Activation of transcription factors NKX2.5 TBX5 GATA4 MEF2C SMARCD3 (BAF60C)

+ MESP1

Eomes Second heart field ISL1 TBX1 FGF8 FGF10

• Fig. 10.3  Activation of cardiac transcription factors. BMP, Bone morphogenetic protein; FGF, fibroblast growth factor.

different developmental time point can contribute to the heart when placed in the appropriate location. 

The Heart Tube Within the cardiac crescent, the primitive heart tube forms. The horseshoe-shaped cardiac crescent forms a tube with two caudolateral inlets, or venous pole, and one craniomedial outlet, or arterial pole. The process starts with the formation of endothelial-lined channels in the mesenchyme that form a plexus that eventually coalesces to form paired endothelial tubes. Folding of the embryo then brings these paired tubes together in the midline where they fuse to form a single endothelial-lined tube with a surrounding cuff of mesenchyme, separated from it by extracellular matrix termed cardiac jelly. The endocardium develops simultaneously with the myocardium and is a specialised endothelial cell type derived from the splanchnic mesoderm that, via an unknown mechanism, differs from the myocardial precursors.11 The linear heart tube subsequently grows by addition of cells from a proliferating pool of precursors located external to it in the heart fields and not by division of myocytes in situ. Being added to the heart, their fate is not fixed, and their identity depends on their eventual location. The cardiac precursors are therefore initially situated cranial and lateral to the future mouth but caudal to the mesoderm that forms the transverse septum and contributes to the formation of the diaphragm. During the process of folding, the cardiac

precursors end up between the mouth on the cranial aspect, the diaphragm at the caudal aspect, and ventral to the foregut, as in the adult situation.12 By folding of the embryo, the lateral parts of the cardiac mesoderm are brought together, forming the ventral part of the heart tube. The inner curvature of the cardiac crescent forms the dorsal side of the tube and is contiguous with the dorsal mesocardium, the attachment of the heart to the body wall. The peripheral part of the cardiac crescent will eventually face the transverse septum and form the venous pole of the heart, and the central part of the crescent, which forms the outflow tract, is contiguous with the pharyngeal mesenchyme. This intimate association of the cardiac and facial region during development explains the high incidence of combined cardiac and facial malformations in syndromic conditions. Retinoic acid appears critical in specifying these posterior precursor cells to become the inflow, or venous parts of the heart, the sinus venosus and atria, 

Contraction The myocardium shows regular contractions by the third week, meaning that the structural requirements for contraction, namely contractile proteins, sarcoplasmic reticulum and gap junctions, are already present in the myocardial cells of the primitive heart tube. It would appear that the initial contractions, at least in the chick embryo, are not essential for tissue oxygen and nutrient supply

CHAPTER 10  Development of the Heart and Cardiovascular System in Relation to Cardiac Abnormalities

91

but likely have an important function providing mechanical stimuli for further development of the heart.2 

Looping of the Heart Tube The myocardium of the primitive heart tube demonstrates TBX2 and TBX3 expression.13 Traditionally, five consecutive segments of the tube are recognised: venous, atrial, left ventricular, right ventricular and arterial. Between each of the segments is a transition zone – the sinoatrial ring, the atrioventricular (AV) canal, the primary heart fold and the ventricular outflow tract. It is important to recognise that the cardiac chambers develop as outgrowths from these segments and that the original segments form the connections of the cardiac chambers rather than the chambers themselves. Looping of the straight heart tube follows activation of a gene cascade that determines right–left symmetry. Left–right differentiation has already started at the time of gastrulation with the formation of the Hensen node. Nodal cilia beat in one direction and cause a gradient of molecules across the bilaminar embryo. These genes are already expressed in the cardiac mesoderm before formation of the heart tube and include NODAL, LEFTY and PITX2, of which PITX2 is the effector gene. Furthermore, the direction of looping is not random but is controlled by genes not yet understood and is not under the control of PITX2. The tube loops to the right (see Fig. 10.2), which causes the transition zones to be brought into close proximity on the inner curvature of the loop. This is absolutely essential for establishing the correct connections of the chambers. Actin polymerisationdriven myocardial cell shape changes have been found to contribute to the bending of the heart tube, but the torsional component of looping is largely caused by forces from its encapsulating membrane, the splanchnopleure.14 The looped heart tube has unidirectional blood flow, which was originally thought to be peristaltic but is now regarded as functioning as a Liebau pump (unidirectional and pulsatile flow resulting when an elastic tube containing fluid is periodically squeezed15). At this stage, the AV cushions function as primitive valves to permit unidirectional flow of blood in the looped heart tube.16 

Development of the Chambers and Outflows Both atria and ventricles develop by ballooning growth from the heart tube (Fig. 10.4), the atria growing from the dorsal aspect and the ventricles from the ventral aspect. Atrial segment growth is bilateral and in parallel; hence, it is possible to develop isomerism (Fig. 10.5). Ventricular segment growth is unilateral and in sequence; therefore development of isomerism is not possible. The initially formed atrial myocardium only gives rise to the trabeculated, atrial appendages in the formed heart. All smoothwalled myocardium found in the full-grown heart is added later during development from cells of the posterior heart field. Histologically, chamber formation becomes evident when the extensive extracellular matrix between endocardium and myocardium, known as cardiac jelly, disappears and trabeculations become evident in both atria and ventricles.12 Numerous genes are involved in this process, the primitive myocardium of the primary heart tube expressing TBX2 and TBX3, and the myocardium that forms the chambers

•

Fig. 10.4  Atrial and ventricular formation. The heart on approximately day 37. The two atrial appendages can be seen on either side of the arterial trunk that is positioned predominantly over the right ventricle. Septation of the trunk is evident by the separate column of blood on the right side. The ventricles can be seen ballooning out from the heart tube.

• Fig. 10.5  A 13-week fetus with left atrial isomerism. There were bilateral superior caval veins, complete atrioventricular septal defect and discordant ventriculoarterial connections with anterior aorta. The heart and lungs are viewed from the front. Two morphologically left atrial appendages are present enclosing the anteriorly situated aorta.

shows specific gene expression (ANF, CX40, CX43) but not TBX2 and TBX3.17 The early markers of chamber formation remain restricted to the original trabeculated myocardium, with NOTCH, ERBB, and Ephrin playing roles in trabeculation. The compact ventricular layer does not express ANF and CX40, but NOTCH, bone morphogenetic protein (BMP), and fibroblast growth factor play roles in compact myocardium development.18 Myocardial ‘compaction’ is a slightly misleading term because it is not a process by which the previously trabeculated myocardium becomes compact but rather one in which the myocardium of the epicardial side of the ventricular wall proliferates to form the compact layer; when the compact outer layer starts to form, proliferation in the ventricular trabeculations ceases.12 This process may be related to mechanical strain because there is a gradient of strain across the ventricular wall, greatest on the inner surface and least on the outer surface. The curvature of the heart therefore depends on cell shape

92

SE C T I O N 3     Fetal Physiology and Pathology

changes at the cellular level caused by a complex interplay on haemodynamic shear stress, contractive wall strain and electrical activity. It remains uncertain precisely how the left and right ventricles achieve their different morphologies, although there are differences in gene expression between the left and right ventricles, with the cardiac transcription factor TBX5 expressed in a gradient tapering off toward the right ventricle.19 Distal to the ventricle is the bulbus cordis. It is divided into three components, the proximal part forming the trabeculated part of the right ventricle, the midpart (conus cordis) forming the outflow tract of both ventricles and the distal third the truncus arteriosus. The bulbus cordis is demarcated externally from the ventricle by the bulboventricular sulcus, and internally this is the site of the interventricular foramen. 

Septation Septation of the ventricles occurs between 5 and 7.5 weeks of gestation. Initially, because the atrial segment is connected to the left ventricular segment, there is no direct connection with the right ventricle, but blood can flow from the atria to the right ventricle during diastole via the interventricular foramen (Fig. 10.6). Similarly, the outlet is connected initially solely to the right ventricle, but blood flows from the left ventricle to the outlet during systole via the interventricular foramen. The ventricular septum grows from the apex of the heart loop between the left and right ventricular segments, and its growth is largely appositional caused by ballooning of both ventricles. The interventricular foramen lies above the interventricular septum.12 

Atrial Septation The atria incorporate the draining veins and form a pair of valves around the sinus venosus. Fusion of the anterior part of these valves creates the septum spurium, which contributes to closure of the atria. The primary atrial septum develops to the right of this and grows downwards towards the fused AV cushions (Fig. 10.7) with the gap between its free edge of the primary septum and the AV cushions called the ostium primum. There is a mesenchymal cap on the edge of the primary septum that is continuous ventrally with the ventral AV cushion and dorsally with the dorsal mesenchymal protrusion and the dorsal AV cushion.20 Before obliteration of the ostium primum by fusion of the septum primum and the AV cushions, an opening forms by fenestration in the primary septum – the ostium secundum. The septum secundum then develops on the right side of the septum primum, beginning at about day 41, by infolding of the muscular wall of the atrium. The septum secundum is thicker and more muscular than the septum primum. It grows downwards, and its anterior part fuses with the endocardial cushions, but a defect remains – the oval fossa.21 The ostium primum is actually beneath the free edge of the septum primum and is obliterated when that edge fuses with the endocardial cushions at about day 38. The sinus venosus is incorporated into the right atrium, the coronary sinus representing its left horn. The right valve of the sinus venosus disappears in its upper part, but the lower part becomes the valve of the inferior caval vein (Eustachian valve) and the valve of the coronary sinus (thebesian valve) (Fig. 10.8). Occasionally, a complete remnant of the right valve persists, termed a Chiari network (Fig. 10.9). This may fill out like a sail and extend through the right AV valve. The left valve of the sinus

• Fig. 10.6  The heart during septation. The parts of the primary heart tube are coloured yellow. They form a tight curve on the inner aspect of the heart. The sinus venous (guarded by its valves) enters the posterior aspect the right atrium. The two atria have ballooned laterally, and the two ventricles ventrally, from this tube. The primary foramen lies between the inner curvature of the primary heart tube and the developing interventricular septum. Through it, because of differential streaming of the blood, blood flows from the right atrium to the right ventricle in diastole and from the left ventricle to the outflow tract in systole (black arrows). The developing endocardial cushions are coloured blue. The dorsal and ventral atrioventricular (AV) cushions divide the inflow from the atria. The primary atrial septum is growing downwards, and its mesenchymal cap will fuse with the AV cushions and the dorsal mesenchymal protrusion to seal the right from the left AV junction. The spiral endocardial cushions in the outflow tract have already fused distally and the zone of closure is moving proximally. Their fusion with the atrioventricular cushions will seal the outflow tracts. (Adapted from Sylva M, van den Hoff MJB, Moorman AFM. Development of the human heart. Am J Med Genet 164A(6):1347–1371, 2014.)

venosus is incorporated into the developing septum secundum but occasionally remnants of it persist as thin threads attached to the right side of the septum. The pulmonary vein enters the left part of the atrium and is incorporated into it. The exact site of development of the pulmonary vein remains controversial. The smooth wall of the body of the left atrium results from incorporation of the pulmonary veins into the atrium. Failure of connection of the pulmonary vein results in total anomalous pulmonary venous connection (Fig. 10.10). The atrial appendages hence represent the original parts of the primitive atrium. The smooth-walled part of the right atrium develops from incorporation of the sinus venosus and is termed the sinus venarum. 

The Interventricular Septum The greater part of the interventricular septum results from a process of apposition because of ballooning of the ventricular chambers, beginning at about day 26. The crest of the interventricular septum is at the site of the primary foramen and retains the genetic expression pattern of the primitive heart tube (expressing TBX3 and NOTCH1), fusing with the dorsal AV cushion.22 

A

C

B

D

• Fig. 10.7  Development of the interatrial septum. A, Diagrammatic view of the heart showing the right and left atria and the left ventricle with posterior atrioventricular (AV) cushion. The primary septum (septum primum) grows between the right and left atria. Its lower border grows towards the AV cushion, the gap between them forming the ostium primum (B).  Fusion of the lower border of the primary septum and the AV cushion eliminates the ostium primum. By the time they fuse, however, fenestrations have appeared in the septum primum to permit right-to-left passage of blood in the atrium (C). These fenestrations coalesce as the ostium secundum. A second septum, the septum secundum, grows to the right of the septum primum and covers the ostium secundum, the septum primum forming the flap valve of the oval fossa (D). The left valve of the sinus venosus fuses with the septum secundum. The upper part of the right valve regresses, but the lower part forms the eustachian and thebesian valves.

•

Fig. 10.8  Interatrial septum viewed from the right side. The structures of the right side of the septum are well seen: oval fossa, coronary sinus, eustachian valve and thebesian valve. In addition, there is a secundum atrial septal defect where the flap valve of the oval fossa does not completely cover the orifice.

• Fig. 10.9  Chiari network. Delivery at 31 weeks’ gestation. Fetal hydrops. Pulmonary stenosis and dysplastic tricuspid valve. The right atrium and right ventricle are exposed to show a diaphanous, baglike membrane covering the right side of the interatrial septum, a remnant of the right valve of the sinus venosus.

94

SE C T I O N 3     Fetal Physiology and Pathology

LUPV LLPV

RUPV RLPV

• •

Fig. 10.10  Total anomalous pulmonary venous connection. A case of right atrial isomerism in a fetus of 22 weeks. There were bilateral superior caval veins, atrioventricular septal defect, transposition, pulmonary atresia with major aortopulmonary collateral arteries, absent arterial duct and asplenia. The pulmonary veins come to a cruciate confluence behind the heart, from which a vein ascends to insert into the left superior caval vein. LLPV, Left lower pulmonary vein; LUPV, left upper pulmonary veins; RLPV, right lower pulmonary veins; RUPV, right upper pulmonary vein.

Fig. 10.11  Atrioventricular (AV) cushions. Left side of the AV junction at about day 37. The ventricle is still highly trabeculated with commencement of formation of an epicardial compact layer. The AV canal between the atrial and ventricular chambers shows dorsal and ventral myxoid masses (cushions) with a gap between them. These will eventually fuse to obliterate the central gap and create separate AV orifices to the right and to the left. The cushions also play a role in completing septation of the atria, the ventricles and the outflow tract. Separate cushions are also evident in the arterial trunk where they are just fusing to divide it.

The Atrioventricular Junction The AV valves develop form the two endocardial cushions. During looping, the cardiac jelly is eliminated from much of the heart tube but persists at the site of the endocardial cushions at the AV junction as well as in the outflow tract. The cushions are originally acellular, but beginning on day 26, the epithelial-to-mesenchymal transition produces cells that populate the cushions. In the first stage of this process, a subset of endocardial cells lining the AV junction and the outflow tract transform into a mesenchymal phenotype mediated by BMP through transforming growth factor β (TGFβ) and Notch produced by the myocardium of the AV junction and invade the cardiac jelly.23 This invasive, proliferating mesenchyme progressively remodels the matrix, and the resultant cellular masses, now called cushions, continue to grow and extend into the lumen (Fig. 10.11). In the AV canal, these cushions form superiorly and inferiorly and fuse in the midline to create right and left AV orifices, differential growth of the right AV canal having brought the right atrium into contact with the right ventricle. Fusion of the cushions with the developing interventricular muscular septum and the atrial primary septum completes septation of the atrial and ventricular chambers. This complex process may be defective and result in ventricular septal defects (perimembranous in this region) (Fig. 10.12). Lateral cushions also develop, attracting cells from the epicardium. The dorsal mesenchymal protrusion, also called the vestibular spine, is contiguous with the dorsal AV cushion and the mesenchymal cap of the primary atrial septum in the atria and is derived from extracardiac cells. Maldevelopment of the dorsal mesenchymal protrusion causes AV septal defect (Figs. 10.13 and 10.14), and absence of the dorsal mesenchymal protrusion is seen in fetuses with Down syndrome (Fig. 10.15).24

• Fig. 10.12  Ventricular septal defect. A heart with tetralogy of Fallot, cut in a simulated echocardiographic long axis view. There is a ventricular septal defect with overriding of the aorta. The crest of the interventricular septum forms the lower part of the defect.

Even before septation, laminar blood flow ensures separation of the right and left streams of blood. The primary foramen is that part of the primitive heart tube from which the ventricles balloon. It is sometimes referred to as the interventricular foramen but is not actually between the two ventricles but rather above them at the inner curvature of the heart (see Fig. 10.6). Blood flows through this foramen from the right atrium to the right ventricle during diastole and from the left ventricle to the aorta during systole. This foramen becomes septated by the membranous septum at about day 45. The AV canal develops ventral and dorsal cushions that separate the canal into right and left parts. The outflow tract is separated by two ridges called the septal and parietal outflow tract ridges (Figs. 10.16 and 10.17). In an adult heart, the membranous part of the ventricular septum is the remnant of the fused AV and outflow tract cushions. It has

CHAPTER 10  Development of the Heart and Cardiovascular System in Relation to Cardiac Abnormalities

•

Fig. 10.13  Complete atrioventricular (AV) septal defect. Heart cut in a simulated four chamber echocardiographic view. There is a common AV junction with a common valve. The large defect lies between the lower border of the interatrial septum and the upper border of the interventricular septum.

95

•

Fig. 10.15  Membranous interventricular septum, trisomy 21 in a male infant aged 9 months who died from respiratory viral infection. There is a secundum atrial septal defect, and histologically, the lungs showed changes of pulmonary arterial hypertension. The right atrium and ventricle have been opened to expose the septal structures. The membranous septum (black dots) is large, and there is discontinuity between the septal and anterior leaflets of the tricuspid valve.

• • Fig. 10.14  Atrioventricular (AV) septal defect in left atrial isomerism. There are bilateral left atrial appendages. There is also situ inversus. There is a complete AV septal defect. There were also bilateral superior caval veins and secundum atrial septal defect and aortic isthmus hypoplasia. The shape of the heart (allowing for situs inversus) shows a striking similarity to a heart at about 35 days (see Fig. 9.10.)

been proposed25 that the morphology of transposition of the great arteries is a defect of laterality akin to the heterotaxy syndromes (Fig. 10.18). 

Valves The septal leaflet of the tricuspid valve and the aortic leaflet of the mitral valve arise from fusion of the dorsal and ventral AV cushions on the right and left sides, respectively.26 The septal leaflet detaches from the myocardium by cellular apoptosis. The mural leaflets of the two AV valves are formed from the lateral AV cushions, and the ventricular space grows behind these mural leaflets.27

Fig. 10.16  Outflow tract in an embryo at 37 days. The ventricular myocardium is trabeculated. The outflow is connected largely to the developing right ventricle. Endocardial cushions are present in the outflow tract that are growing toward one another but have not yet fused.

The semilunar valves form from the parietal and septal outflow tract cushions and two intercalated ridges. The parietal outflow tract cushion gives rise to the right aortic and pulmonary valve leaflets. The septal outflow tract cushion gives rise to the left aortic and pulmonary leaflets, and the right and left intercalated ridges give rise to the posterior aortic and anterior pulmonary leaflets. The cushion at the distal margin undergoes apoptosis to achieve a cusp. The AV and semilunar valves then mature by remodelling of their matrix. 

Outflow Tract The outflow tract connects the developing ventricles to the aortic sac that is connected to the symmetrical pharyngeal arch arteries. Septation starts at day 32 and occurs distally to proximally (see Figs. 10.16 and 10.17) with the cushions arranged in a spiral fashion. The outflow tract myocardium becomes incorporated into the

96

SE C T I O N 3     Fetal Physiology and Pathology

•

Fig. 10.17  Septation of the truncus arteriosus in an embryo at approximately day 37. The truncus is cut in cross section lying above the ventricular mass as it travels backwards. Four endocardial cushions are present, two major and two lesser ones. The developing interventricular septum can be seen in addition to the interventricular foramen lying superior to it.

•

Fig. 10.19  Truncus arteriosus. A common arterial trunk arises from the heart from which the aorta and pulmonary arteries arise. Of necessity, there is a ventricular septal defect. The defect arises from failure of fusion of the outflow tract cushions.

developing right ventricle. The outflow tract is connected via the aortic sac to arch arteries 3, 4 and 6. A protrusion of pharyngeal mesoderm, the aortopulmonary septum, grows into the aortic sac and connects distally to the spiralling outflow tract ridges, which separates the sixth and fourth pharyngeal arch arteries. The outflow tracts therefore have a complex origin, partly from the primary heart tube and partly from ingrowth of cells from two distinct sources, including the cardiac neural crest and the second heart field. The complex interaction of all these tissues gives rise to the ventricular outflow tracts, the arterial valves and the intrapericardial parts of the aorta and pulmonary trunk (Fig. 10.19).28 

Coronary Circulation The coronary arteries and veins develop by both vasculogenesis (the formation of vessels in situ) and angiogenesis (the formation of new vessels by sprouting from existing vessels) from cells that grow over the myocardium from the proepicardium (Fig. 10.20). Both the endothelium and the medial smooth muscle of the coronary arteries derive from this source (Fig. 10.21). These vessels link up and grow to join with the aorta (Fig. 10.22).29 The pericardium develops as a sac around the developing heart tube. Initially, the tube is connected to the posterior mediastinum by the dorsal mesocardium, but this breaks down, permitting the folding of the heart tube on which subsequent development is so critically dependent. 

Conduction Tissue • Fig. 10.18  Transposition of the great arteries. The heart is cut in a simulated echocardiographic long-axis view. The aorta arises anteriorly from the right ventricle and the pulmonary trunk posteriorly from the left ventricle. The normal spiral relation of the great arteries to each other is abolished. Instead they ascend in parallel. The pathogenesis of the defect is regarded by some on morphological and epidemiological grounds as akin to the heterotaxy syndromes in which there is disturbance of the normal left–right patterning.

The conduction tissue develops from the myocardium of the primitive heart tube, being found in the transitional zones.30 Biomechanical factors play a critical role in induction and patterning of the cardiac conduction system.2 The formation of an insulating fibrous and fatty tissue plane between atrial and ventricular myocardium occurs only after completion of septation, beginning at 7 weeks and largely complete by 12 weeks of development. 

CHAPTER 10  Development of the Heart and Cardiovascular System in Relation to Cardiac Abnormalities

97

•

Fig. 10.20  Derivation of the epicardium. A schematic representation of the developing heart showing the location of the proepicardial organ in the pericardial cavity adjacent to the septum transversum. The cells migrate over the surface of the heart and undergo epithelial-to-mesenchymal transformation to form the epicardium, the stroma of the myocardium and the coronary arteries. (Adapted from Pérez-Pomares JM, de la Pompa JL, Franco D, et al. Congenital coronary artery anomalies: a bridge from embryology to anatomy and pathophysiology—a position statement of the development, anatomy, and pathology ESC Working Group. Cardiovasc Res 109(2):204–216, 2016.)

Pulmonary Veins The pulmonary vein develops and connects to the heart after the formation of the initial heart tube and start of chamber formation. Mesenchyme dorsal to the heart differentiates into a vascular plexus surrounding the embryonic foregut. At around day 30, the cranial component of this plexus connects as a solitary pulmonary vein to the heart through the dorsal mesocardium in the midline and cranial to the AV node. Failure of the vein to connect with the atrium will result in anomalous pulmonary venous connection (see Fig. 10.10). Although a midline structure, eventually, the pulmonary vein will drain into the left atrium because the primary atrial septum develops at the right side. Following its connection to the developing left atrium, the pulmonary vein and its bifurcations acquire a sleeve of myocardium, which differentiate de novo. The transcription factor PITX2c plays a crucial role in the differentiation of the pulmonary vein myocardium.31 The muscularised pulmonary vein is gradually incorporated into the left atrium, up to its second division, resulting in four pulmonary orifices in the left atrium. The pulmonary myocardial sleeves may extend upstream of the pulmonary orifices. 

The Venous System There are three pairs of major veins in the embryo in the fifth week.

•

Fig. 10.21  Origin of the coronary circulation, showing the triple origin of the coronary circulation. The coronary veins develop by budding from the endothelium of the sinus venosus (orange). These small veins ramify in the mesenchyme of the epicardium. The epicardial cells, transformed to mesenchyme (purple), form the tunica media and adventitia of the coronary arteries. The endothelium of the coronary arteries derives largely from the endocardium that extends through the trabeculations of the developing myocardium of the ventricles and atria as sinsuoids (green). (Adapted from Pérez-Pomares JM, de la Pompa JL, Franco D, et al. Congenital coronary artery anomalies: a bridge from embryology to anatomy and pathophysiology—a position statement of the development, anatomy, and pathology ESC Working Group. Cardiovasc Res 109(2):204–216, 2016.))

Vitelline Veins (Omphalomesenteric Veins) These paired veins carry blood from the yolk sac to the sinus venosus. Before joining the sinus medially, they break into a plexus around the duodenum and cross the septum transversum. Hepatocytes growing into the septum induce the hepatic sinusoids. The proximal right vitelline vein persists as the hepatic part of the inferior vena cava, its distal part becomes the superior mesenteric vein and the anastomotic network around the duodenum becomes the portal vein. The proximal part of the left vitelline vein disappears 

Umbilical Veins Paired veins join the sinus lateral to the vitelline veins and medial to the cardinal veins. With growth of the liver, they form anastomoses with the hepatic sinusoids. The proximal parts of both right and left veins disappear, and the distal left umbilical vein persists and drains to the hepatic sinusoids. A direct anastomosis develops between the left umbilical vein and the proximal right vitelline vein – the venous duct (ductus venosus). 

98

SE C T I O N 3     Fetal Physiology and Pathology

•

Fig. 10.22  Origin of the coronary artery from the aorta. Section of the developing aortic root of a mouse embryo showing the connection of the coronary artery with the aortic lumen. The aortic valve still comprises cushions that have not undergone remodelling.

Cardinal Veins The paired common cardinal veins join the sinus most laterally and are formed by the confluence of anterior cardinal veins (draining the head) and posterior cardinal veins. From the fifth to the seventh week, further veins form: the subcardinal veins draining mostly the kidneys, the supracradinal veins draining the body wall by the intercostal veins following regression of the posterior cardinal veins, and the sacrocardinal veins draining the lower limbs. Anastomoses develop between the right and left sides. The brachiocephalic vein is formed of the anastomosis between the two anterior cardinal veins. The superior caval vein is formed from the proximal right anterior cardinal vein and the right common cardinal vein. The anastomosis between the two subcardinal veins becomes the left renal vein and the left subcardinal vein and then regresses with its distal part, becoming the left gonadal vein. The right subcardinal vein becomes the renal segment of the inferior caval vein, and it connects proximally with the hepatic inferior caval vein derived from the right vitelline vein (Fig. 10.23). The anastomosis between the sacrocardinal veins becomes the left common iliac vein. The right supracardinal vein becomes the supracardinal segment of the inferior caval vein. The azygos vein derives from the right supracardinal vein and part of the posterior cardinal vein. On the left side, the supracardinal vein between the fourth and seventh intercostal veins becomes the haemiazygos vein that drains to the azygos vein (Fig. 10.24). Initially, the veins draining to the heart are embedded in the mesenchyme at the venous pole of the heart with expansion of the pericardial cavity excavating the connecting veins from this mesenchyme. By this process, the common cardinal veins, which are the confluence of the left and right superior and inferior cardinal veins, become incorporated within the pericardial cavity. They then acquire a sleeve of myocardium, and the confluence of the systemic veins is then called the sinus venosus, or left and right sinus horns, which both eventually connect to the right atrium. 

• Fig. 10.23  Embryonic origin of Inferior caval vein. The inferior caval vein is viewed from behind. The aorta is to its left. The iliac veins and inferior vena cava bifurcation derive from the post cardinal veins (orange). The segment between the renal veins and the bifurcation derives from the supra cardinal vein (yellow). The segment between the liver and the renal veins derives from the subcardinal vein (green). The short segment between the liver and the right atrium derives from the right vitelline vein (red).

The Arterial System Folding of the embryo during the fourth week (days 22–24) causes the paired dorsal aortae attached to the cranial end of the heart to form a pair of dorsoventral loops – the first aortic arches. The paired dorsal aortae fuse below the level of the fourth thoracic segment and become connected with the umbilical arteries. Between days 26 and 29, four additional pairs of aortic arches develop in succession from cranial to caudal within the mesenchyme of the pharyngeal arches, connecting the aortic sac at the superior end of the truncus arteriosus to the dorsal aortae – arches 2, 3, 4 and 6 (Fig. 10.25). No fifth aortic arch ever develops. The first two pairs of arch arteries regress while the later arch arteries are forming. It is therefore the aortic arch arteries 3, 4 and 6 that give rise to the arteries of the head, neck and thorax (Table 10.1).32

CHAPTER 10  Development of the Heart and Cardiovascular System in Relation to Cardiac Abnormalities

A

B

• Fig. 10.24  Azygos continuation of the inferior caval vein. Photograph (A) and drawing (B) of the main features. Termination of pregnancy at 20 weeks’ gestation for left atrial isomerism. The thoracoabdominal organs are viewed from behind. The aorta and caval vein ascend together through the diaphragm, the vein as the azygos vein that enters the superior caval vein. In addition to left atrial isomerism, there were complete atrioventricular septal defect, bilateral superior caval veins and situs inversus with polysplenia. The hepatic veins drained independently to the right atrium.

A

B • Fig. 10.25  A, Schematic representation of the aortic arches viewed from the left side. The head is to the left and the lower body to the right. The arch arteries are symmetrically paired and develop form cranial to caudal. No fifth arch ever develops. Not all the arches are present at the same time. The first and second have regressed by the time the sixth arch develops.  B, Schematic representation of the aortic arches following remodelling showing the persistence of some and involution of others. As in A, the specimen is viewed from the left side with the head to the left of the field. The third arch derivatives are symmetrical, but the derivatives of the fourth and sixth arches are asymmetrical.

99

100

SE C T I O N 3     Fetal Physiology and Pathology

TABLE Arterial Derivatives of the Embryonic Aortic 10.1 Arches

Embryonic Arch

Arterial Derivative

I

Maxillary artery

II

Hyoid artery Stapedial artery

III

Common carotid artery First part of the internal carotid artery

Dorsal aorta gives rise to remainder of internal carotid

IV

Proximal right subclavian artery Aortic arch between the LCCA and LSCA

LSCA derives from the seventh intersegmental artery

VI

Branch pulmonary arteries and arterial duct

Notes

•

Fig. 10.27  Retro-oesophageal right subclavian artery. The right subclavian artery derives from the seventh intersegmental artery. Normally, it connects to the ventral part of the fourth arch artery. When it connects instead to the right dorsal aorta, because it is posteriorly situated, it courses behind the gut and airway to reach its usual course.

LCCA, left common carotid artery; LSCA, left subclavian artery.

  

•

•

Fig. 10.28  Retro-oesophageal right subclavian artery. The normal right subclavian artery develops from the connection of the seventh intersegmental artery with the right fourth arch artery. In retro-oesophageal right subclavian artery, this does not happen. Instead the seventh right intersegmental artery takes origin from the right dorsal aorta. In this photograph, the thoracic contents are viewed from behind, and the right subclavian artery is seen to take origin from the descending aorta on the left side and to run behind the oesophagus to reach its normal location.

The dorsal aorta develops three sets of branches: 1. A series of ventral branches that supply the gut derivatives derived from a network of vitelline arteries 2. Lateral branches that supply retroperitoneal structures such as adrenals, kidneys and gonads 3. Dorsolateral intersegmental branches that penetrate between the somite derivatives The third arch on both right and left sides becomes the common carotid and first part of the internal carotid artery (see Fig. 10.25). The segments of dorsal aorta connecting arches 3 and 4 regress,

leaving the third aortic arch to supply blood to the head. The fourth and sixth arches undergo asymmetrical remodelling to supply the upper extremities, dorsal aorta and lungs. The aortic sac gives rise to the proximal aortic arch and brachiocephalic artery. (Fig. 10.26). The left fourth arch becomes the aortic arch between the left common carotid and left subclavian arteries and the most cranial part of the descending aorta. A retro-oesophageal right subclavian artery arises when the seventh intersegmental artery that normally connects with the ventral part of the right fourth arch connects instead to the persistent right dorsal aorta (Fig. 10.27). A retro-oesophageal right subclavian artery is a common finding in trisomy 21 (Fig. 10.28). The right and left subclavian arteries derive from the seventh intersegmental arteries. The sixth arch arteries provide the pulmonary arteries, and the left arch provides the arterial duct. Persistence of remnants of the aortic arches can cause vascular rings that enclose the oesophagus and trachea (Figs. 10.29 and 10.30). There are multiple patterns described. Abnormal involution can also lead to interruption of the aortic arch (Fig. 10.31). 

Fig. 10.26  Derivatives of the aortic arch arteries. The truncus arteriosus gives rise to the most proximal segments of the aorta and pulmonary trunk (orange). The aortic sac gives rise to the ascending aorta and brachiocephalic artery (green). The third aortic arches give rise to the internal carotid arteries. The fourth aortic arch gives rise on the right side to the first part of the subclavian artery and on the left to the segment of aortic arch between the left common carotid and left subclavian arteries (purple). The distal right subclavian artery and all the left subclavian artery derive from the seventh intersegmental arteries (yellow). The pulmonary arteries and arterial duct derive from the sixth aortic arch arteries (blue). The remainder of the descending aorta (uncoloured) derives from the left dorsal aorta.

CHAPTER 10  Development of the Heart and Cardiovascular System in Relation to Cardiac Abnormalities

101

•

Fig. 10.29  Vascular ring. The ascending aorta gives rise to left and right common carotid arteries and the right subclavian artery. It then arches over the hilum of the right lung, and the descending aorta lies on the right side. The pulmonary trunk gives rise to the right and left pulmonary arteries, with a left-sided arterial duct connecting the left subclavian artery to the left pulmonary artery. The left subclavian artery arises from the descending aorta via a socalled retro-oesophageal diverticulum (diverticulum of Kommerell). There is thus a complete vascular ring which surrounds the trachea and oesophagus; viewed posteriorly, the vessels display a Y-configuration formed by the aortic arch on the right and the left subclavian artery or diverticulum of Kommerell on the left. The descending thoracic aorta more distally is situated in the midline, posterior to the oesophagus. No right-sided arterial duct was identified, and there was no evidence of coarctation.

A

B

• Fig. 10.30  Vascular ring in a male fetus aged 20 weeks. A, The thoracic contents are viewed from behind. There is a complete arterial ring around the oesophagus and trachea. There is a right aortic arch with right-sided aorta but a left arterial duct and an isolated left subclavian artery. B, Isolated heart with the trachea and oesophagus removed. The ring is formed by the right aortic arch and the left-sided arterial duct. The left subclavian artery arises from the duct.

102

SE C T I O N 3     Fetal Physiology and Pathology

*

• Fig. 10.31  Interrupted aortic arch. The great vessels arise normally from the heart. The aorta gives rise to brachiocephalic and left common carotid arteries, but the aortic isthmus is absent (asterisk), and the descending aorta and left subclavian artery are supplied from the pulmonary trunk via the arterial duct. The patient was a neonate who collapsed suddenly and died. At postmortem, a ventricular septal defect and aortic stenosis were also seen.

Conclusion The human heart develops over a 4-week period from a mass of mesenchymal cells in the ventral embryo. An endothelial-lined tube enveloped by muscle forms first and begins rhythmic contraction. The tube loops to the right, and from its walls, the atrial and ventricular chambers balloon out. Mesenchymal protrusions into the lumen effect separation of the right and left sides in addition to forming the AV and arterial valves. The conduction system develops from the primitive myocardium of the original heart

tube. A complex series of arteries and veins develops sequentially and remodels to form the definitive arterial and venous system. Defects in the process can cause defects of laterality (isomerism and transposition) or failure of septation (atrial, AV and ventricular septal defects). Secondary haemodynamic changes can greatly modify and exacerbate the original defect. Access the complete reference list online at ExpertConsult.com. Self-assessment questions available at ExpertConsult.com.

References 1. Bruneau BG. Signaling and transcriptional networks in heart development and regeneration. Cold Spring Harb Perspect Biol. 2013;5:1–18. 2. Lindsey SE, Butcher JT, Yalcin HC. Mechanical regulation of cardiac development. Front Physiol. 2014;5:318. 3. Su W, Zhu P, Wang R, et al. Congenital heart diseases and their association with the variant distribution features on susceptibility genes. Clin Genet. 2017;91:349–354. 4. Gut P, Reischauer S, Stainier DYR, Arnaout R. Little fish, big data: zebrafish as a model for cardiovascular and metabolic disease. Physiol Rev. 2017;97:889–938. 5. Kulesa PM, McKinney MC, McLennan R. Developmental imaging: the avian embryo hatches to the challenge. Birth Defects Res C Embryo Today. 2013;99:121–133. 6. Krishnan A, Samtani R, Dhanantwari P, et  al. A detailed comparison of mouse and human cardiac development. Pediatr Res. 2014;76:500–507. 7. Rana MS, Christoffels VM, Moorman AFM. A molecular and genetic outline of cardiac morphogenesis. Acta Physiol. 2013;207:588–615. 8. Chung IM, Rajakumar G. Genetics of congenital heart defects: the NKX2–5 gene, a key player. Genes (Basel). 2016;7: pii, E6. 9. Lough J, Sugi Y. Endoderm and heart development. Dev Dyn. 2000;217:327–342. 10. Abu-Issa R. Heart fields: spatial polarity and temporal dynamics. Anat Rec (Hoboken). 2014;297:175–182. 11. Haack T, Abdelilah-Seyfried S. The force within: endocardial development, mechanotransduction and signalling during cardiac morphogenesis. Development. 2016;143:373–386. 12. Sylva M, van den Hoff MJB, Moorman AFM. Development of the human heart. Am J Med

Genet Am J Med Genet A. 2014;164A(6): 1347–1371. 13. Miquerol L, Kelly RG. Organogenesis of the vertebrate heart. Wiley Interdiscip Rev Dev Biol. 2013;2:17–29. 14. Taber LA, Voronov DA, Ramasubramanian A. The role of mechanical forces in the torsional component of cardiac looping. Ann N Y Acad Sci. 2010;1188:103–110. 15. Manner J, Wessel A, Yelbuz TM. How does the tubular embryonic heart work? Looking for the physical mechanism generating unidirectional blood flow in the valveless embryonic heart tube. Dev Dyn. 2010;239:1035–1046. 16. Butcher JT, McQuinn TC, Sedmera D, et  al. Transitions in early embryonic atrioventricular valvular function correspond with changes in cushion biomechanics that are predictable by tissue composition. Circ Res. 2007;100:1503–1511. 17. Moorman AFM, Christoffels VM. Cardiac chamber formation: development, genes and evolution. Physiol Rev. 2003;83:1223–1267. 18. Samsa LA, Yang B, Liu J. Embryonic cardiac chamber maturation: trabeculation, conduction, and cardiomyocyte proliferation. Am J Med Genet C Semin Med Genet. 2013;163C:157–168. 19. Boogerd CJ, Evans SM. TBX5 and NuRD divide the heart. Dev Cell. 2016;36:242–244. 20. Briggs LE, Kakarla J, Wessels A. The pathogenesis of atrial and atrioventricular septal defects with special emphasis on the role of the dorsal mesenchymal protrusion. Differentiation. 2012;84:117–130. 21. Jensen B, Spicer DE, Sheppard MN, Anderson RH. Development of the atrial septum in relation to postnatal anatomy and interatrial communications. Heart. 2017;103:456–462. 22. Anderson RH, Brown NA, Mohun TJ. Insights regarding the normal and abnormal formation of the atrial and ventricular septal structures. Clin Anat. 2016;29:290–304.

23. MacGrogan D, Luna-Zurita L, de la Pompa JL. Notch signaling in cardiac valve development and disease. Birth Defects Res A Clin Mol Teratol. 2011;91:449–459. 24. Briggs LE, Kakarla J, Wessels A. The pathogenesis of atrial and atrioventricular septal defects with special emphasis on the role of the dorsal mesenchymal protrusion. Differentiation. 2012;84:117–130. 25. Unolt M, Putotto C, Silvestri LM, et al. Transposition of great arteries: new insights into the pathogenesis. Front Pediatr. 2013;1:11. 26. Hinton RB, Yutzey KE. Heart valve structure and function in development and disease. Annu Rev Physiol. 2011;73:29–46. 27. Combs MD, Yutzey KE. Heart valve development: regulatory networks in development and disease. Circ Res. 2009;105:408–421. 28. Anderson RH, Mori S, Spicer DE, et al. Development and morphology of the ventricular outflow tracts. World J Pediatr Congenit Heart Surg. 2016;7:561–577. 29. Pérez-Pomares JM, de la Pompa JL, Franco D, et  al. Congenital coronary artery anomalies: a bridge from embryology to anatomy and pathophysiology—a position statement of the development, anatomy, and pathology ESC working group. Cardiovasc Res. 2016;109(2): 204–216. 30. van Weerd JH, Christoffels VM. The formation and function of the cardiac conduction system. Development. 2016;143:197–210. 31. Mommersteeg MT, Brown NA, Prall OW, et  al. Pitx2c and Nkx2-5 are required for the formation and identity of the pulmonary myocardium. Circ Res. 2007;101:902–909. 32. Yashiro K, Shiratori H, Yashiro K, et al. Haemodynamics determined by a genetic programme govern asymmetric development of the aortic arch. Nature. 2007;450:285–288.

102.e1

11

Lung Growth and Maturation RICHARD HARDING, ANNIE R.A. MCDOUGALL AND STUART B. HOOPER

KEY POINTS • S urvival at birth depends upon the lung attaining an adequate size and degree of structural maturity during fetal life. This chapter deals with mechanisms underlying normal and impaired lung growth and lung maturation before birth. • The airways of the fetal lung contain a liquid that is actively secreted by the epithelium; this ‘lung liquid’ causes the lung to develop in an expanded state, which is necessary for normal lung growth and maturation. • The degree of lung expansion in a fetus is determined by the lung’s physical environment, including intrathoracic space, fetal breathing movements (FBMs) and amniotic fluid volume. Mechanical stress in lung tissue stimulates gene networks, leading to tissue growth and differentiation. The long-term absence of the physiological stretch stimulus leads to lung hypoplasia. • Clearance of lung liquid begins with the onset of labour caused by (i) imposed fetal postural changes that cause loss of lung liquid via the nose and mouth and (ii) active reabsorption across the lung epithelium. Luminal liquid remaining after birth is cleared because of transpulmonary pressure gradients generated by inspiration. • Pulmonary blood flow (PBF) is generally low during fetal life but can increase with FBMs. At birth, pulmonary vascular resistance decreases markedly, thereby permitting increased blood flow through the lungs, which is necessary for adequate gas exchange. • Lung aeration at birth underpins the cardiovascular transition at birth, including the marked increase in PBF. With the loss of umbilical venous return at birth, the increase in pulmonary venous return takes over the critical role of supplying preload for the left ventricle. • Maturation of the lung in preparation for birth involves extracellular matrix remodelling, alveolar epithelial cell differentiation and surfactant production. These changes are driven by mechanical stress in lung tissue and corticosteroid signalling.

Introduction In fetal life, the lungs play no role in gas exchange, but at birth, they must immediately take over from the placenta the critical role of gas exchange. This transition is normally uneventful, which is remarkable given that before birth, the lungs are liquid filled with a low blood flow. For the lung to function as an organ of gas

exchange at birth, it must cease the secretion of fetal lung liquid, and the airways must be cleared of luminal liquid; the lungs must produce adequate amounts of surfactant; and pulmonary vascular resistance (PVR) must be reduced, allowing them to receive the entire output of the right ventricle. During normal fetal development, the lung becomes progressively prepared for these dramatic changes in physiology at birth. However, if lung growth or maturation in utero is impaired or if an infant is born before term, the newborn infant may develop respiratory distress syndrome (RDS). This chapter focuses on the processes controlling prenatal lung growth and maturation and highlights the physiological changes that underpin the transition to newborn life. Some of the more common respiratory complications in neonates and their fetal origins are discussed together with strategies for their treatment. 

Stages of Lung Development Pulmonary morphologists recognise five or six major stages in human lung development1 (Table 11.1).

Embryonic Stage (4–7 Weeks) The lung first appears as an outgrowth of the primitive foregut (i.e., endodermal tissue) at 22 to 26 days postconception. This bud divides to form the left and right bronchi, which then undergo dichotomous branching to form the major units of the bronchial tree. During early embryonic development, epithelial cells that are endodermal in origin form the developing ‘airways’ and grow into the surrounding tissue, which is derived from splanchnic mesoderm. This mesodermal tissue gives rise to the mesenchymal cells that ultimately form the nonepithelial structures of the lung, including blood and lymph vessels, airway cartilage and smooth muscle, fibrous tissue and other components of the lung parenchyma. 

Pseudoglandular Stage (5–17 Weeks) During the pseudoglandular stage, the lung resembles a typical exocrine gland. The major bronchi and associated functional units of the lung (i.e., acini) progressively form, accompanied by branches of the pulmonary arterial tree. As a result, each major ‘airway’ is accompanied by a branch of the pulmonary artery. The formation of each acinus (respiratory unit) results from repeated branching of the distal extremities of blind-ending tubes or ‘airways’ composed of epithelial cells (Fig. 11.1). This process of branching (branching morphogenesis) is induced by airway epithelial cells interacting with adjacent mesenchymal cells, which are supplied by a loose network of capillaries (see Fig. 11.1). Airway epithelial cells gradually differentiate (in a centrifugal direction) 103

104

SE C T I O N 3     Fetal Physiology and Pathology

TABLE 11.1 Stages of Lung Development in the Human

Stage

Gestational Age

Major Events

Embryonic

4–7 wk

Appearance of ventral bud in foregut. Epithelial tube branches and grows into surrounding mesenchyme. Vascular connec­ tions formed.

Pseudo­ glandular

5–17 wk

Development of bronchial tree, par­ alleled by formation of vascular tree. Lung periphery contains parenchymal precursors.

Canalicular

16–26 wk

Addition of further generations of airways and vascular tree. Differentiation of type I and type II epithelial cells. Formation of thin air–blood barrier. Start of surfactant production.

lung tissue volume. During the terminal sac stage, the development of secondary septa begins; these outgrowths from primary septa will eventually subdivide the terminal sac into multiple alveoli (see Fig. 11.1). The primitive primary septa, which separate adjacent saccules, are thicker than secondary septa and contain a double capillary network rather than the single capillary layer of the mature alveolus. Elastic fibres are formed by myofibroblasts within secondary septa and are deposited at their tips, thereby contributing to the inherent elastic (recoil) properties of the lung. The epithelial cells become differentiated into type I and type II epithelial cells. As a result of these structural changes, the separation between luminal ‘air’ and capillary blood becomes smaller, thereby enhancing the ability of the lung to exchange respiratory gases after birth. 

Alveolar Stage (36 Weeks of Gestation to 1–2 Years)

into specific cell types: ciliated cells (by 11–13 weeks), goblet cells and mucous glands. 

During the alveolar stage, terminal sacs become subdivided by the outgrowth of secondary septa from the primary septa to form alveoli. Initially, these alveoli resemble shallow cups, but they deepen because of elongation of the secondary septa. The alveolar walls and the epithelial cells lining them become thinner, leading to the formation of definitive alveoli. The mean alveolar diameter increases greatly, from about 30 μm at 30 weeks to about 150 μm at 40 weeks. The final stage of alveolar maturation involves the restructuring of the capillary network, such that the more primitive double capillary network lining the terminal sacs and alveoli is transformed into a single layer of capillaries1 (see Fig. 11.1); this marks the existence of definitive alveoli. By the time of term birth, the human lung contains 20 to 50 million alveoli. An adult human lung contains approximately 300 million alveoli, indicating that most are formed postnatally. The alveolar stage of lung development is thought to continue for at least 1 to 2 years after birth, although some alveoli may continue to be formed later in life. In species born at an earlier stage of development (e.g., rats and mice), the alveolar stage begins after birth; therefore, at birth, gas exchange occurs across terminal sacs. 

Canalicular Stage (16–26 Weeks)

Pulmonary Circulation

During the canalicular stage, the airways widen and lengthen, and mesenchymal tissue surrounding the distal airways becomes attenuated (see Fig. 11.1). This process (canalisation) results in a substantial increase in the ratio of lumen volume to tissue volume. During the canalicular stage, the functional units of the lung are formed, consisting of terminal bronchioles ending with expansions that subsequently form terminal sacs (primitive alveoli). A network of blood capillaries develops around the terminal air sacs, increasing the proximity of blood capillaries to the epithelial surface; this marks the beginning of the air–blood interface that is required for effective gas exchange (see Fig. 11.1). Thus the late canalicular stage is the earliest at which the lungs can support independent life. 

Structural Development

Saccular stage

25–40 wk (term)

Formation of additional airway generations. Dilation of prospec­ tive gas-exchanging airspaces. Maturation of surfactant system.

Alveolar stage

36 wk–18 mo

Start of alveolar formation by outgrowth of secondary septa.

Microvascular maturation

Birth–3 yr

Change from double- to singlecapillary network. Reduction in interstitial tissue mass; fusion of capillaries; preferential growth of single-layered capillary network areas.

Reproduced in modified form, with permission, from Burri P. In: Hanson MA, Spencer JAD, Rodeck CH, Walters DV, eds. Fetus and Neonate. Physiology and Clinical Implications. Cambridge, United Kingdom: Cambridge University Press; 1994:3–19.

  

Terminal Sac Stage (28–40 Weeks) The terminal sac, or saccular, stage of lung development sees a progressive enlargement of the distal ‘air spaces’. This enlargement results from further attenuation of perisaccular mesenchymal tissue and leads to a further increase in luminal volume relative to

The structural development of the pulmonary vasculature has recently been reviewed in detail.2,3 The lung develops with two anatomically and functionally distinct vascular systems: the pulmonary system, which supplies the gas-exchanging tissue (alveoli), and the bronchial system, which perfuses the non–gasexchanging tissue of the lung. The arteries of the bronchial circulation give rise to capillaries which supply the walls of the bronchi and bronchioles but do not extend to the most peripheral gasexchanging parts of the bronchial tree; bronchial venous blood returns via the pulmonary veins because of anastomoses between the bronchiolar and pulmonary veins. The pulmonary arteries develop a muscular wall except near the lung periphery, where the arteries are only partially muscularised. Pulmonary veins show a branching pattern similar to that of arteries but do not follow the arteries and airways; rather, they tend to run at right angles in the mesenchyme. Arterioles are virtually absent in the adult pulmonary circulation; therefore, pulmonary blood flow (PBF) is determined largely

CHAPTER 11  Lung Growth and Maturation

c c

c

c c

c

A

B

c c

c c

c c

C

D •

Fig. 11.1 Diagram showing development of lung parenchyma and its microvasculature. A, Pseudoglandular stage, during which epithelial tubes lined by columnar epithelial cells invade the mesenchyme, which contains a loose network of blood capillaries (C). The remaining panels show further development of structures enclosed by the frame in A. B, Canalicular stage, showing differentiation of ‘airspace’ epithelium and expansion of airspaces resulting in attenuation of mesenchyme; capillaries are rearranged around the epithelial tubes so that walls between ‘airspaces’ contain a double layer of capillaries. A thin ‘air’–blood interface develops, and types I and II epithelial cells become apparent. C, Terminal sac and alveolar stages, showing development of secondary septa from primary septa; septa are primitive in that they contain a double capillary network and a central layer of connective tissue. D, Mature lung, showing thin interalveolar walls containing a single capillary layer. (Reproduced with permission from Burri PH. Fetal and postnatal development of the lung. Ann Rev Physiol. 1984;46:617–628.)

105

106

SE C T I O N 3     Fetal Physiology and Pathology

Section of RBC Blood–gas barrier

Capillary endothelial cell Type I alveolar epithelial cell

Fused basement membrane

Alveolus

Capillary

Nucleus of type I alveolar epithelial cell

Alveolus 1 m

Alveolus

• Fig. 11.2  An electron micrograph of an alveolus and an adjacent capillary, demonstrating the very thin barrier that separates the airspace from the capillary lumen (air–blood barrier). Note that this barrier consists of the attenuated cytoplasm of an alveolar epithelial cell and a capillary endothelial cell, which are separated by their respective basement membranes that have fused (see inset). In this micrograph, the attenuated cytoplasmic extension of the type I cell indicated extends around the entire alveolus. RBC, Red blood cell.

by the resistance of the alveolar capillaries. In fetuses and newborns, however, the smooth muscle surrounding the small pulmonary arteries is thicker than in an adult lung, relative to diameter, and extends farther down the vascular tree. This likely contributes to the high vascular resistance of the fetal lung and may be a consequence of the high fetal pulmonary artery pressure (relative to postnatally). In the first few weeks after birth, the arterial smooth muscle thins, leading to a reduction in arterial wall thickness, likely because of a reduction in pulmonary arterial pressure following the functional separation of the pulmonary and systemic circulations. The creation of an efficient gas exchange surface within the lung depends upon the development of a dense capillary bed in close proximity to the epithelium of the terminal sacs or alveoli. Early in development, capillaries form a loose network adjacent to the immature airspaces, but the two structures are usually separated by other cells and extracellular matrix (ECM) components. During the canalicular stage, the number of capillaries increases greatly, and they come into close contact with the epithelium of the primitive air sacs. By the terminal sac stage, the capillaries form a dense network around these sacs and, with further attenuation of tissue between adjacent sacs, the basement membrane underlying the capillary endothelial cells fuses with the basement membrane underlying the alveolar epithelial cells (Fig. 11.2). The sites of basement membrane fusion are initially focal, but they expand as the lung matures, providing a very thin (∼0.2 μm) barrier for gas exchange. 

Functional Development of the Pulmonary Circulation This topic has recently been reviewed in depth.3 At midgestation, only 3% to 4% of total cardiac output perfuses the lung, and by late gestation, this has risen to only 8% to 10%.3 In a fetus, most (∼88%) of the right ventricular output is diverted away from the lungs to the descending aorta via the ductus arteriosus. Mean pulmonary arterial pressure in a near-term fetus is about 55 mm Hg, which is about 5 mm Hg higher than mean aortic pressure, thereby maintaining flow from the pulmonary to the systemic circulation through the ductus arteriosus. Although PVR declines progressively during fetal life due to a large increase in total crosssectional area of the pulmonary vascular bed, it remains much higher (up to eightfold) just before birth than immediately after birth.3 In fetuses, PVR is influenced by a range of factors, including the physical and oxygen environments of pulmonary vessels and the presence of vasoactive agents. Because this topic has been extensively reviewed,3,4 only a brief outline will be provided here. In fetal sheep, PVR is reduced by vigorous fetal breathing movements (FBMs)5 and is closely related to lung liquid volume.6 Increasing the volume, and hence pressure, of liquid within the terminal air sacs likely compresses the small pulmonary vessels (mainly capillaries), thereby increasing PVR.6 The low Po2 of blood perfusing the fetal lungs also contributes to a high PVR. In postnatal lambs, perfusion of the lungs with blood having a Po2 similar to in fetuses greatly increases PVR.7 In fetal sheep,

CHAPTER 11  Lung Growth and Maturation

A

107

B

• Fig. 11.3  A, Simultaneous phase-contrast x-ray images and angiograms of a newborn rabbit after unilateral ventilation of the right lung, showing that global increases in pulmonary blood flow are independent of lung aeration. The flow of iodine (contrast reagent; black) through vessels is equal in both the aerated right lung and nonaerated left lung. B, Phase-contrast x-ray image of a spontaneously breathing newborn rabbit in which the air–liquid boundary is visible. Complete aeration of the lungs has been achieved (white speckle), down to the most distal gas-exchange regions (inset); single alveoli can be seen when one airway is in projection. The role of inspiration in clearing lung liquid has been demonstrated using this technique, showing that lung liquid can be completely cleared from the airways during the first three to five breaths caused by the transpulmonary hydrostatic pressures generated during inspiration.

lowering and raising the Po2 of arterial blood (i.e., hypoxia and hyperoxia) causes increases and decreases in PVR, respectively.3 The mechanism by which oxygen tension influences PVR is unknown but may involve the release of prostacyclin (PGI2) and endothelium-derived nitric oxide (NO), both of which affect vascular smooth muscle. 

Changes at Birth Lung aeration is the primary trigger for the decrease in PVR at birth, resulting in an 8- to 10-fold increase in PBF.6 Although the increase in PBF is enhanced by increased oxygenation, it is not dependent on an increase in oxygenation8,9 and occurs even if the lungs are ventilated with 100% nitrogen.7 Furthermore, as partial lung aeration leads to a global increase in PBF, increasing equally in both aerated and nonaerated regions (Fig. 11.3), the increase in PBF is not spatially related to aerated lung regions.10 This can lead to a large ventilation/perfusion mismatch in the lung if it only partially aerates at birth, which as argued later, is likely to be more beneficial than harmful for the transitioning infant.

The mechanisms underlying the large fall in PVR at birth are complex and probably involve alterations in the physical environment, the oxygen environment and the balance between vasodilator and vasoconstrictor substances. Because the alveolar epithelium and capillary endothelium are mechanically coupled (see Fig. 11.2), the creation at birth of an air–liquid interface in the alveoli and the partial recoil of the lungs (see later) likely alter the geometry of small pulmonary vessels, causing them to dilate. Rhythmic expansion of the lungs at birth, increased oxygenation and release of vasodilators such as PGI2, bradykinin (a potent vasodilator in the fetus) and NO, are thought to mediate pulmonary vasodilation after birth. Indeed, inhibition of NO synthesis before birth attenuates the birth-induced increase in PBF and results in pulmonary hypertension in the newborn.3 None of the above-mentioned mechanisms can readily explain the global increase in PBF induced by partial lung aeration because the increase occurs equally in aerated and nonaerated lung regions and can be induced by ventilation with 100% N2 (see Fig. 11.3).7,10 Although the mechanism is unknown, it has been suggested that the clearance of airway liquid into the surrounding

108

SE C T I O N 3     Fetal Physiology and Pathology

perialveolar tissue could activate J-receptors which signal via vagal afferents to effect global pulmonary vasodilation.10 The increase in PBF at birth plays a critical role in the cardiovascular transition at birth. Before birth, because PBF is low, pre­ load for the left ventricle is predominantly derived from umbilical venous return, which flows via the ductus venosus, inferior vena cava and foramen ovale to enter the left atrium.11 Thus if the umbilical cord is clamped before the lungs have aerated and PBF has increased, the left ventricle will be deprived of preload until the lungs aerate and PBF increases.12,13 This can cause a sustained decrease in cardiac output (by up to 50%) after birth and places the infant at high risk for systemic hypotension and hypoxic-ischemic brain injury. This is because an increase and redistribution of cardiac output are the primary mechanisms that protect the brain from oxygen deficiency during hypoxic-asphyxic episodes.14 On the other hand, aerating the lung and increasing PBF before the umbilical cord is clamped allows the supply of preload blood to the left ventricle to immediately switch from umbilical venous to pulmonary venous return without any decrease in supply.12 As a result, the characteristic decrease in cardiac output (often evidenced as a decrease in heart rate) is prevented when the onset of ventilation precedes umbilical cord clamping.12 Furthermore, in view of the vital role that a high PBF plays after birth in providing preload for the left ventricle and maintaining cardiac output, it is logical for the increase in PBF to be independent of, and not linked (at least initially), to the degree of lung aeration. 

Fetal Breathing Movements Episodes of breathing-like movements occur intermittently throughout much of gestation in healthy mammalian fetuses.15 These movements, termed ‘fetal breathing movements’, share key features with postnatal breathing and are thought to be an early expression of coordinated, rhythmical activity in the brainstem regions responsible for the control of breathing. FBMs involve rhythmic ‘inspiratory’ activation of the diaphragm and dilator muscles of the larynx.16 Expiratory muscles (e.g., intercostal muscles, upper airway constrictors) are not significantly active during periods of unstimulated FBM. Similar to postnatal breathing, FBMs are stimulated by increased arterial PaCO2 and by decreased pH in the blood and cerebrospinal fluid, probably via stimulation of central chemoreceptors.17 FBMs are also influenced by fetal behavioural states; they principally occur in association with a state resembling rapid eye movement (REM) sleep and are absent during the state resembling quiet (slow-wave or non-REM sleep.15 Whether the fetus is ever in a state of wakefulness is controversial; however, short periods of a fetal state of heightened activity resembling wakefulness (low-voltage electrocortical activity, eye movements, fetal swallowing, head and neck movements) are usually accompanied by FBM. The suppression of FBM during fetal quiet (non-REM) sleep is of considerable interest because this suppression must cease after birth when breathing becomes continuous, regardless of sleep– wake states. It is currently thought that breathing becomes continuous after birth as a result of increased CO2 production and removal from putative inhibitory agents released by the placenta. Fetal breathing movements differ from postnatal breathing in that the ‘tidal volume’ is very small, and each ‘breath’ is essentially isovolumic; the low tidal volume of a fetus can be attributed to the high viscosity (and inertia) of water relative to air, and the high resistance of moving liquid through the respiratory tract.18 In ovine fetuses, the ‘tidal volume’ is normally less than 1 mL19 compared with 40 to 50 mL (8–10 mL/kg) in newborn lambs20;

TABLE Composition of Fetal Lung Liquid in Comparison 11.2 to Fetal Arterial Blood and Amniotic Fluid

Parameter

Arterial Blood

Lung Liquid

Amniotic Fluid

Na+

150 ± 0.7

150 ± 1.3

113 ± 6.5

(mmol/L)

K+

(mmol/L)

4.8 ± 0.2

6.3 ± 0.7

7.6 ± 0.8

Cl–

(mmol/L)

107 ± 1.0

157.1 ± 4.1

87 ± 5

3.3 ± 0.1

0.8 ± 0.1

1.6 ± 0.1

24 ± 1.2

2.8 ± 0.3

19 ± 3

Urea

291 ± 2

7.9 ± 2.7

10.5 ± 2.4

Osmolality (mOsm)

291 ± 2

294 ± 2

265 ± 2

Protein (mg/100 mL)

4090 ± 260

27 ± 2

100 ± 10

pH

7.34 ± 0.04

6.27 ± 0.5

7.02 ± 0.09

Ca2+

(mmol/L)

HCO3−

(mmol/L)

Values are derived from Adamson TM, Boyd RD, Platt HS, Strang LB. Composition of alveolar liquid in the foetal lamb. J Physiol. 1969;204:159–168.

  

a similar difference exists in humans. That is, the change in lung luminal volume associated with individual FBM in late gestation is normally less than 1% of resting (baseline) luminal volume. Thus FBM resemble postnatal breathing with an obstructed upper airway; this presumably gives rise to the paradoxical nature of FBM during which the chest wall retracts during inspiratory efforts and the abdominal wall moves out.21,22 Fetal breathing movements are readily detected by ultrasound and, as a component of the fetal biophysical profile,23 have been used to assess fetal health. The incidence of FBMs is reduced in fetuses that are subjected to intrauterine stresses such as hypoxemia, hypoglycaemia, intrauterine infection, increased levels of prostaglandins and labour.15,24 FBMs may be absent or impaired in fetuses with congenital abnormalities of the nervous system or skeletal muscle. Maternal drug use can also abolish or attenuate FBM; for example, alcohol consumption, tobacco smoking and common sedatives and narcotics can inhibit FBM. Fetal breathing movements are now known to be critical for normal lung development; indeed, the prolonged absence of FBM results in hypoplastic lungs, in which the lungs are small and structurally immature.25,26 If severe, lung hypoplasia can result in respiratory insufficiency or death in the newborn. FBMs play a critical role in maintaining the fetal lungs in an expanded state by opposing the inherent tendency of the fetal lung to recoil. Lung expansion during fetal life is known to be necessary for the normal growth and structural maturation of the lungs; the role of FBMs in maintaining lung expansion is discussed in more detail later. 

Fetal Lung Liquid Control of Fetal Lung Liquid Secretion The future air spaces of the fetal lung contain a liquid that is secreted by the pulmonary epithelium. This unique liquid (fetal lung liquid) is not inhaled amniotic fluid because it accumulates in the lungs when the trachea is obstructed,16 and it has an ionic composition very different to that of amniotic fluid27 (Table 11.2). Measurements of unidirectional ion fluxes in fetal

CHAPTER 11  Lung Growth and Maturation

Control of Fetal Lung Liquid Volume For most of gestation, the fetal lung develops in an expanded state, and the volume of liquid within the future airways increases markedly over the last half of gestation.48 Over the last third of ovine gestation, lung liquid volume typically increases from 25 to 30 mL/kg to 45 to 50 mL/kg near term (145–150 days) (Fig. 11.4). In contrast, the corresponding luminal volume of the air-filled

19

50 Resting lung volume (mL/kg)

sheep lungs indicate that lung liquid secretion results from the net movement of chloride and sodium ions across the pulmonary epithelium into the ‘airway’ lumen.28 This generates a transepithelial osmotic gradient that promotes the movement of water into the lung lumen. It is thought that Na+,K+-ATPase, located on the basolateral surface of pulmonary epithelial cells, provides the electrochemical gradient for Na+ to enter the cell coupled to Cl–. The Cl– then exits the cell across its apical membrane and enters the lung lumen down the transmembrane electrochemical gradient. The net movement of Cl– into the lung lumen provides an electrical gradient for Na+ to enter the lumen as well; together these ionic movements create an osmotic gradient for the movement of water from the cell into the lung lumen.28 After being secreted, fetal lung liquid leaves the lung via the trachea and enters the pharynx, from where it is either swallowed or enters the amniotic sac.29,30 Although there are no data on lung liquid secretion for human fetuses, fetal sheep secrete lung liquid at 3 to 4 mL/kg/h during the last third of gestation before the onset of labour.31 The rate of secretion is controlled by endocrine, metabolic and physical factors. Both adrenaline, acting via β-adrenergic receptors,32,33 and arginine vasopressin34 are potent inhibitors of lung liquid secretion in  vivo, possibly via activation of adenylate cyclase leading to an increase in intracellular cyclic adenosine monophosphate (cAMP) concentrations.35 Other hormones known to affect fetal lung liquid secretion include cortisol and prolactin, both of which increases lung liquid secretion in vivo.36-38 Because lung liquid secretion is an active process, it is inhibited by hypoxaemia (acute or chronic),39,40 an effect which is likely to be mediated by a reduction in oxygen availability or associated changes in tissue pH rather than an increase in circulating adrenaline and vasopressin.33,41,42 Physical factors such as the pulmonary luminal liquid pressure also influence the secretion of fetal lung liquid. A reduction in fetal lung liquid volume, and hence a reduction in luminal pressure, increases lung liquid secretion rates.43-45 In contrast, sustained increases in fetal lung expansion, which increase luminal pressure, lead to either a reduction46 or cessation of lung liquid secretion.5,47 The driving force for the net movement of water across the lung epithelium must be governed by the sum of all forces affecting liquid movement. Under normal conditions, a small hydrostatic pressure exists across the lungs because pressure within the lung lumen is 1 to 2 mm Hg greater than amniotic sac pressure.16 This small pressure gradient likely opposes the osmotic pressure resulting from the movement of chloride ions. The osmotic pressure promoting lung liquid secretion must exceed the opposing hydrostatic pressure for liquid to cross the epithelium into the lung lumen. Thus reductions or increases in the intraluminal pressure, resulting from alterations in lung liquid volume, would be expected to increase or reduce net lung liquid production rates by altering the magnitude of the opposing hydrostatic pressure (i.e., without directly affecting the ionic mechanism of lung liquid secretion). 

109

38 36

40

47 46 36

9 14

30

13 14 14

20 10

0

120 130 140 Gestational age (days)

Birth

10 20 30 Postnatal age (days)

40

•

Fig. 11.4  The volume of fetal lung liquid, measured by dye dilution, during the last 40 days of gestation in chronically catheterised fetal sheep in utero. Measurements of functional residual capacity in postnatal lambs, made using a He-dilution technique, have been included for comparison.20 Numbers represent the number of measurements made at each gestational or postnatal age.

lung (functional residual capacity (FRC)) is 15 to 20 mL/kg immediately after birth in newborn rabbits49 and 20 to 25 mL/kg in newborn lambs at 1 to 2 days of age.20 Thus, during late gestation, the fetal lung is hyperexpanded relative to the postnatal airfilled lung (see Fig. 11.4). The reduction in lung luminal volume (i.e., FRC) at birth is likely due to the formation at birth of an air–liquid interface, and hence surface tension, which constitutes the major part of the postnatal lung’s elastic recoil. Although the presence of surfactant greatly reduces surface tension, it does not eliminate it and, as a consequence, the lung tends to pull away from the chest wall after birth. This increased recoil after birth results in the formation of a subatmospheric pressure in both the intrapleural and the perialveolar interstitial tissue space which is not present in a fetus.13,16 During fetal life, lung liquid volume is largely maintained by factors that control transpulmonary pressure and the efflux of liquid from the lungs rather than by alterations in secretion rate.16 The rate of efflux of lung liquid is controlled by transpulmonary pressure and the resistance of the upper airway, particularly the muscles of the larynx.18,50 During ‘nonbreathing’ periods in the fetus (i.e., during fetal ‘apnea’), tonic activity in the laryngeal constrictor muscles restricts lung liquid efflux and thereby opposes the lung’s elastic recoil (Fig. 11.5). During periods of FBM, the glottis dilates because of phasic activity of the laryngeal dilator muscles, which reduces the resistance to lung liquid efflux. Consequently, the combined effect of a lowered resistance to lung liquid efflux and the lung’s elastic recoil increases liquid loss during periods of FBM.30 However, the efflux of lung liquid during FBM episodes is opposed by simultaneous rhythmic contractions of the diaphragm muscle,43,44 which limit the loss of lung liquid during FBM episodes31 (see Fig. 11.5). Thus the high degree of lung expansion in the fetus is apparently caused by (i) the low level of elastic recoil (owing to the absence of an air–liquid interface), (ii) the high resistance to lung liquid efflux offered by the larynx during non-FBM periods and (iii) contractions of the diaphragm muscle during FBM episodes which oppose lung liquid efflux associated with rhythmic dilation of the glottis and reduced upper airway resistance. 

110

SE C T I O N 3     Fetal Physiology and Pathology

Glottis adducted High resistance to liquid efflux

Intraluminal distending pressure 1–2 mm Hg

Lung liquid

Dilated glottis Low resistance to liquid efflux

Intraluminal pressure –0 mm Hg

Lung liquid

A B • Fig. 11.5  Control of fetal lung liquid volume during (A) periods of apnea and (B) periods of fetal breathing movements (FBMs). During apnea, the glottis is actively adducted, which restricts the efflux of lung liquid and promotes its accumulation within the future airways, thereby maintaining an intraluminal distending pressure of 1 to 2 mm Hg above ambient pressure (i.e., amniotic sac pressure). During periods of FBM, the glottis phasically dilates, which greatly reduces the resistance to lung liquid efflux. As a result, liquid leaves the lungs at a higher rate, causing a reduction in lung liquid volume and the distending pressure (at end-expiration) reduces to ambient pressure.

Clearance of Lung Liquid at Birth Fetal lung liquid must be cleared from the airways at birth so that effective pulmonary gas exchange can be established. This process begins before birth and continues for some time after birth. Studies in fetal sheep show that lung liquid clearance begins with the onset of labour48 and that in healthy fetuses with normal amniotic fluid volumes, much of the liquid is cleared during labour and after birth. It was previously considered that increased circulating concentrations of adrenaline and arginine vasopressin (AVP) in fetal plasma37,38 are primarily responsible for clearing airway liquid by activating amiloride-blockable sodium channels on the luminal surface of pulmonary epithelial cells.51 Activation of these channels increases Na+ and Na+-linked Cl– flux away from the lung lumen into the interstitium, thereby reversing the osmotic gradient across the epithelium and leading to lung liquid reabsorption.51 However, this mechanism is unlikely to be the only mechanism, and recent evidence suggests that it has minimal impact on airway liquid clearance at birth.52 Indeed, any factor that increases transpulmonary pressure can contribute to the loss of liquid during labour. For instance, in unstressed fetal sheep at term, large amounts of lung liquid (∼50%) are lost during the early stages of labour before fetal plasma adrenaline and AVP concentrations increase.48 This reduction in lung liquid volume is probably caused by compression of the lungs resulting from postural changes imposed on the fetus when the uterine muscle shortens during labour; it has been established that fetal trunk flexion causes substantial loss of lung liquid.53 Phase contrast x-ray imaging studies in rabbits have demonstrated that after birth, any liquid remaining in the airways is cleared because of transpulmonary hydrostatic pressures generated during inspiration.52,54 Using this imaging technique, the air–­liquid boundary is clearly visible and was observed to move

through the airways towards the distal gas exchange regions only during inspiration (see Fig. 11.3). During expiration, the air–liquid interface tended to move proximally, resulting in a gradual decline in FRC between breaths.52,54 However, the subsequent inspiration rapidly recleared this liquid, resulting in very rapid lung aeration.54 Indeed, rabbit pups at term were observed to completely clear their airways of liquid within 3 to 5 breaths (∼20 sec), resulting in the rapid formation of an FRC of about 15 mL/kg. Although the liquid was found to clear very rapidly from the airways, clearance from the surrounding perialveolar tissue is much slower (∼4 hours).55 Interestingly, the coexistence of airway liquid residing in the lung tissue and the presence of a gas volume filling the airways immediately after birth results in expansion of the chest wall.52 This finding underlines the importance of having a compliant chest wall at birth. The movement of airway liquid into perialveolar lung tissue after birth is, in itself, known to increase interstitial tissue pressure,55 which must increase the potential for liquid to reenter the airways at FRC. This likely explains the gradual reduction in FRC between breaths in spontaneously breathing newborn rabbits.52,54 However, if the chest wall was less compliant, interstitial tissue pressures resulting from airway liquid clearance would increase to a higher level, which would increase the pressure gradient for liquid to reflood the airways at FRC. 

Lung Growth Regulation of Fetal Lung Growth The degree of lung expansion in the fetus, and hence the degree of lung tissue stretch, plays a critical role in the growth and maturation of the fetal lung.56 Thus most clinical conditions that lead to fetal lung hypoplasia do so by causing a prolonged reduction in

CHAPTER 11  Lung Growth and Maturation

lung expansion.16,31 These include disorders that prevent the fetal lung from expanding (e.g., diaphragmatic hernias, pleural fluid accumulation), cause compression of the fetal chest (e.g., oligohydramnios) and impair fetal skeletal muscle activity or bone (rib cage) development. The critical importance of fetal lung expansion in regulating lung development was first demonstrated by experiments showing profound changes in lung growth and maturation after prolonged, experimentally induced alterations in lung liquid volume. Prolonged drainage of fetal lung liquid, which chronically reduces the degree of lung expansion, causes a cessation of fetal lung growth57 and severe lung hypoplasia.45,58 In contrast, chronically increasing the degree of fetal lung expansion (by obstructing the fetal trachea) markedly increases fetal lung growth and enhances alveolarisation.57-59 The lung growth response to tracheal obstruction is rapid and can cause an almost doubling in total lung cell number within 7 days.57 The growth response, in terms of cell proliferation rates, follows a specific time-course, with maximum rates being detected at 1 to 2 days, which return to control levels by 10 days.47 Thus the processes leading to an acceleration in fetal lung growth after an increase in lung expansion are only active within a relatively narrow window of time. 

Mechanisms Relating Lung Stretch to Lung Growth The cellular and molecular processes mediating changes in fetal lung growth in response to altered lung expansion are localised and apparently restricted to expanded or deflated lung regions.59 This response is not unique to the lung as mechanical forces are well known to affect DNA synthesis as well as the phenotypic expression, via the activation or repression of genes, of many different types of cells.60 However, the mechanisms by which mechanical forces are translated into cellular responses (mechanotransduction) are poorly understood. It is likely that the distortion of lung tissue associated with a change in lung expansion is transmitted via the ECM and causes changes in cell shape or tension within the cytoskeleton. This stimulus may be translated into a cellular response because of direct activation of stretch-sensitive ion channels or to the direct activation of second messenger systems (e.g., tyrosine kinases and phospholipases) associated with the proteins that interlink ECM receptors (e.g., integrins) with the cytoskeleton.61 It is also possible that distortion of a cell may directly activate or inactivate genes or DNA synthesis via changes in tension or orientation of the cytoskeleton or nucleoskeleton.62,63 This may regulate the access of transcription factors to DNA via altering chromatin structure and acetylation of gene promoters.64 It is possible that alterations in gene expression for a variety of growth factors contribute to the pulmonary growth response induced by changes in lung expansion, possibly by integrating and propagating the response. Indeed, alterations in fetal lung expansion induce corresponding changes in insulin-like growth factor-II (IGF-II) gene expression in lung tissue.43,57 IGF-II has potent mitogenic and differentiating activities and is thought to play an important role in fetal growth. Similarly, intermittent stretch of pulmonary epithelial cells in culture is a potent stimulus for pulmonary DNA synthesis65 and increases platelet-derived growth factor (PDGF) gene expression.65 Because the effect of phasic stretch on DNA synthesis in cultured pulmonary epithelial cells can be blocked by antisense oligonucleotides for PDGF, PDGF must play a crucial role in this response.66 However, an

111

in vivo study has failed to demonstrate an increase in PDGF and IGF-II expression when cell proliferation rates are elevated in response to an increase in fetal lung expansion.67 Furthermore, a differential gene expression analysis, designed to identify genes activated and repressed by increases in fetal lung expansion, failed to identify a number of other growth factors thought to mediate expansion-induced fetal lung growth.68 However, numerous other genes were identified which are likely to be activated directly by the mechanical stimulus and play a vital role in this process.68,69 

Fetal Lung Hypoplasia Lung hypoplasia at birth is a graded phenomenon and if severe can increase the risk for neonatal morbidity and mortality.70 Fetal lung hypoplasia has multiple causes, including congenital diaphragmatic hernia (CDH), oligohydramnios, fetal hydrothorax and congenital cystic adenomatoid malformations (CCAM), as well as fetal muscular and skeletal abnormalities.70 It is likely that fetal lung hypoplasia associated with these prenatal conditions has a common mechanism, namely a prolonged reduction in fetal lung expansion. That is, the lung hypoplasia most probably results from the absence of a growth stimulus (i.e., tissue stretch) rather than from an active inhibition of tissue growth. The hypoplastic fetal lung is not simply small but is also structurally immature. For example, hypoplastic lungs contain reduced numbers of airways and alveoli and71,72 have a reduced proportion of airspace, reduced elastin development, narrower airways and altered vascular development.73 The maturation of the respiratory epithelium is impaired, as evidenced by the persistence of cuboidal cells, particularly in the peripheral parts of the acinus.74 This structural immaturity is also associated with a reduced size and effectiveness of the gas-exchange surface, resulting in impaired gas exchange that contributes to the neonatal respiratory compromise. Another important factor that limits gas exchange is increased PVR in hypoplastic lungs; this is likely a result of altered structural and functional development of the pulmonary vascular bed.73 The severity and range of pathological changes in the lungs will depend on the duration and degree of reduced lung expansion,75 and it is likely that some of these will persist into postnatal life owing to the limited capacity for lung regeneration after birth. Because fetal lung growth is so sensitive to alterations in lung expansion, tracheal obstruction has been used therapeutically to reverse lung growth deficits in human fetuses with severe CDH.76,77 In utero, a balloon is deployed into the fetal trachea via ultrasound-guided endoscopy. Trials so far have indicated that fetal endoscopic tracheal occlusion significantly decreases mortality rates in babies born with CDH77-79 via reversal of the severe lung hypoplasia normally seen in these babies. However, the available evidence from animal studies suggests that a number of unwanted side effects need to be considered. In particular, tracheal obstruction leads to altered proportions of epithelial cell types in the terminal airways, resulting in fewer type II cells58,80,81 and hence reduced surfactant protein A, B, and C gene expression.82 For this reason, the balloon placed in the trachea is deflated before delivery, which has been shown to restore type II cell numbers and surfactant protein expression.81 However, more information is needed regarding cardiorespiratory function in newborns with CDH, whether or not they have been exposed to tracheal obstruction in utero, because current recommendations are based upon expert opinion.83 

SE C T I O N 3     Fetal Physiology and Pathology

Lung Maturation Structural Maturation of the Lung The unique architecture of the lung is largely dependent upon its ECM and on cell-to-cell and cell-substrate adhesion properties. Components of the ECM are synthesised by a variety of cell types and together they provide the structural scaffolding that supports the lung cells.84 Consequently, the ECM plays an integral role in lung development from the early in utero stages through to postnatal life. Indeed, different components of the ECM are considered to be critically involved with cell migration, branching morphogenesis, cellular proliferation and cytodifferentiation as well as determining tissue compliance.85 The ECM of the lungs is composed of collagen (principally types I, III, V and VI), elastin, glycoproteins (e.g., fibronectin and laminin) and proteoglycans.85 At the level of the peripheral airways, these components form the epithelial and endothelial basement membranes and the structural fibres that course through the interstitium located at the interalveolar septa; these structural fibres connect with axial fibres running in parallel with major conducting airways and blood vessels and are further braced by fibres projecting in from the pleura. Versican is one of the most abundant proteoglycans located within the perisaccular parenchyma of the developing lung.86 Its high ionic charge density promotes the retention of water within tissue, which contributes to tissue volume and has a major influence on tissue viscoelastic properties. As versican content decreases in parallel with the age-related decrease in the volume of tissue in the peripheral lung during late gestation, a loss of versican from the perialveolar tissue compartment may contribute to the reduction in tissue volume and the thinning of interalveolar walls.86 Thus, major alterations in lung architecture, such as those that occur during development, likely involve remodelling of the ECM. The increase in fetal plasma cortisol concentration before parturition is thought to play an important role in maturing the lung by influencing its architecture, its tissue compliance, development of the vascular bed, differentiation of epithelial cells and the synthesis of surfactant.87 These changes lead to an increase in potential airspace volume, a reduction in gas-diffusing distance and an increase in compliance of the air-filled lung. Indeed, over the last third of gestation in fetal sheep, there is a large increase in lung luminal volume (see Fig. 11.4), which is associated with increased alveolar surface area and reduced interalveolar tissue volume.88 The findings that fetal infusions of corticosteroids accelerate these changes in lung architecture but that fetal adrenalectomy or hypophysectomy (which removes or reduces the source of endogenous fetal corticosteroids) retards them36,37 indicate that corticosteroids are intimately involved in the structural modification of the lung during late gestation. Furthermore, mice with a targeted disruption of the glucocorticoid receptor gene die at birth because of respiratory failure89; their lungs are morphologically immature, and hypercellular with abnormal development of the terminal airways.90,91 Collectively, these findings indicate that a progressive increase in circulating concentrations of fetal corticosteroids plays an important role in promoting structural changes within the lung. This concept is consistent with numerous studies demonstrating that antenatal corticosteroid treatment greatly increases lung compliance92,93 and ventilatory efficiency94 in prematurely delivered fetuses. One mechanism by which corticosteroids stimulate structural maturation of the fetal lung is by inhibiting the proliferation of interstitial mesenchymal cells,90,95,96 which promotes thinning of

Proportion of alveolar epithelial cells %

112

100

Birth

80

Type I AECs Type II AECs Undifferentiated cells %

60 40 20 0 –20 80 d,100 d,120 d,140 d, 2 wk, 8 wk, Gestational age Postnatal age

2 yr

• Fig. 11.6  Changes in the relative proportions of undifferentiated (inverted triangles), type I (blue circles) and type II (yellow circles) alveolar epithelial cells in sheep during the last third of gestation and up to 2 years of age. (Redrawn from Flecknoe SJ, Wallace MJ, Cock ML, et al. Changes in alveolar epithelial cell proportions during fetal and postnatal development in sheep. Am J Physiol Lung Cell Mol Physiol. 2003;285:L664–L670.)

the interstitial tissue and decreases in the thickness of the alveolar wall and the air–blood barrier. Glucocorticoids also promote structural maturation of the lung via remodelling of the ECM.96 The collagen and elastin contents of the lung parenchyma97,98 increase with the exponential increase in circulating cortisol concentrations and lung luminal volumes in the fetal sheep lungs.37 By regulating interstitial cell proliferation and ECM remodelling, corticosteroids are able to influence structural development of the lung and thus its ability to function as a gas exchange organ after birth. 

Epithelial Cell Differentiation The success of pulmonary gas exchange after birth depends on many factors. These include a large surface area for gas exchange, adequate blood flow through alveolar capillaries, a thin air–blood barrier and a high degree of lung compliance. Many of these factors are dependent upon the maturation of pulmonary epithelial cells. During early stages of lung development, epithelial cells are either columnar (pseudoglandular stage) or cuboidal (canalicular stage), are unable to synthesise surfactant and form a thick barrier to gas exchange. During the canalicular stage (by 22–24 weeks), alveolar epithelial cells (AEC) begin to differentiate into thinner type I cells, which have a thin attenuated cytoplasm with a large surface area for gas exchange and cuboidal type II cells which synthesise, store and release pulmonary surfactant.99 During the early stages of lung development, all epithelial cells within the terminal airways are undifferentiated99,100 and gradually differentiate into both cell types as development progresses (Fig. 11.6). Although it is widely considered that both type I and type II cells arise as daughter cells from proliferating type II cells, recent data indicate that this concept may not be correct for the following reasons: (i) in sheep fetuses, type I cells can be detected at an earlier period in gestation than type II cells,88,100 and (ii) increased fetal lung expansion induces type II to type I cell transdifferentiation, but reductions in fetal lung expansion induce type I to type II AEC transdifferentiation81(see Fig. 11.6). The mechanisms that regulate the differentiation of pulmonary epithelial cells are largely unknown but are influenced by corticosteroids,91,101 the degree of fetal lung expansion (i.e., tissue

CHAPTER 11  Lung Growth and Maturation

stress),58,80,81 structural development of the ECM102 and endothelial cells.103 Studies in sheep and mice show that the absence of glucocorticoid signalling increases numbers of type II and undifferentiated epithelial cells and reduces the number of type I cells.91,101 Similar changes are observed in fetal sheep studies after prolonged reductions in lung expansion, which increase the proportion of type II cells and reduce the proportion of type I cells.80 Conversely, increasing the degree of lung expansion reduces the proportion of type II epithelial cells and increases the proportion of type I cells.80 Similar results have been observed in  vitro in lung explants and monolayer cultures of pulmonary epithelial cells exposed to static stretch, which increases markers of type I cells and decreases markers of type II cells.104,105 Given the known role of glucocorticoid signalling in structural maturation of the lungs,89,90,95 it is possible that the absence of glucocorticoid signalling alters AEC differentiation secondary to changes in the structural maturity of the lungs which reduces the ability of the lungs to expand.91 In addition, differentiation of type I cells appears to depend on their close proximity to the developing vascular endothelium.103 

Pulmonary Surfactant Pulmonary surfactant is essential for postnatal lung function because it stabilises alveoli, making the lung easier to expand, thereby decreasing the work of breathing and enhancing gas exchange. Pulmonary surfactant is a complex, surface active mixture of phospholipids and proteins (∼90% lipid and 10% protein) that is synthesised within type II epithelial cells.106 After being secreted, surfactant forms a monolayer of tightly packed lipid molecules at the air–liquid interface of the liquid film that lines the alveoli. This monolayer displaces water molecules from the interface, thereby greatly lowering the surface tension. Surfactant deficiency is common in preterm infants because of immaturity and reduced numbers of type II alveolar epithelial cells. The lack of surfactant can lead to RDS (or hyaline membrane disease), which is characterised by laboured breathing and progressive cyanosis because of inadequate gas exchange.107,108 In preterm infants, the effects of surfactant deficiency may be exacerbated by structural immaturity of the lungs. Antenatal glucocorticoid treatment enhances surfactant

113

synthesis and structural maturation of the lungs,109 both of which are essential for normal lung function after birth. 

Conclusions Survival at birth depends upon the lung being sufficiently large and structurally mature to enable it to immediately take over the critical role of gas exchange. The physiological and pathophysiological processes affecting lung growth and development before birth involve both endocrine and physical factors. The fetal lung is not collapsed, but the ‘airways’ contain a liquid secreted by the lung epithelium; the degree to which this liquid expands the lung is a major determinant of lung tissue growth and maturation. The level of fetal lung expansion is determined by the lung’s physical environment, including intrathoracic space, fetal posture and FBMs. Lung tissue stretch stimulates gene networks, leading to tissue growth and differentiation. Lung hypoplasia results if the fetal lung is chronically underexpanded. • Clearance of liquid from the airways begins with the onset of labour due to postural changes imposed on the fetus causing lung liquid efflux via the nose and mouth, as well as active liquid reabsorption. Lung liquid remaining after birth is cleared as a result of the transpulmonary pressure gradient generated by inspiration. • PBF is generally low during fetal life but can increase transiently with FBMs. At birth, pulmonary vascular resistance markedly decreases, permitting the high PBF essential for adequate gas exchange. • The increase in PBF at birth is triggered by lung aeration, which in turn underpins the cardiovascular transition at birth. With the loss of umbilical venous return, the increase in pulmonary venous return takes over the critical role of supplying preload blood to the left ventricle. • Maturation of the lung in preparation for birth involves ECM remodelling, alveolar epithelial cell differentiation and surfactant production. These changes are driven by mechanical stress on the lung tissue and corticosteroid signalling. Access the complete reference list online at ExpertConsult.com. Self-assessment questions available at ExpertConsult.com.

References

1. Burri PH. Fetal and postnatal development of the lung. Ann Rev Physiol. 1984;46:617–628. 2. Jones R, Reid LM. Development of the pulmonary vasculature. In: Harding R, Pinkerton KE, Plopper CG, eds. The Lung: Development, Aging and the Environment. London: Elsevier Academic Press; 2004:81. 3. Dodson RB, Galambos C, Abman SH. Developmental physiology of the pulmonary circulation. In: Harding R, Pinkerton KE, Plopper CG, eds. The Lung: Development, Aging and the Environment. London: Elsevier Academic Press; 2004:121–136. 4. Tod ML, Cassin S. In: Crystal RG, West DB, Weibel ER, Barnes PJ, eds. The Lung: Scientific Foundations. Philadelphia: Lippincott-Raven; 1997:2129. 5. Polglase GR, Wallace MJ, Grant DA, Hooper SB. Influence of fetal breathing movements on pulmonary hemodynamics in fetal sheep. Pediatr Res. 2004;56:932–938. 6. Hooper SB. Role of luminal volume changes in the increase in pulmonary blood flow at birth in sheep. Exp Physiol. 1998;83:833–842. 7. Lang JA, Pearson JT, Binder-Hesch C, et  al. Increase in pulmonary blood flow at birth: role of oxygen and lung aeration. J Physiol. 2016;594:1389–1398. 8. Sobotka KS, Hooper SB, Allison BJ, et al. An initial sustained inflation improves the respiratory and cardiovascular transition at birth in preterm lambs. Pediatr Res. 2011;70:56–60. 9. Teitel DF, Iwamoto HS, Rudolph AM. Changes in the pulmonary circulation during birth-related events. Pediatr Res. 1990;27: 372–378. 10. Lang JA, Pearson JT, te Pas AB, et al. Ventilation/perfusion mismatch during lung aeration at birth. J Appl Physiol. 2014;117:535–543. 11. Rudolph AM. Distribution and regulation of blood flow in the fetal and neonatal lamb. Circ Res. 1985;57:811–821. 12.  Bhatt S, Alison BJ, Wallace EM, et al. Delaying cord clamping until ventilation onset improves cardiovascular function at birth in preterm lambs. J Physiol. 2013;591:2113–2126. 13. Crossley KJ, Allison BJ, Polglase GR, et  al. Dynamic changes in the direction of blood flow through the ductus arteriosus at birth. J Physiol. 2009;587:4695–4704. 14. Bocking AD, Gagnon R, White SE, et  al. Circulatory responses to prolonged hypoxemia in fetal sheep. Am J Obstet Gynecol. 1988;159:1418–1424. 15. Harding R. In: Crystal RG, West DB, Weibel ER, Barnes PJ, eds. The Lung: Scientific Foundations. Philadelphia: Lippincott-Raven; 1997:2093. 16.  Harding R, Hooper SB. Regulation of lung expansion and lung growth before birth. J Appl Physiol. 1996;81:209–224. 17. Moore PJ, Hanson MA. Control of breathing: central influences. In: Hanson MA, Spencer JAD, Rodeck CH, Walters DV, eds. Fetus and Neonate: Breathing. Cambridge, United Kingdom: Cambridge University Press; 1994:109. 18.  Harding R, Bocking AD, Sigger JN. Influence of upper respiratory tract on liquid flow to and from fetal lungs. J Appl Physiol. 1986;61:68–74. 19. Maloney JE, Adamson TM, Brodecky AV, et  al. Diaphragmatic activity and lung liquid flow in the unanesthetized fetal sheep. J Appl Physiol. 1975;39:423–428.

20. Jakubowska AE, Billings K, Johns DP, et  al. Respiratory function in lambs after prolonged oligohydramnios during late gestation. Pediatr Res. 1993;34:611–617. 21.  Harding R, Liggins GC. Changes in thoracic dimensions induced by breathing movements in fetal sheep. Reprod Fertil Dev. 1996;8:117–124. 22. Patrick J, Natale R, Richardson B. Patterns of human fetal breathing activity at 34 to 35 weeks’ gestational age. Am J Obstet Gynecol. 1978;132:507–513. 23. Bocking AD, Gagnon R. In: Thorburn GD, Harding R, eds. Textbook of Fetal Physiology. Oxford, United Kingdom: Oxford University Press; 1994:186. 24. Hooper SB, Harding R. In: Harding R, Bocking AD, eds. Fetal Growth and Development. Cambridge, United Kingdom: Cambridge University Press; 2001:114. 25. Liggins GC, Vilos GA, Campos GA, et  al. The effect of spinal cord transection on lung development in fetal sheep. J Dev Physiol. 1981;3:267–274. 26. Wigglesworth JS, Desai R. Effect on lung growth of cervical cord section in the rabbit fetus. Early Hum Dev. 1979;3:51–65. 27.  Adamson TM, Boyd RD, Platt HS, Strang LB. Composition of alveolar liquid in the foetal lamb. J Physiol. 1969;204:159–168. 28. Olver RE, Strang LB. Ion fluxes across the pulmonary epithelium and the secretion of lung liquid in the foetal lamb. J Physiol. 1974;241:327–357. 29. Brace RA, Wlodek ME, McCrabb GJ, Harding R. Swallowing and urine flow responses of ovine fetuses to 24 h of hypoxia. Am J Physiol. 1994;266:R1345–R1352. 30. Harding R, Sigger JN, Wickham PJ, Bocking AD. The regulation of flow of pulmonary fluid in fetal sheep. Respir Physiol. 1984;57: 47–59. 31. Hooper SB, Harding R. Fetal lung liquid: a major determinant of the growth and functional development of the fetal lung. Clin Exp Pharmacol Physiol. 1995;22:235–247. 32. Brown MJ, Olver R, Ramsden CA, et al. Effects of adrenaline and of spontaneous labour on the secretion and absorption of lung liquid in the fetal lamb. J Physiol. 1983;344:137–152. 33. Hooper SB, Harding R. Effect of betaadrenergic blockade on lung liquid secretion during fetal asphyxia. Am J Physiol. 1989;257: R705–R710. 34. Wallace MJ, Hooper SB, Harding R. Regulation of lung liquid secretion by arginine vasopressin in fetal sheep. Am J Physiol. 1990;258:R104–R111. 35. Walters DV, Ramsden CA, Olver RE. Dibutyryl cAMP induces a gestation-dependent absorption of fetal lung liquid. J Appl Physiol. 1990;68:2054–2059. 36. Wallace MJ, Hooper SB, Harding R. Effects of elevated fetal cortisol concentrations on the volume, secretion, and reabsorption of lung liquid. Am J Physiol. 1995;269:R881– R887. 37. Wallace MJ, Hooper SB, Harding R. Role of the adrenal glands in the maturation of lung liquid secretory mechanisms in fetal sheep. Am J Physiol. 1996;270:R33–R40. 38.  Cassin S, Perks AM. Studies of factors which stimulate lung fluid secretion in fetal goats. J Dev Physiol. 1982;4:311–325. 39. Hooper SB, Dickson KA, Harding R. Lung liquid secretion, flow and volume in response

to moderate asphyxia in fetal sheep. J Dev Physiol. 1988;10:473–485. 40. Hooper SB, Harding R. Changes in lung liquid dynamics induced by prolonged fetal hypoxemia. J Appl Physiol. 1990;69:127–135. 41. Hooper SB, Wallace MJ, Harding R. Amiloride blocks the inhibition of fetal lung liquid secretion caused by AVP but not by asphyxia. J Appl Physiol. 1993;74:111–115. 42. Wallace MJ, Hooper SB, McCrabb GJ, Harding R. Acidaemia enhances the inhibitory effect of hypoxia on fetal lung liquid secretion in sheep. Reprod Fertil Dev. 1996;8: 327–333. 43. Harding R, Hooper SB, Han VK. Abolition of fetal breathing movements by spinal cord transection leads to reductions in fetal lung liquid volume, lung growth, and IGF-II gene expression. Pediatr Res. 1993;34:148–153. 44. Miller AA, Hooper SB, Harding R. Role of fetal breathing movements in control of fetal lung distension. J Appl Physiol. 1993;75: 2711–2717. 45. Nardo L, Hooper SB, Harding R. Lung hypoplasia can be reversed by short-term obstruction of the trachea in fetal sheep. Pediatr Res. 1995;38:690–696. 46. Perks AM, Cassin S. The rate of production of lung liquid in fetal goats, and the effect of expansion of the lungs. J Dev Physiol. 1985;7:149–160. 47. Nardo L, Hooper SB, Harding R. Stimulation of lung growth by tracheal obstruction in fetal sheep: relation to luminal pressure and lung liquid volume. Pediatr Res. 1998;43: 184–190. 48. Lines A, Hooper SB, Harding R. Lung liquid production rates and volumes do not decrease before labor in healthy fetal sheep. J Appl Physiol. 1997;82:927–932. 49. Siew ML, Te Pas AB, Wallace MJ, et al. Positive end-expiratory pressure enhances development of a functional residual capacity in preterm rabbits ventilated from birth. J Appl Physiol. 2009;106:1487–1493. 50. Harding R, Bocking AD, Sigger JN. Upper airway resistances in fetal sheep: the influence of breathing activity. J Appl Physiol. 1986;60: 160–165. 51. Olver RE, Ramsden CA, Strang LB, Walters DV. The role of amiloride-blockable sodium transport in adrenaline-induced lung liquid reabsorption in the fetal lamb. J Physiol. 1986;376:321–340. 52. Hooper SB, Kitchen MJ, Siew ML, et  al. Imaging lung aeration and lung liquid clearance at birth. FASEB J. 2007;21:3329–3337. 53. Harding R, Hooper SB, Dickson KA. A mechanism leading to reduced lung expansion and lung hypoplasia in fetal sheep during oligohydramnios. Am J Obstet Gynecol. 1990;163:1904–1913. 54. Siew ML, Wallace MJ, Kitchen MJ, et  al. Inspiration regulates the rate and temporal pattern of lung liquid clearance and lung aeration at birth. J Appl Physiol. 2009;106:1888–1895. 55. Miserocchi G, Poskurica BH, Del Fabbro M. Pulmonary interstitial pressure in anesthetized paralyzed newborn rabbits. J Appl Physiol. 1994;77:2260–2268. 56.  Hooper SB, Wallace MJ. Role of the physicochemical environment in lung development. Clin Exp Pharmacol Physiol. 2006;33:273–279. 57. Hooper SB, Han VK, Harding R. Changes in lung expansion alter pulmonary DNA

113.e1

113.e2

References

synthesis and IGF-II gene expression in fetal sheep. Am J Physiol. 1993;265:L403–L409. 58.  Alcorn D, Adamson TM, Lambert TF, et  al. Morphological effects of chronic tracheal ligation and drainage in the fetal lamb lung. J Anat. 1977;123:649–660. 59. Moessinger AC, Harding R, Adamson TM, et  al. Role of lung fluid volume in growth and maturation of the fetal sheep lung. J Clin Invest. 1990;86:1270–1277. 60. Vandenburgh HH. Mechanical forces and their second messengers in stimulating cell growth in vitro. Am J Physiol. 1992;262:R350– R355. 61. Ingber DE. Cellular mechanotransduction: putting all the pieces together again. FASEB J. 2006;20:811–827. 62. Dahl KN, Ribeiro AJ, Lammerding J. Nuclear shape, mechanics, and mechanotransduction. Circ Res. 2008;102:1307–1318. 63. Pajerowski JD, Dahl KN, Zhong FL, et al. Physical plasticity of the nucleus in stem cell differentiation. Proc Natl Acad Sci U S A. 2007;104: 15619–15624. 64. Lelievre SA. Contributions of extracellular matrix signaling and tissue architecture to nuclear mechanisms and spatial organization of gene expression control. Biochim Biophys Acta. 2009;1790:925–935. 65. Liu M, Xu J, Liu J, et al. Mechanical strainenhanced fetal lung cell proliferation is mediated by phospholipase C and D and protein kinase C. Am J Physiol. 1995;268:L729–L738. 66. Liu M, Liu J, Buch S, et al. Antisense oligonucleotides for PDGF-B and its receptor inhibit mechanical strain-induced fetal lung cell growth. Am J Physiol. 1995;269:L178–L184. 67. Wallace MJ, Thiel AM, Lines AM, et al. Role of platelet-derived growth factor-B, vascular endothelial growth factor, insulin-like growth factor-II, mitogen-activated protein kinase and transforming growth factor-beta1 in expansion-induced lung growth in fetal sheep. Reprod Fertil Dev. 2006;18:655–665. 68. Sozo F, Wallace MJ, Zahra VA, et  al. Gene expression profiling during increased fetal lung expansion identifies genes likely to regulate development of the distal airways. Physiol Genomics. 2006;24:105–113. 69. McDougall AR, Hooper SB, Zahra VA, et al. The oncogene Trop2 regulates fetal lung cell proliferation. Am J Physiol Lung Cell Mol Physiol. 2011;301:L478–L489. 70. Harding R, Albuquerque C. In: Gaultier C, Bourbon J, Post M, eds. Lung Development. Oxford, United Kingdom: Oxford University Press; 1999:364–394. 71. Askenazi SS, Perlman M. Pulmonary hypoplasia: lung weight and radial alveolar count as criteria of diagnosis. Arch Dis Child. 1979;54:614–618. 72. Thibeault DW, Beatty Jr EC, Hall RT, et al. Neonatal pulmonary hypoplasia with premature rupture of fetal membranes and oligohydramnios. J Pediatrics. 1985;107:273–277. 73. Suzuki K, Hooper SB, Cock ML, Harding R. Effect of lung hypoplasia on birth-related changes in the pulmonary circulation in sheep. Pediatr Res. 2005;57:530–536. 74. Wigglesworth JS. Pathology of the lung in the fetus and neonate, with particular reference to problems of growth and maturation. Histopathology. 1987;11:671–689. 75. Moessinger AC, Collins MH, Blanc WA, et al. Oligohydramnios-induced lung hypoplasia:

the influence of timing and duration in gestation. Pediatr Res. 1986;20:951–954. 76. Deprest JA, Nicolaides K, Gratacos E. Fetal surgery for congenital diaphragmatic hernia is back from never gone. Fetal Diag Ther. 2011;29:6–17. 77.  Al-Maary J, Eastwood MP, Russo FM, et  al. Fetal tracheal occlusion for severe pulmonary hypoplasia in isolated congenital diaphragmatic hernia: a systematic review and meta-analysis of survival. Ann Surg. 2016;264(6):929–933. 78. Ruano R, Ali RA, Patel P, et  al. Fetal endoscopic tracheal occlusion for congenital diaphragmatic hernia: indications, outcomes, and future directions. Obstet Gynecol Survey. 2014;69:147–158. 79.  Ruano R, da Silva MM, Campos JA, et al. Fetal pulmonary response after fetoscopic tracheal occlusion for severe isolated congenital diaphragmatic hernia. Obstet Gynecol. 2012;119:93–101. 80. Flecknoe S, Harding R, Maritz G, Hooper SB. Increased lung expansion alters the proportions of type I and type II alveolar epithelial cells in fetal sheep. Am J Physiol Lung Cell Mol Physiol. 2000;278:L1180–L1185. 81.  Flecknoe SJ, Wallace MJ, Harding R, Hooper SB. Determination of alveolar epithelial cell phenotypes in fetal sheep: evidence for the involvement of basal lung expansion. J Physiol. 2002;542: 245–253. 82. Lines A, Nardo L, Phillips ID, et  al. Alterations in lung expansion affect surfactant protein A, B, and C mRNA levels in fetal sheep. Am J Physiol. 1999;276:L239–L245. 83. Snoek KG, Reiss IK, Greenough A, et  al. CDH EURO Consortium. Standardized Postnatal Management of Infants with Congenital Diaphragmatic Hernia in Europe: the CDH EURO Consortium Consensus—2015 update. Neonatology. 2016;110:66–74. 84. Schellenberg JC. In: Johnston BM, Gluckman PD, eds. Respiratory Control and Lung Development in the Fetus and Newborn. Ithaca, NY: Perinatology Press; 1986:3–62. 85. Mizikova I, Morty RE. The extracellular matrix in bronchopulmonary dysplasia: target and source. Front Med (Lausanne). 2015;2:91. 86. Faggian J, Fosang AJ, Zieba M, et al. Changes in versican and chondroitin sulfate proteoglycans during structural development of the lung. Am J Physiol Regul Integr Comp Physiol. 2007;293:R784–R792. 87. Hillman NH, Kallapur SG, Jobe AH. Physiology of transition from intrauterine to extrauterine life. Clin Perinatol. 2012;39:769–783. 88. Alcorn DG, Adamson TM, Maloney J, Robinson PM. A morphologic and morphometric analysis of fetal lung development in the sheep. Anat Rec. 1981;201:655–667. 89. Cole TJ, Blendy JA, Monaghan AP, et  al. Molecular genetic analysis of glucocorticoid signaling during mouse development. Steroids. 1995;60:93–96. 90. Bird AD, Tan KH, Olsson PF, et al. Identification of glucocorticoid-regulated genes that control cell proliferation during murine respiratory development. J Physiol. 2007;585:187–201. 91. Cole TJ, Solomon NM, Van Driel R, et  al. Altered epithelial cell proportions in the fetal lung of glucocorticoid receptor null mice. Am J Respir Cell Mol Biol. 2004;30:613–619. 92. Jobe AH, Polk D, Ikegami M, et  al. Lung responses to ultrasound-guided fetal treatments with corticosteroids in preterm lambs. J Appl Physiol. 1993;75:2099–2105.

93. Liggins GC, Schellenberg JC, Finberg K, et al. The effects of ACTH1-24 or cortisol on pulmonary maturation in the adrenalectomized ovine fetus. J Dev Physiol. 1985;7:105–111. 94. Polk DH, Ikegami M, Jobe AH, et  al. Postnatal lung function in preterm lambs: effects of a single exposure to betamethasone and thyroid hormones. Am J Obstet Gynecol. 1995;172:872–881. 95. Bird AD, Choo YL, Hooper SB, et  al. Mesenchymal glucocorticoid receptor regulates development of multiple cell layers of the mouse lung. Am J Respir Cell Mol Biol. 2014;50(2):419–428. 96. Habermehl D, Parkitna JR, Kaden S, et  al. Glucocorticoid activity during lung maturation is essential in mesenchymal and less in alveolar epithelial cells. Mol Endocrinol. 2011;25:1280–1288. 97. Schellenberg JC, Liggins GC. Elastin and collagen in the fetal sheep lung. I. Ontogenesis. Pediatr Res. 1987;22:335–338. 98. Shibahara SU, Davidson JM, Smith K, Crystal RG. Modulation of tropoelastin production and elastin messenger ribonucleic acid activity in developing sheep lung. Biochemistry. 1981;20:6577–6584. 99. Possmayer F. In: Jones CT, ed. The Biochemical Development of the Fetus and Neonate. St. Louis: Elsevier; 1982:337–391. 100. Flecknoe SJ, Wallace MJ, Cock ML, et  al. Changes in alveolar epithelial cell proportions during fetal and postnatal development in sheep. Am J Physiol Lung Cell Mol Physiol. 2003;285:L664–L670. 101. Crone RK, Davies P, Liggins GC, Reid L. The effects of hypophysectomy, thyroidectomy, and postoperative infusion of cortisol or adrenocorticotrophin on the structure of the ovine fetal lung. J Dev Physiol. 1983;5:281–288. 102. Honda T, Ishida K, Hayama M, et al. Type II pneumocytes are preferentially located along thick elastic fibers forming the framework of human alveoli. Anat Rec. 2000;258:34–38. 103. Hermanns MI, Fuchs S, Bock M, et al. Primary human coculture model of alveolo-capillary unit to study mechanisms of injury to peripheral lung. Cell Tissue Res. 2009;336:91–105. 104. Foster CD, Varghese LS, Skalina RB, et  al. In  vitro transdifferentiation of human fetal type II cells toward a type I-like cell. Pediatr Res. 2007;61:404–409. 105. Gutierrez JA, Ertsey R, Scavo LM, et  al. Mechanical distention modulates alveolar epithelial cell phenotypic expression by transcriptional regulation. Am J Respir Cell Mol Biol. 1999;21:223–229. 106. Orgeig S, Morrison JL, Sullivan LC, Daniels CB. In: Harding R, Pinkerton KE, eds. The Lung: Development, Aging and the Environment. London: Academic Press; 2015:183–210. 107. Whitsett J. In: Crystal RG, West DB, Weibel ER, Barnes PJ, eds. The Lung: Scientific Foundations. Philadelphia: LippincottRaven; 1997:2167. 108. Jobe AH, Ikegami M. Lung development and function in preterm infants in the surfactant treatment era. Ann Rev Physiol. 2000;62:825–846. 109. Liggins GC, Schellenberg JC. In: Johnston BM, Gluckman PD, eds. Respiratory Control and Lung Development in the Fetus and Newborn. Ithaca, NY: Perinatology Press; 1986:107–133.

12

Development of the Kidneys and Urinary Tract in Relation to Renal Anomalies PAUL J.D. WINYARD

KEY POINTS • D  evelopment of the kidneys (nephrogenesis) occurs between the 5th and 32nd weeks of human gestation when the ureteric bud interacts with metanephric mesenchyme, which undergoes mesenchymal–epithelial conversion to form glomeruli and tubules and renal stroma, with coordinated vascular development and signalling being critical. • Nephron number is the major factor determining long-term kidney function. The development of the nephrons is finalised by the 32nd week; the average nephron number is about 900,000 per kidney; smaller babies with fewer nephrons have an increased long-term risk for hypertension and kidney failure. • There is an increased risk for hypertension in ex-premature children and young people, with a possible renal link to steroid use. • Congenital anomalies of the kidney and urinary tract (CAKUTs), such as aberrant renal development and urinary tract obstruction, have the potential to decrease the number of nephrons, and postnatal processes such as cyst formation, inflammation or infection can have similar effects on renal function by destroying mature nephrons. • There are several known causes of CAKUTs, including genetic defects; urinary tract obstruction; and maternal environment, diet and teratogens, although most CAKUT remains unexplained.

hypoplasia. Potter described this sequence with bilateral renal agenesis but other causes include bilateral multicystic, dysplastic or polycystic kidneys or lower urinary tract obstruction with posterior urethral valves or urethral atresia, all of which represent the severe end of the spectrum of congenital anomalies of the kidney and urinary tract (CAKUTs). Urine is produced in the kidneys by nephrons, with filtration of blood in the glomerulus, modification of the filtrate as it passes through tubules, loop of Henle and collecting duct, before transition through the renal pelvis into the ureters. Nephron number is determined by the 32nd week of gestation, by which point the kidneys can regulate fluid balance, electrolytes and acid–base balance. However, full renal function does not develop until birth, when renal blood flow increases, and then postnatally as nephrons elongate and mature. The fetal kidneys only receive around 3% to 5% of cardiac output compared with around 20% for the mature organs, and nephrons lack many specialised transporters in early developmental stages. Moreover, only dilute urine can be produced because the medulla is relatively small, and there is reduced aquaporin expression, which prevents development of a full medullary osmotic gradient and reabsorption of water, respectively. Such renal immaturity is unimportant if the mother has normal renal function because the placenta is an efficient biological dialysis machine to balance fetal biochemistry. This should be taken into consideration when considering early delivery of fetuses with renal dysfunction because it is much easier to dialyse a 3-kg rather than a 1.5-kg baby even without factoring in increased risk for respiratory and other prematurity-related problems. 

Introduction

Timeline of Kidney Development

Kidneys that produce urine and a lower urinary tract that permits urine flow into the amniotic fluid are essential for normal human in utero development. Kidneys generate urine from around the 12th week of gestation, which comprises the majority of the amniotic fluid from the second trimester and more than 90% by late gestation. Failure to either generate enough urine or expel it into the amniotic sac causes the eponymous ‘Potter sequence’ of severe oligohydramnios with limb and craniofacial malformations, such as clubbed feet, contractures, a flattened ‘parrot-beak’ nose, a recessed chin and low-set ears, accompanied by pulmonary

Humans pass through three stages of renal development during nephrogenesis: the pronephros, mesonephros and metanephros, which arise sequentially on the dorsal body wall.1 Hence, those with normal development will have had six distinct kidneys before birth, with excretory function improving significantly at each stage. Whereas the pronephros and mesonephros regress and disappear in the fetus, the metanephros matures into the fully functioning definitive kidney. The pronephros is the functioning kidney of adult hagfish and some amphibians, as is the mesonephros in adult lampreys, some fishes and amphibians. Conservation of

114

CHAPTER 12  Development of the Kidneys and Urinary Tract in Relation to Renal Anomalies

TABLE Comparative Timing of Human and Mouse 12.1 Nephrogenesisa

Human (Postconception Days Unless Stated)

Mouse (Postconception Days)

Appears

22

9

Regresses

25

10

Appears

24

10

Regresses

Structure Pronephros

Mesonephros

16 wk

14

Metanephros

32

11.5

First glomeruli

8 wk

14

End of nephrogenesis

32 wk

7 after birth

Length of gestation

40 wk

20

aRats’

  

timing is about 1 day longer or later than mice.

gene function across species means that valuable information pertinent to human development can still be gleaned from these different stages in animals; many recent investigations, for example, involve functional experiments in zebrafish larvae which have a pronephros containing just two glomeruli.2 The timing of key events in human kidney development is outlined in Table 12.1. The equivalent stages are also listed for mice, the most frequently used models of nephrogenesis. There is a distinct difference in later stages, however, because murine nephrogenesis continues after birth; this allows experimental surgical and pharmacologic interventions, but extrapolation of results may not always be applicable to humans, in whom nephrogenesis completes in the protected in utero environment (unless born prematurely).

The Pronephros The human pronephros is first visible at the 10-somite stage, around 22 days postconception, which is morphologically equivalent to E9 in mice.1 It comprises a small group of nephrotomes with segmental condensations, grooves and vesicles between the second and sixth somites. Extrapolation from animal studies suggest that the pronephros does filter fluid, although human data are lacking. The pronephric duct develops from the intermediate mesoderm lateral to the notochord adjacent to the ninth somite. The duct elongates caudally and reaches the cloaca by day 26. It is renamed the mesonephric, or Wolffian duct, as mesonephric tubules develop. The nephrotomes and pronephric part of the duct involute and cannot be identified by day 25. 

The Mesonephros In humans, the long sausage-shaped mesonephros develops from around 24 days postconception with a duct that grows in a caudal direction connected to adjacent tubules. Mesonephric tubules originate from intermediate mesoderm medial to the duct by ‘mesenchymal–epithelial’ transformation, a process which is subsequently reiterated during nephron formation in metanephric development. In humans, a total of around 40 mesonephric

Epithelium

115

Mesenchyme

Mesenchyme Collecting ducts

Early parts of nephron

Stromal differentiation

Normal, fully developed kidney

•

Fig. 12.1  Cellular model of normal kidney development. Note that vascular development occurs concurrently via a combination of migration and in situ differentiation

tubules are produced (several per somite), but the cranial tubules regress at the same time as caudal ones are forming; hence, there are maximum of around 30 pairs at any time. Each mesonephric ‘nephron’ has a medial cup-shaped sac encasing a knot of capillaries, functionally equivalent to Bowman’s capsule and glomerulus of the mature kidney. This connects to segments of the tubule that histologically resemble mature proximal and distal tubules but lack a loop of Henle. The human mesonephros is thought to produce small quantities of urine between weeks 6 and 10 that drains via the mesonephric duct, but again there is little direct evidence for this and much is extrapolated from sheep and cattle.3 The mouse metanephros organ is rudimentary and has poorly differentiated glomeruli. The mesonephros disappears by 16 weeks, except in male fetuses, in whom the proximal segments of some caudal mesonephric tubules contribute to the efferent ducts of the epididymis whilst the mesonephric duct is incorporated into ductular parts of the epididymis, the seminal vesicle and ejaculatory duct. 

The Metanephros The adult human kidney develops from the metanephros, which consists of two major cell types at its inception: the epithelial cells of the ureteric bud and the mesenchymal cells of the metanephric mesenchyme. A series of reciprocal interactions between epithelia and mesenchymal cells cause the ureteric bud to branch sequentially to form the ureter, renal pelvis, calyces and collecting tubules whilst the mesenchyme has a more varied fate; most focus has been on the portion of mesenchyme that undergoes epithelial conversion into nephrons, but other mesenchymal cells differentiate into interstitial cells or stroma in the mature kidney (Fig. 12.1). Threeway induction between epithelia, tubular- and stromal-progenitor mesenchyme appears to be of importance in generating a normal kidney.4,5 Metanephric kidney development starts by day 28 in humans, when the ureteric bud sprouts from the distal part of the mesonephric duct. By day 32, the tip (ampulla) of the bud penetrates the metanephric blastema, a specialised area of sacral intermediate mesenchyme, and this condenses around the growing ampulla to generate the metanephros. The first glomeruli form by 8 weeks, and nephrogenesis continues in the outer rim of the cortex until somewhere between the 32nd and 36th week; this was originally suggested to continue longer,6 but more recent studies suggest 32 weeks.7,8 Many of the nephrons generated in early weeks of nephrogenesis only have transient function before

116

SE C T I O N 3     Fetal Physiology and Pathology

being lost by apoptosis during increase in kidney size9; effectively, their initial ‘cortical’ position is overrun and remodelled as medulla growth expands outwards. In mice, the ureteric bud enters the metanephric mesenchyme by embryonic day 10.5, the first glomeruli form by embryonic day 14 and nephrogenesis continues for up to 1 week after birth10 (earlier reports incorrectly state 14 days). New nephrons are never generated after completion of nephrogenesis, although strategies to restart nephron formation are actively being researched as an obvious treatment for both developmental and acquired kidney diseases.11 Kidney development is not complete at this point, however, because nephron segments elongate, the medulla expands and segment-specific differentiation continues, whilst blood supply increases, and glomerular filtration rate (GFR) increases over the first 18 months of life. 

The Ureteric Bud and Collecting Duct Lineage The ureteric bud grows into the metanephric blastema and the adjacent mesenchymal cells begin to condense around its tip. Signalling from the mesenchyme stimulates bifurcation of the ampulla to form a ‘t-shape’, and this process of growth and branching occurs repeatedly during nephrogenesis to generate a tree-like collecting duct system. Around 20 rounds of branching occur in humans, double that of mice.12 The bud tips connect to the distal part of developing nephrons while more central parts differentiate into collecting ducts that drain successively into minor calyces, major calyces, the renal pelvis and then the ureter.6 

Differentiation of the Mesenchyme Renal stroma differentiates from one subset of the metanephric mesenchyme whilst nephrons are induced in mesenchymal cells adjacent to each ampullary tip of the ureteric bud. The mesenchyme is initially loosely arranged, but the cells destined to become nephrons grow closely together and compact or condense around the bud tips before undergoing phenotypic transformation into epithelial renal vesicles. Each vesicle elongates to form a comma shape, which folds back on itself to become an S-shaped body.13 The proximal S-shape develops into the glomerulus whilst the distal portion elongates and differentiates into all nephron segments from proximal convoluted tubule to distal convoluted tubule. It was suggested that sequential phases of nephron formation occurred with an initial bud branch–to–nephron ratio of 1 to 1, and then several branches per bud in an arcade extending outwards as the kidney grows and finally the terminal bud branch attached to as many as five nephrons.6 This theory was based on postmortem dissection rather than repeated, quantitative studies, but it appears plausible with recent mouse work also suggesting several distinct phases.14 

Development of the Vasculature Blood is supplied to the kidneys via the renal arteries, which give rise to distinct microcirculations in glomerular capillaries, cortical vessels and vasa rectae which pass alongside loops of Henle into the medulla. Renal vessels develop from a combination of vasculogenesis, in which mesenchyme differentiates in situ to form capillary endothelia, and angiogenesis, which involves ingrowth of existing capillaries.15 Renal vascular resistance is high during fetal life, predominantly controlled via the renin–angiotensin system

(RAS) and renal nerves, so the kidneys only receive 3% to 5% of cardiac output.16 Measurement of GFR is contentious in neonates but appears low at birth at around 20 mL/min/1.73 m2 in term infants (or 15 mL/min/1.73 m2 in infants with low birth weight).17 Overall renal blood flow rises as systemic blood pressure increases and renal resistance falls after birth with the kidneys receiving 10% of the cardiac output by the end of the first postnatal week. GFR increases two- to threefold during the first month of life but does not reach adult levels until 18 months to 2 years of age.18 

Final Nephron Number Final nephron number is important because this determines renal function into adulthood, before GFR starts to decline from around age 40 onwards.19 However, because the kidneys have the ability to adapt and compensate, it is possible for individuals with a wide range of nephron numbers to appear to have the same level of renal function as assessed by plasma creatinine or estimated GFR. The search for an accurate estimate of nephron number has evolved through many different techniques in the past 25 years with a broad range suggested of 0.6 to 1.3 million per kidney.20-22 The current gold-standard counting method is unbiased stereology, but this is time consuming and expensive and, more important, only possible when the kidney is dissected.23 Magnetic resonance imaging-based techniques are being developed for live estimates and seem to work in mice,24 with prospective clinical uses planned in future. Using unbiased stereology, a more accurate range with a mean of around 900,000 nephrons per kidney has now been reported across many populations. Strikingly, as a population, Australian aboriginals have a much lower mean of around 680,00025; this group has a high prevalence of both renal disease and hypertension, and similar links between low nephron count and primary hypertension are reported in other populations.26 Reiterating the possible high variability within a population without obvious immediate renal dysfunction, nephron number spanned a 12-fold range between 210,000 and 2.7 million in a large study of 176 African Americans.27 Two hypotheses outlined by Barker and colleagues28 in Southampton, United Kingdom, and Brenner in Boston, Massachusetts, United States, have linked nephron number with in utero development and associated predisposition to progressive renal disease. Barker and colleagues28 initially demonstrated an inverse correlation between systolic blood pressure and birth weight and proposed that ‘the intrauterine environment influences blood pressure during adult life’. Their group (and many others) have subsequently confirmed the link between birth weight and hypertension but also associated risks of cardiovascular disease, type 2 diabetes and obesity.29-31 An important mechanism appears to be epigenetic modification of diverse tissues, including kidney, liver and pancreas.32,33 Direct proof linking the ‘Barker hypothesis’ with nephron number comes from maternal undernutrition or protein restriction and diabetic experimental models with consequent decreased nephron numbers, often linked to hypertension.34-37 Although originally based on the deleterious effects of increased dietary protein,38 the ‘Brenner hypothesis’ is that glomerular hyperfiltration causes progressive glomerular sclerosis, proteinuria and nephron loss, which then exacerbates hyperfiltration in the surviving functional glomeruli, leading to a recurring cycle of nephron dropout and increased stress on remaining

CHAPTER 12  Development of the Kidneys and Urinary Tract in Relation to Renal Anomalies

Genetic factors Impaired urinary flow, obstruction

Maternal environment, diet, teratogens

Mesenchyme Epithelium Mesenchyme Abnormal branching to primitive ducts

Primitive nephrons Cysts

Aberrant collecting system

Increased stroma

Formation of Failed nephron metaplastic cartilage differentiation

Nephron deficit

•

Fig. 12.2  Conceptual cellular model underlying congenital anomalies of the kidney and urinary tract.

glomeruli. Hyperfiltration is inevitable when a low nephron number has been caused by disease or induced by fetal programming39,40; hence, it is important to identify potentially affected babies and follow up looking for proteinuria and hypertension so that early treatment can be given. 

Types of Renal Anomalies Renal anomalies are often detected on antenatal ultrasound examination, but many are incidental changes such as minor dilatation of the renal pelvis, usually nonpathological.41 The differential diagnosis of anomalies is reviewed in Chapter 33, but a brief outline is given here in relation to their pathogenesis. Agenesis, or absent kidney, is often associated with aberrant or absent ureters; hence, a potential underlying mechanism has been suggested as early failure of ureteric bud branching. Agenesis may be isolated, but it can also occur as part of multiorgan disorders such as Branchio-Oto-Renal, Kallmann, Fraser and DiGeorge syndromes.42 Bilateral agenesis has an incidence of 1 in 5,000 to 10,000, whilst unilateral is much commoner at 1 in 1,000. Dysplasia includes conditions in which the kidney fails to undergo normal development and differentiation, with abnormal structure and which may contain metaplastic tissues such as cartilage.43 These pathological processes are depicted schematically in Fig. 12.2, along with normal and abnormal histology in Fig. 12.3. There is a spectrum of dysplasia from large kidneys distended with cysts, such as ‘multicystic dysplastic kidneys’, which are often attached to atretic ureters, or small echobright organs with a few rudimentary tubules that resemble ‘frustrated’ ureteric bud branches.6 Dysplasia can occur as an isolated anomaly or in a multi-organ syndrome, such as the renal cysts and diabetes (RCAD) or renal-coloboma syndromes.44,45 Around a third have an associated abnormal contralateral kidney, often with vesicoureteric reflux. The incidence of multicystic dysplastic kidney is around 1 in 2500,46although

117

this may be an underestimate because many involute and can be completely reabsorbed, which leads to an incorrect diagnosis of agenesis when detected later. Hypoplasia is defined pathologically as a kidney weighing less than 50% of expected,42 but the term is often used loosely to imply significantly fewer nephrons than normal. Dysplasia cannot be present, so all of the nephrons should appear normal, and undifferentiated tissues are not present. Again, this appears to represent a spectrum with some kidneys appearing grossly normal, albeit small on scan, whilst others are tiny. This raises a frequent question as to whether kidney size correlates with nephron number. The broad answer is yes, in that, given a normal radiologic appearance with no cysts or other structural abnormalities, a large kidney is likely to have more nephrons than a small one. However, this in itself does not necessarily mean that GFR will be different initially, but the smaller kidneys are likely to develop more severe kidney disease in the long term.47,48 Duplex kidneys represent some degree of duplication of the renal pelvis and ureter. This finding is relatively common, occurring in about 5% of unselected autopsies. The range of anatomy includes simple bifurcation of the extrarenal renal pelvis through complete duplication with two distinct (but contiguous) kidneys, separate ureters and two ureterovesical openings.49 Many cases are asymptomatic, although up to half may develop complications requiring treatment; these classically include obstruction to the upper part and reflux into the lower moiety. Ectopic kidneys and malrotation represent abnormal renal position. The kidneys should relatively ‘ascend’ to a progressively more rostral position during development, starting in the sacral region and ending between the 12th thoracic and 3rd lumbar vertebral bodies. There is also associated rotation such that the renal pelvis changes from an anterior- to a medial-facing orientation by term. Failure to ascend completely is relatively common, around 1 in 800 on routine renal imaging, and is usually associated with retention of a more anterior-facing renal pelvis. Occasionally, in crossed ectopia, the kidney is on the wrong side of the body as well as in the wrong position and may be fused to the contralateral kidney in cross-fused ectopia. Ectopic kidneys are often dysplastic and may also be associated with reflux or hydronephrosis or obstruction because of abnormal ureteric positioning and length. Kidneys can also be fused in a horseshoe kidney (1 in 600); these are usually situated lower than normal and, again, have an increased risk for vesicoureteric reflux or hydronephrosis. 

Causes of Human Renal Anomalies In the era of next generation sequencing and rapid gene screening, there is a temptation to focus on genetic causes of CAKUTs, but recognised mutations still account for only a small fraction of cases.50-52 This raises the possibility that most anomalies are a consequence of polygenic inheritance, perhaps with individually recessive genes in combination in compound heterozygotes to generate a CAKUT phenotype.53 Lower urinary tract obstruction and maternal environment may also cause malformations, both alone and in combination with genetic factors. Finally, random stochastic events remain likely, but they are almost impossible to prove. A general cause-and-effect schematic is depicted for dysplastic kidneys in Fig. 12.2. This demonstrates that one or multiple perturbations, including genetic, obstructive and environmental factors have the same net effect by perturbing epithelial–mesenchymal

118

SE C T I O N 3     Fetal Physiology and Pathology

B

A

D

C

E

•

Fig. 12.3  Histology of developing and dysplastic kidneys. 11-week (A) 12-week (B and C) gestation normal kidneys. D and E, Dysplastic kidney. A, Low-power view demonstrating ureteric buds radiating out from the centre (lower right) of the metanephros. Intermediate (B) and higher (C) magnification of nephrogenic cortex: multiple ureteric buds visible with condensing mesenchyme adjacent to tips. Each deeper layer shows progressive nephron differentiation from comma-shapes, through early to mature glomeruli. D, Dysplastic kidney demonstrating lack of normal renal tissues; instead there is stromal proliferation with a few primitive epithelia tubules and cysts. E, String of tortuous blood vessels (bottom left towards top right), demonstrating vascular as well as nephron abnormalities in dysplastic kidneys.

interactions leading to abnormal kidney development. This chapter briefly lists a few of the common causes, but readers are referred to recent reviews for a more detailed account.54-56

Genetic Factors i. TCF2 gene. Although the list of genes linked to kidney malformations is increasing rapidly, there is one that repeatedly occurs: the TCF2 gene, encoding the transcription factor hepatocyte nuclear factor-1β (HNF1β). This was originally described in the RCAD syndrome,57 which accounts for up to one third of antenatal presentations with ‘bright’ kidneys and 10% of CAKUTs overall.58,59 The wide spectrum of renal malformations in RCAD ranges from grossly cystic dysplastic kidneys through hypoplasia to agenesis.60 Females may also

have uterine abnormalities. An important issue is that both cysts and diabetes can develop at different ages; hence, repeated enquiry about new family diagnoses is worthwhile at follow up. ii. PAX–EYA–SIX transcription factor genes. Altered expression of the transcription factor PAX2 has been linked to a spectrum from renal agenesis (absent PAX2), through hypoplasia (reduced levels) to cystogenesis (overexpression).61-63 PAX2 mutations cause the ‘renal-coloboma’ syndrome with optic nerve colobomas, renal anomalies and vesicoureteric reflux,64 although other genes have now been implicated, too.65 PAX genes interact with several other transcription factors, including members of the SIX and EYA families. Many of these have been linked to renal malformations, including EYA1 in the branchio-oto-renal syndrome and SIX1 and SIX2 in familial CAKUTs.66,67

CHAPTER 12  Development of the Kidneys and Urinary Tract in Relation to Renal Anomalies

iii. GDNF/RET system. Glial cell line-derived neurotrophic factor (GDNF) and its receptor, RET, are critical factors determining initial outgrowth of the ureteric bud from the mesonephric duct and then in regulating ureteric bud branching in the metanephros.68 Mutations were originally described in multiple endocrine neoplasia but are now also recognised in CAKUTs.69 Because it is important to develop just one ureteric bud at the right time and in the right place, there are a number of negative regulators of GDNF/RET signalling, including the SLIT2-ROBO pathway, Semaphorin and Sprouty genes. Mutations in many of these cause duplex kidneys and other CAKUTs in mice, and these are now also being reported in human malformations.70-72 Common polymorphisms, rather than rare mutations, may also affect kidney development. Examples include PAX2 and RET in which smaller newborn kidneys have been reported for certain common variants, with the presumption that these will have lower nephron numbers.73,74 Polymorphisms of genes in the renin angiotensin system (RAS) have also been linked to variation in kidney size at birth.75 

Impaired Urinary Flow and Obstruction Dysplastic kidneys represent the largest group cause of chronic kidney disease in childhood, with urinary tract obstruction a close second.76,77 Examples include: posterior urethral valves (1 in 2500 boys), urethral atresia, and pelviureteric and ureterovesical junction obstruction. Moreover, the most severely perturbed nephrogenesis seen in multicystic dysplastic kidneys is classically associated with an obstructed ureter. Experimental obstruction of the developing urinary tract has been known to generate renal malformations for more than 40 years, and many of the same patterns of perturbed gene expression are seen as in nonobstructed human CAKUTs.56,78,79 It is worth noting that most of the cases labelled as ‘obstruction’ do not have a complete blockage; rather, they have a narrowing or restriction of the renal outflow tract that impairs flow. This is important because a complete block within the tract will cause severe oligo- or an-hydramnios and Potter sequence. Renal pathology can be reduced in animal models of obstruction by treatment with growth factors such as insulin-like growth factor and epidermal growth factor, but it is currently difficult to determine how such therapies might be selectively delivered to the kidney in human fetuses in utero. There are conflicting data on whether correcting early lower tract obstruction in utero allows renal function to recover. There are good results in large animals but poorer outcomes in mice and rats. The human data are limited to relatively small cases series, but even the largest clinical trial (PLUTO - percutaneous shunting in lower urinary tract obstruction) demonstrated poor efficacy of in utero vesicoamniotic shunt: survival seemed more likely with a shunt, but the size and direction of effect were uncertain; chances of surviving with good renal function were low.80 

Maternal Environment, Diet, and Teratogens An aberrant maternal in utero environment, diet or teratogens can cause both reduced nephron numbers and structural kidney defects. The first evidence that maternal diet affected the adult health of the offspring came from the World War II Dutch famine cohort; these mothers were grossly malnourished by limited food access, and a large follow-up study demonstrated

119

both an increased risk for hypertension and decreased glucose tolerance.81,82 Similar hypertension was noted in births after the siege of Leningrad and has been replicated in many other cohorts.83 These effects are now believed to result from malnutrition causing reduced nephron number, with extensive data in animals implicating protein restriction.34,35,84 Other maternal factors leading to lower birth weight (e.g., placental insufficiency, smoking and many chronic conditions) also predispose to adult hypertension, presumably again via reduced nephron numbers.85 Being born with such a deficit can then be exacerbated postnatally by overfeeding; this has been shown in laboratory models of protein restriction and then excess, but there is a worry that the same ‘biological experiment’ will be reiterated with obesogenic diet in humans.86,87 Diverse medication and chemicals have the potential to disrupt nephrogenesis.88 They include exogenous drugs and endogenous factors which become pathogenic when present in abnormal quantities. A good example is the RAS: the RAS is important in normal renal growth89 and may affect nephron number,75 and mutations are one cause of autosomal recessive renal tubular dysgenesis,90 whilst angiotensin-converting enzyme inhibitors and receptor blockers in pregnancy can cause fetal tubular dysgenesis (and skull malformations).91 Intriguingly, maternal protein restriction suppresses the neonatal RAS in rats and predisposes to hypertension, demonstrating how several factors might act in concert.92 High glucose levels in mothers with diabetes are associated with an increased incidence of kidney and lower urinary tract malformations, as well as abnormalities in the nervous, cardiovascular and skeletal systems.31,93 This effect may be multifactorial, however, because diabetes is associated with caudal regression syndrome in fetuses. Furthermore, one should always consider the possibility that mother and fetus may share HNF1β mutations that cause CAKUTs in the renal cysts and diabetes syndrome.59 Supplementary vitamins are routinely recommended during pregnancy but should be taken with caution because too much can be as deleterious as too little. A classic example is retinoic acid, a natural metabolite of vitamin A, which perturbs nephrogenesis if depleted or in excess.94 A polymorphism of the ALDH1A2 gene involved in retinoic acid metabolism causes a 22% increase in newborn kidney size95; whether this relates to increased nephron number is unproven. Vitamin D deficiency is very common worldwide and has been linked to lower birth weight with adverse pregnancy outcomes.96 Impact on nephrogenesis is less certain, however, with one study in rats showing that vitamin D deficiency generated a 20% increase in nephron number.97 A recent study from the Netherlands sought to identify maternal risk factors in 562 children with CAKUTs and 2139 healthy control participants.98 Specific use of folic acid rather than multivitamins increased the likelihood of CAKUTs, particularly duplex collecting systems and vesicoureteral reflux, which is interesting because high-dose folic acid can be used to induce experimental kidney disease in laboratory mice.99 Maternal obesity and pregnancy-associated diabetes were also associated with CAKUTs, with the latter particularly linked to posterior urethral valves (odds ratio, 2.6; 95% confidence interval, 1.1–5.9).98 

Potential Adverse Effects of Prematurity Based on a study measuring nephron number in 11 human fetuses between 15 and 40 weeks’ gestation20 (and acknowledging the small sample size), it appears that less than one third of the final nephron complement have formed before 24 weeks of

120

SE C T I O N 3     Fetal Physiology and Pathology

gestation. Premature babies born at that gestation have to rely exclusively on their own kidneys for excretion and electrolyte homeostasis, and the kidney is suddenly exposed to significantly increased renal blood flow. This causes hyperfiltration in already formed nephrons and some disruption of developing nephrons. Additive deleterious effects would also be predicted from nephrotoxic drugs, suboptimal nutrition and possible infections. In premature babies, therefore, one would expect fewer nephrons to form, with an increased rate of damage and loss of those that have already been generated (via mechanism underlying the Brenner hypothesis). Kidney function may appear initially normal in premature infants because urine output and creatinine remain within normal limits initially. However, these measures are too crude to accurately predict long-term renal function, and there should be ongoing screening for hypertension and kidney failure into adulthood.100-102 

Conclusions Development of the kidneys with concomitant contribution of urine to the amniotic fluid is essential for normal fetal development. Renal anomalies vary from gross structural malformations such as multicystic dysplastic kidneys through subtle reductions in nephron number, undetectable at birth but predisposing to hypertension in later life. Many texts focus on genetics as the main cause of such anomalies, but known mutations account for fewer than 20% of cases at present. Obstruction of the urinary tract, maternal environment, diet and teratogens may be just as important, along with chance, stochastic maldevelopment. Children who have, or are at risk for having, a low nephron number need regular follow up to identify and treat hypertension early. Access the complete reference list online at ExpertConsult.com. Self-assessment questions available at ExpertConsult.com.

References

1. Vize PD, Woolf AS, Bard JBL, eds. The Kidney: From Normal Development to Congenital Disease. London, UK: Academic Press; 2003. 2. Drummond IA, Davidson AJ. Zebrafish kidney development. Methods Cell Biol. 2010;100:233–260. 3. Moritz KM, Wintour EM. Functional development of the meso- and metanephros. Pediatr Nephrol. 1999;13(2):171–178. 4. Hatini V, Huh SO, Herzlinger D, et al. Essential role of stromal mesenchyme in kidney morphogenesis revealed by targeted disruption of Winged Helix transcription factor BF-2. Genes Dev. 1996;10(12):1467–1478. 5. Fetting JL, Guay JA, Karolak MJ, et al. FOXD1 promotes nephron progenitor differentiation by repressing decorin in the embryonic kidney. Development. 2014;141(1):17–27. 6. Potter EL. Normal and Abnormal Development of the Kidney. Chicago: Year Book Medical Publishers; 1972. 7. Jennette JC, Olson JL, Silva FG, D’Agati VD. Heptinstall’s Pathology of the Kidney. Philadelphia: Lippincott Williams & Wilkins (LWW); 2015. 8. McMahon AP. Development of the mammalian kidney. Curr Topics Dev Biol. 2016;117:31–64. 9. Winyard PJD, Nauta J, Lirenman DS, et  al. Deregulation of cell survival in cystic and dysplastic renal development. Kidney Int. 1996;49(1):135–146. 10. Rumballe BA, Georgas KM, Combes AN, et al. Nephron formation adopts a novel spatial topology at cessation of nephrogenesis. Dev Biol. 2011;360(1):110–122. 11. Little MH, Kairath P. Regenerative medicine in kidney disease. Kidney Int. 2016;90(2):289– 299. 12. Ekblom P. Embryology and prenatal development. In: Holliday MA, Barrett TM, Avner ED, eds. Pediatric Nephrology. Baltimore: Williams and Wilkins; 1994:2–18. 13. Saxen L. Organogenesis of the Kidney. Cambridge, United Kingdom: Cambridge University Press; 1987. 14. Short KM, Combes AN, Lefevre J, et  al. Global quantification of tissue dynamics in the developing mouse kidney. Dev Cell. 2014;29(2):188–202. 15. Woolf AS, Gnudi L, Long DA. Roles of angiopoietins in kidney development and disease. J Am Soc Nephrol. 2009;20(2):239–244. 16. Drukker A, Guignard JP. Renal aspects of the term and preterm infant: a selective update. Curr Opin Pediatr. 2002;14(2):175–182. 17. Filler G, Guerrero-Kanan R, Alvarez-Elias AC. Assessment of glomerular filtration rate in the neonate: is creatinine the best tool? Curr Opin Pediatr. 2016;28(2):173–179. 18. Schwartz GJ, Work DF. Measurement and estimation of GFR in children and adolescents. Clin J Am Soc Nephrol. 2009;4(11):1832– 1843. 19. Denic A, Glassock RJ, Rule AD. Structural and functional changes with the aging kidney. Adv Chronic Kidney Dis. 2016;23(1):19–28. 20. Hinchliffe SA, Sargent PH, Howard CV, et al. Human intrauterine renal growth expressed in absolute number of glomeruli assessed by the disector method and Cavalieri principle. Lab Invest. 1991;64(6):777–784. 21. Merlet-Benichou C, Gilbert T, Vilar J, et  al. Nephron number: variability is the

rule. Causes and consequences. Lab Invest. 1999;79(5):515–527. 22. Bertram JF, Douglas-Denton RN, Diouf B, et  al. Human nephron number: implications for health and disease. Pediatr Nephrol. 2011;26(9):1529–1533. 23. Puelles VG, Bertram JF. Counting glomeruli and podocytes: rationale and methodologies. Curr Opin Nephrol Hypertens. 2015;24(3):224–230. 24. Baldelomar EJ, Charlton JR, Beeman SC, et  al. Phenotyping by magnetic resonance imaging nondestructively measures glomerular number and volume distribution in mice with and without nephron reduction. Kidney Int. 2016;89(2):498–505. 25. Hoy WE, Hughson MD, Singh GR, et  al. Reduced nephron number and glomerulomegaly in Australian Aborigines: a group at high risk for renal disease and hypertension. Kidney Int. 2006;70(1):104–110. 26. Keller G, Zimmer G, Mall G, et al. Nephron number in patients with primary hypertension. N Engl J Med. 2003;348(2):101–108. 27. Puelles VG, Hoy WE, Hughson MD, et  al. Glomerular number and size variability and risk for kidney disease. Curr Opin Nephrol Hypertens. 2011;20(1):7–15. 28. Barker DJ, Osmond C, Golding J, et  al. Growth in utero, blood pressure in childhood and adult life, and mortality from cardiovascular disease. BMJ. 1989;298(6673):564–567. 29. Barker DJ. The origins of the developmental origins theory. J Intern Med. 2007;261(5):412– 417. 30. Heindel JJ, Vandenberg LN. Developmental origins of health and disease: a paradigm for understanding disease cause and prevention. Curr Opin Pediatr. 2015;27(2):248–253. 31. Hokke S, Arias N, Armitage JA, et al. Maternal glucose intolerance reduces offspring nephron endowment and increases glomerular volume in adult offspring. Diabetes Metab Res Rev. 2016;32(8):816–882. 32. Godfrey KM, Costello PM, Lillycrop KA. Development, epigenetics and metabolic programming. Nestle Nutr Inst Workshop Ser. 2016;85:71–80. 33. Eriksson JG. Developmental Origins of Health and Disease—from a small body size at birth to epigenetics. Ann Med. 2016;48(6):456– 467. 34. Langley-Evans SC, Welham SJ, Jackson AA. Fetal exposure to a maternal low protein diet impairs nephrogenesis and promotes hypertension in the rat. Life Sci. 1999;64(11):965– 974. 35. Welham SJ, Wade A, Woolf AS. Protein restriction in pregnancy is associated with increased apoptosis of mesenchymal cells at the start of rat metanephrogenesis. Kidney Int. 2002;61(4):1231–1242. 36. Villar-Martini VC, Carvalho JJ, Neves MF, et al. Hypertension and kidney alterations in rat offspring from low protein pregnancies. J Hypertens Suppl. 2009;27(6):S47–S51. 37. Watkins AJ, Lucas ES, Torrens C, et  al. Maternal low-protein diet during mouse preimplantation development induces vascular dysfunction and altered renin-angiotensinsystem homeostasis in the offspring. Br J Nutr. 2010;103(12):1762–1770. 38. Brenner BM, Meyer TW, Hostetter TH. Dietary protein intake and the progressive nature of kidney disease: the role of

hemodynamically mediated glomerular injury in the pathogenesis of progressive glomerular sclerosis in aging, renal ablation, and intrinsic renal disease. N Engl J Med. 1982;307(11):652–659. 39. Brenner BM, Garcia DL, Anderson S. Glomeruli and blood pressure. Less of one, more the other? Am J Hypertens. 1988;1(4 Pt 1):335– 347. 40. Luyckx VA, Brenner BM. Birth weight, malnutrition and kidney-associated outcomes—a global concern. Nat Rev Nephrol. 2015;11(3):135–149. 41. Hothi DK, Wade AS, Gilbert R, Winyard PJ. Mild fetal renal pelvis dilatation: much ado about nothing? Clin J Am Soc Nephrol. 2009;4(1):168–177. 42. Liapis H, Winyard PJD. Cystic diseases and developmental kidney defects. In: Jennette JC, Olson JL, Silva FG, D’Agati VD, eds. Heptinstall’s Pathology of the Kidney. Philadelphia: Wolters Kluwer; 2015:119–172. 43. Winyard P, Chitty LS. Dysplastic kidneys. Semin Fetal Neonatal Med. 2008;13(3):142– 151. 44. Martinovic-Bouriel J, Benachi A, Bonniere M, et al. PAX2 mutations in fetal renal hypodysplasia. Am J Med Genet A. 2010;152A(4):830– 835. 45. Clissold RL, Hamilton AJ, Hattersley AT, et al. HNF1B-associated renal and extra-renal disease-an expanding clinical spectrum. Nat Rev Nephrol. 2015;11(2):102–112. 46. Winding L, Loane M, Wellesley D, et al. Prenatal diagnosis and epidemiology of multicystic kidney dysplasia in Europe. Prenat Diagn. 2014;34(11):1093–1098. 47. Kandasamy Y, Smith R, Wright IM, Lumbers ER. Reduced nephron endowment in the neonates of Indigenous Australian peoples. J Dev Orig Health Dis. 2014;5(1):31–35. 48. Matsell DG, Cojocaru D, Matsell EW, Eddy AA. The impact of small kidneys. Pediatr Nephrol. 2015;30(9):1501–1509. 49. Doery AJ, Ang E, Ditchfield MR. Duplex kidney: not just a drooping lily. J Med Imaging Radiat Oncol. 2015;59(2):149–153. 50. McPherson E. Renal anomalies in families of individuals with congenital solitary kidney. Genet Med. 2007;9(5):298–302. 51. Weber S, Moriniere V, Knuppel T, et  al. Prevalence of mutations in renal developmental genes in children with renal hypodysplasia: results of the ESCAPE study. J Am Soc Nephrol. 2006;17(10):2864–2870. 52. Hwang DY, Dworschak GC, Kohl S, et  al. Mutations in 12 known dominant diseasecausing genes clarify many congenital anomalies of the kidney and urinary tract. Kidney Int. 2014;85(6):1429–1433. 53. Kohl S, Hwang DY, Dworschak GC, et  al. Mild recessive mutations in six Fraser syndrome-related genes cause isolated congenital anomalies of the kidney and urinary tract. J Am Soc Nephrol. 2014;25(9):1917–1922. 54. Rodriguez MM. Congenital anomalies of the kidney and the urinary tract (CAKUT). Fetal Pediatr Pathol. 2014;33(5-6):293–320. 55. Nicolaou N, Renkema KY, Bongers EM, et al. Genetic, environmental, and epigenetic factors involved in CAKUT. Nat Rev Nephrol. 2015;11(12):720–731. 56. Chevalier RL. Prognostic factors and biomarkers of congenital obstructive nephropathy. Pediatr Nephrol. 2016;31(9):1411–1420.

120.e1

120.e2

References

57. Bohn S, Thomas H, Turan G, et al. Distinct molecular and morphogenetic properties of mutations in the human HNF1beta gene that lead to defective kidney development. J Am Soc Nephrol. 2003;14(8):2033–2041. 58. Decramer S, Parant O, Beaufils S, et al. Anomalies of the TCF2 gene are the main cause of fetal bilateral hyperechogenic kidneys. J Am Soc Nephrol. 2007;18(3):923–933. 59. Raaijmakers A, Corveleyn A, Devriendt K, et al. Criteria for HNF1B analysis in patients with congenital abnormalities of kidney and urinary tract. Nephrol Dial Transplant. 2015;30(5):835–842. 60. Bockenhauer D, Jaureguiberry G. HNF1Bassociated clinical phenotypes: the kidney and beyond. Pediatr Nephrol. 2016;31(5):707– 714. 61. Dressler GR, Wilkinson JE, Rothenpieler UW, et  al. Deregulation of Pax-2 expression in transgenic mice generates severe kidney abnormalities. Nature. 1993;362:65–67. 62. Winyard PJD, Risdon RA, Sams VR, et  al. The PAX2 transcription factor is expressed in cystic and hyperproliferative dysplastic epithelia in human kidney malformations. J Clin Invest. 1996;98:451–459. 63.  Sharma R, Sanchez-Ferras O, Bouchard M. Pax genes in renal development, disease and regeneration. Semin Cell Dev Biol. 2015;44:97–106. 64. Sanyanusin P, Schimmentl LA, McNoe LA, et al. Mutations of the PAX2 gene in a family with optic nerve colobomas, renal anomalies and vesicoureteral reflux. Nat Genet. 1995;9:358–364. 65. Okumura T, Furuichi K, Higashide T, et  al. Association of PAX2 and other gene mutations with the clinical manifestations of renal coloboma syndrome. PLoS One. 2015;10(11):e0142843. 66. Ruf RG, Xu PX, Silvius D, et al. SIX1 mutations cause branchio-oto-renal syndrome by disruption of EYA1-SIX1-DNA complexes. Proc Natl Acad Sci U S A. 2004;101(21):8090– 8095. 67. Weber S. Novel genetic aspects of congenital anomalies of kidney and urinary tract. Curr Opin Pediatr. 2012;24(2):212–218. 68. Costantini F, Kopan R. Patterning a complex organ: branching morphogenesis and nephron segmentation in kidney development. Dev Cell. 2010;18(5):698–712. 69. Chatterjee R, Ramos E, Hoffman M, et  al. Traditional and targeted exome sequencing reveals common, rare and novel functional deleterious variants in RET-signaling complex in a cohort of living US patients with urinary tract malformations. Hum Genet. 2012;131(11):1725–1738. 70. Basson MA, Akbulut S, Watson-Johnson J, et al. Sprouty1 is a critical regulator of GDNF/ RET-mediated kidney induction. Dev Cell. 2005;8(2):229–239. 71. Hwang DY, Kohl S, Fan X, et al. Mutations of the SLIT2-ROBO2 pathway genes SLIT2 and SRGAP1 confer risk for congenital anomalies of the kidney and urinary tract. Hum Genet. 2015;134(8):905–916.

72. Reidy K, Tufro A. Semaphorins in kidney development and disease: modulators of ureteric bud branching, vascular morphogenesis, and podocyte-endothelial crosstalk. Pediatr Nephrol. 2011;26(9):1407–1412. 73. Zhang Z, Quinlan J, Hoy W, et  al. A common RET variant is associated with reduced newborn kidney size and function. J Am Soc Nephrol. 2008;19(10):2027–2034. 74. Quinlan J, Lemire M, Hudson T, et  al. A common variant of the PAX2 gene is associated with reduced newborn kidney size. J Am Soc Nephrol. 2007;18(6):1915–1921. 75.  Kaczmarczyk M, Kuprjanowicz A, Loniewska B, et  al. Epistatic interaction between common AGT G(-6)A (rs5051) and AGTR1 A1166C (rs5186) variants contributes to variation in kidney size at birth. Gene. 2015;572(1):72– 78. 76. Pruthi R, O’Brien C, Casula A, et  al. UK Renal Registry 16th Annual Report: Chapter 7 Demography of the UK Paediatric Renal Replacement Therapy population in 2012. Nephron Clin Pract. 2013;125:127–138. 77. Woolf AS, Stuart HM, Newman WG. Genetics of human congenital urinary bladder disease. Pediatr Nephrol. 2014;29(3):353–360. 78. Yang SP, Woolf AS, Quinn F, Winyard PJD. Deregulation of renal transforming growth factor-á1 after experimental short-term ureteric obstruction in fetal sheep. Am J Pathol. 2001;159:109–117. 79. Holst BD, Goomer RS, Wood IC, et al. Binding and activation of the promoter for the neural cell adhesion molecule by Pax-8. J Biol Chem. 1994;269(35):22245–22252. 80. Morris RK, Malin GL, Quinlan-Jones E, et al. Percutaneous vesicoamniotic shunting versus conservative management for fetal lower urinary tract obstruction (PLUTO): a randomised trial. Lancet. 2013;382(9903):1496– 1506. 81. Roseboom TJ, van der Meulen JH, Ravelli AC, et  al. Blood pressure in adults after prenatal exposure to famine. J Hypertens. 1999;17(3):325–330. 82.  Ravelli AC, van der Meulen JH, Michels RP, et al. Glucose tolerance in adults after prenatal exposure to famine. Lancet. 1998;351(9097):173– 177. 83. Sparen P, Vagero D, Shestov DB, et al. Long term mortality after severe starvation during the siege of Leningrad: prospective cohort study. BMJ. 2004;328(7430):11. 84. Wood-Bradley RJ, Barrand S, Giot A, Armitage JA. Understanding the role of maternal diet on kidney development; an opportunity to improve cardiovascular and renal health for future generations. Nutrients. 2015;7(3):1881–1905. 85. Law CM, Shiell AW. Is blood pressure inversely related to birth weight? The strength of evidence from a systematic review of the literature. J Hypertens. 1996;14(8):935–941. 86. Yim HE, Yoo KH. Early life obesity and chronic kidney disease in later life. Pediatr Nephrol. 2015;30(8):1255–1263. 87. Tsuboi N, Utsunomiya Y, Hosoya T. Obesity-related glomerulopathy and the

nephron complement. Nephrol Dial Transplant. 2013;28(suppl 4):108–113. iv. 88. Shepard TH. Catalog of Teratogenic Agents. Baltimore: The Johns Hopkins University Press; 2010. 89. Pope JC, Nishimura H, Ichikawa I. Role of angiotensin in the development of the kidney and urinary tract. Nephrologie. 1998;19(7):433–436. 90. Gribouval O, Gonzales M, Neuhaus T, et al. Mutations in genes in the renin-angiotensin system are associated with autosomal recessive renal tubular dysgenesis. Nat Genet. 2005;37(9):964–968. 91. Barr Jr M, Cohen Jr MM. ACE inhibitor fetopathy and hypocalvaria: the kidney-skull connection. Teratology. 1991;44(5):485–495. 92. Woods LL, Ingelfinger JR, Nyengaard JR, Rasch R. Maternal protein restriction suppresses the newborn renin-angiotensin system and programs adult hypertension in rats. Pediatr Res. 2001;49(4):460–467. 93. Woolf AS. Environmental influences on renal tract development: a focus on maternal diet and the glucocorticoid hypothesis. Klin Padiatr. 2011;223(suppl 1):S10–S17. 94. Lee LM, Leung CY, Tang WW, et  al. A paradoxical teratogenic mechanism for retinoic acid. Proc Natl Acad Sci U S A. 2012;109(34):13668–13673. 95. El KR, Manolescu DC, Lakhal-Chaieb L, et  al. A human ALDH1A2 gene variant is associated with increased newborn kidney size and serum retinoic acid. Kidney Int. 2010;78(1):96–102. 96. Reichetzeder C, Chen H, Foller M, et  al. Maternal vitamin D deficiency and fetal programming—lessons learned from humans and mice. Kidney Blood Press Res. 2014;39(4):315– 329. 97. Maka N, Makrakis J, Parkington HC, et  al. Vitamin D deficiency during pregnancy and lactation stimulates nephrogenesis in rat offspring. Pediatr Nephrol. 2008;23(1):55–61. 98. Groen In’t Woud S, Renkema KY, Schreuder MF, et  al. Maternal risk factors involved in specific congenital anomalies of the kidney and urinary tract: a case-control study. Birth Defects Res A Clin Mol Teratol. 2016;106(7):596–603. 99. Kolatsi-Joannou M, Price KL, Winyard PJ, Long DA. Modified citrus pectin reduces galectin-3 expression and disease severity in experimental acute kidney injury. PLoS One. 2011;6(4):e18683. 100. Carmody JB, Charlton JR. Short-term gestation, long-term risk: prematurity and chronic kidney disease. Pediatrics. 2013;131(6):1168– 1179. 101. Viswanathan S, Kumar D, Sykes C, et  al. Making a diagnosis of hypertension and defining treatment threshold in very low birth weight infants’ need revision? J Renal Inj Prev. 2016;5(2):55–60. 102. de Jong F, Monuteaux MC, van Elburg RM, et  al. Systematic review and meta-analysis of preterm birth and later systolic blood pressure. Hypertension. 2012;59(2):226–234.

13

The Perinatal Postmortem Examination J. CIARAN HUTCHINSON, SUSAN C . SHELMERDINE AND NEIL J. SEBIRE

KEY POINTS • P  erinatal autopsy (examination after death) fulfils several roles, including determination or clarification of the underlying diagnosis, answering specific questions raised by parents and clinicians, quality assurance, governance and public health aspects, and improved understanding of disease mechanisms through research. • Examination after death may involve a spectrum of investigations, including placental pathology, genetic testing, postmortem imaging and internal organ examination or sampling. • Parents should be informed regarding their options, with the extent of investigation determined by parental acceptability and appropriateness for specific clinical circumstances. • Examination after death should be individualised according to the clinical features present and parental consent requirements. • Placental examination should be considered in all cases of pregnancy complications even if postmortem fetal examination is declined. • Future advances in imaging and laboratory medicine, such as the widespread introduction of various ‘omic’ technologies, are likely to significantly change the approach to investigation after death.

Overview Examination after perinatal death may be a difficult area for obstetricians and fetal medicine practitioners, many of whom may otherwise have little interaction with specialist pathology services. Therefore, the aim of this chapter is not to present detailed findings of issues in this field but rather to provide practical guidance for interaction of clinicians and pathologists to maximise utility of the various facets related to perinatal autopsy examination from consent through to technical aspects of the process itself and likely future advances in the area. Some aspects of the major categories of pathology that may be disclosed through postmortem investigations are also covered; detailed explanations of these conditions can be found in specialist embryology and perinatal pathology textbooks. 

Introduction Autopsy: Greek: ‘autos’ (self ) + ‘optos’ (seen); or ‘autoptēs’ (eyewitness)

The role of the perinatal autopsy has come under increasing scrutiny from medical professionals and the public. Advances in medical imaging, increasing use of antenatal genetic testing and controversies associated with human tissue retention have combined with shifting population demographics and changing public attitudes, resulting in a reduction in acceptability of traditional autopsy and the encouragement of development of potentially more acceptable, contemporary approaches.1 Research into parental attitudes to autopsy has revealed that traditional postmortem examination is becoming less acceptable,2 especially among certain ethnic and religious groups,3-5 with autopsy rates falling in most countries (Table 13.1). In addition to moral or religious reasons, parents have aversion to large incisions because of perceptions that the fetus or infant has ‘suffered enough’. From a clinical perspective, there is also a perception that autopsy reports vary in adequacy, alongside perceived difficulties in negotiating a highly specific informed consent process.6 Although great insights into normal developmental processes and pathogenesis of congenital anomalies have resulted from perinatal autopsy findings, contemporary obstetric practice has changed, with introduction of widespread first and second trimester antenatal ultrasound screening resulting in accurate antenatal detection of a wide range of fetal abnormalities.7 Consequently, the role of examination of after death is also changing, with detection of unexpected major fetal anomalies now less frequent, but the range of antenatal fetal interventions and complexity of associated pathologies increasing. In addition, mechanistic data derived from many years of autopsy practice, which have provided improved understanding of numerous obstetric complications, is decreasingly likely to generate new insights in the absence of the introduction of novel approaches. Although the concept of the autopsy examination as a means of medical audit and governance remains important (e.g., for terminations of pregnancy or after complex medical treatment8), the additional direct clinical benefit from autopsy examination of otherwise uncomplicated cases remains uncertain if there are no specific additional clinical questions to be addressed. The concept of examination after death must therefore develop in parallel with changes in antenatal care and technologies if it is to continue to make important contributions to research and clinical care. The concept of ‘investigation after death’ may therefore more accurately reflect the future of this approach, with personalised investigations performed targeted to address the specific issues of particular cases to improve the quality of information gained and increase parental acceptability.9 Investigation after death is unusual in that many factors surrounding the decisions made and approaches used are primarily 121

122

SE C T I O N 3     Fetal Physiology and Pathology

TABLE Number of Postmortem Examinations Offered and Consented to by Type of Death (Stillbirth, Neonatal Death, 13.1A Extended Perinatal Death): United Kingdom and Crown Dependencies, for Births in 2014 STILLBIRTHSa

NEONATAL DEATHSa

EXTENDED PERINATAL DEATHSa

Number

(%)

Number

(%)

Number

(%)

Not offered

50

(1.6)

137

(10.0)

187

(4.1)

Not known if offered

67

(2.1)

155

(11.3)

222

(4.8)

1503

(46.6)

628

(45.7)

2131

(46.3)

83

(2.6)

54

(3.9)

137

(3.0)

120

(3.7)

28

(2.0)

148

(3.2)

1402

(43.5)

372

(27.1)

1774

(38.6)

Postmortem Status

Offered but no consent Offered but unknown consent Offered and limited consent Offered and full consent aExcluding

termination of pregnancy and births 99% 97.4% 87.5%

>99% 97.4% 93.8%

>99% 96.4% >99%

>99.1% 98.3% 98.2%

Gestation

10 wk

10 wk

10 wk

9 wk

10 wk

Twin gestation

Yes

Yes

Yes

No

Yes

MPS, Massively parallel sequencing; SNP, single nucleotide polymorphism

  

Gestational age. The rate of placental cell apoptosis and the fetal DNA concentration increase as gestation advances50,58,59 (see Fig. 21.2). At 10 weeks’ gestation, the median fetal fraction is approximately 10% and rises by 0.1% per week between 10 and 20 weeks of gestation followed by a 10-fold increase to 1% per week from 21 weeks to term.59 Approximately 2% of pregnancies less than 21 weeks’ gestation have a fetal fraction below 4%, which is considered too low to be used for aneuploid screening.  Maternal weight. There is a significant negative correlation between fetal fraction and maternal weight59 from a mean of about 12% for a maternal weight of 60 kg to 6% for a weight of 120 kg.60 Accordingly, the proportion of pregnancies with a fetal fraction less than 4% increases with increasing maternal weight.59 For example, 20% of women over 250 lb will have a value less than 4% as will 50% at more than 350 lb.61 Obesity may be associated with a lower fetal fraction caused by a dilutional effect from an increased maternal circulatory volume59-61 but is more likely related to an increase in maternal cfDNA from a higher adipocyte turnover rate.62  Fetal aneuploidies. Specific fetal chromosome abnormalities may alter the fetal fraction.63-65 Specifically, in fetuses with Down syndrome, the DNA fragments result in a higher proportion of fetal DNA than in disomic fetuses.63-66 This observation has also been observed for other fetal aneuploidies such as trisomy 13 and sex chromosomal abnormalities such as 47,XXY but perhaps less significantly. On the other hand, other studies have shown fetal fraction to be lower with cases of trisomy 18 and monosomy X, possibly related to a smaller placental size.65,67  Twin gestations. The median fetal fraction per fetus in twin pregnancies is lower than for singleton gestations.62 For instance, Srinivasan and colleagues found the overall fetal fraction per fetus to be reduced by up to 50% in both mono- and dizygotic twins,68 resulting in a clinically significant higher proportion of twin gestations with a fetal fraction too low for accurate assessment.  Other factors. As illustrated by their correlation with threedimensional ultrasound measurements, serum concentrations of β-hCG and PAPP-A are an indirect measure of placental mass69 so that the fetal fraction increases in a linear fashion with these

analytes independent of maternal weight and other maternal characteristics. Fetal fraction is not affected by other prenatal analyte results, maternal age, fetal sex, previous blood donations, race, smoking or ultrasound measurements such as NT or crown–rump length.60–71 

Analysis of cell-free DNA for the prediction of fetal cytogenetic abnormalities. Fetal DNA represents only about 10% of

the total cfDNA in the maternal plasma, requiring that laboratory methods to identify any small increase or decrease associated with a fetal chromosomal aneuploidy must be robust. Today, more than 10 companies are offering this noninvasive prenatal screening (NIPS) test worldwide, and seven of them distribute from the United States.72 Each company is using one of three different testing approaches and offering different testing options (Table 21.2), which are described next. 

Techniques Using Cell-Free DNA to Detect Common Fetal Aneuploidies Massively Parallel Sequencing and Next-Generation Sequencing Shotgun or Random Massively Parallel Sequencing. The tech­

nology underlying massively parallel sequencing (MPS) is illustrated in Fig. 21.3. In summary, fragments of both maternal and fetal DNA are clonally amplified and then each clone is sequenced by the addition of fluorescently labelled nucleic acids. One million to 43 billion reads of 40 to 500 base pairs (bp) each can be obtained per run.73,74 Using bioinformatics, the reads are aligned to a reference copy of the human genome and the chromosomal source of the fragment identified. The basic goal of shotgun MPS for aneuploidy detection is to sequence all informative chromosome regions and then count the DNA fragments assigned to a single-locus.75,76 In this method, maternal and fetal fragments of 150 to 200 bp in size are sequenced simultaneously and approximately the first 36 bp are aligned and mapped to their chromosome of origin. Because the entire human

CHAPTER 21  Noninvasive Screening for Cytogenetic Disorders (Fetal Aneuploidy Including Microdeletions)

207

DNA fragments in maternal plasma

Bioinformatics alignment

36 bp AAGCT... CTAGT... TAGGC... GCATG...

Sequence and align

Chr1 Chr7 ChrX Chr13 Chr1 Chr21 Chr18 ChrY and so on...

nth sequence

Sequence counting Chromosome

1

2

3

4

5

6

7

8

9

10 11 12 13 14 15 16 17 18 19 20 21 22

X

Y

• Fig. 21.3  Mass parallel (shotgun) sequencing analysis of fetal DNA. The process of mass parallel nextgeneration sequencing and sequence counting to the analysis of maternal cell-free DNA for fetal aneuploidy. Cell-free DNA fragments between 120 and 200 kb are sequenced. Bioinformation analysis assigns sequences to their genomic origins, and the number of sequences per genomic assignment is counted. chr, Chromosome. (Adapted from Zhong, X, Holzgreve, W. Circulating cell-free DNA in women’s medicine. Glob Libr Womens Med 2009; DOI 10.3843/GLOWM.10270.)

genome sequence is known, MPS maps between 12 and 25 million fragments per sample to discrete loci on each chromosome. The number of fragment reads from each specific chromosome are then quantified and compared with values from a normal reference chromosome. A relative excess or deficiency in the number of counts on the chromosome of interest compared with a reference chromosome will determine the risk for aneuploidy (trisomy or monosomy) for that specific chromosome. This difference is usually expressed as a Z-score.75-78 A large number of reads is needed because the difference between aneuploidy and euploidy is small (∼1.05 in 1 for a trisomy) because on average only 10% of the DNA fragments will come from an aneuploid fetus and 90% from the euploid mother. The difference is even smaller with lower fetal fractions. 

Targeted or Directed Massively Parallel Sequencing Massively parallel sequencing is highly accurate but relatively inefficient if screening only for the common aneuploidies because the majority of the fragment reads come from chromosomes that are never analysed. Targeted MPS improves efficiency by including a presequencing step that selectively amplifies only fragments from the chromosomes of interest, thereby creating mapped reads that are specific to these chromosomes alone. A small number of single nucleotide polymorphisms (SNPs) are also analysed, allowing calculation of the fetal fraction. This chromosome-selective sequencing is referred to as digital analysis of selected regions (DANSR) and is a more efficient use of sequencing to simultaneously quantify hundreds of loci on selected chromosomes.52,79 After the fragments are counted, a ‘fetal-fraction optimised risk for trisomy evaluation’ (FORTE) algorithm is used to calculate the risk for aneuploidy.52,79 In addition to the actual fragment counts, the FORTE algorithm includes the a priori maternal age-related risks and the fetal fraction to provide an estimate of

patient-specific risk for trisomy. Using this approach, fewer than 400 loci per chromosome are sufficient to enable aneuploidy discrimination.79 Using the DANSR assay and FORTE algorithm, targeted MPS has a sensitivity approaching 100% for detection of trisomy 21 and a sensitivity of 98% for detection of trisomy 18.80 The main advantage is a substantial reduction in the overall sequence data and thus a decrease in costs.55 The increased depth of sequencing of DANSR may also result in better discrimination between euploid and aneuploid cases. 

Single Nucleotide Polymorphism The third method for noninvasive prenatal testing (NIPT) for fetal aneuploidy analyses SNPs and determines the relative quantitative contributions of maternal and fetal DNA in the plasma (Fig. 21.4). SNPs are highly polymorphic DNA sequence differences in which only a single nucleotide varies among the population and can be easily determined.81 The fetus will have SNPs from its father at multiple alleles that differ from those transmitted from its mother.53 The SNP approach selectively amplifies and sequences nearly 20,000 polymorphic loci on chromosomes of interest (13, 18, 21, X and Y), mathematically subtracts out the contribution of the maternal SNPs and then uses a proprietary algorithm for analysis, referred to as the next-generation aneuploidy test using SNPs (NATUS) algorithm.53,82 This technology does not compare the sequencing results from specific chromosomes with control chromosomes but instead uses complex computer bioinformatics and a Bayesian-based maximum likelihood statistical method to determine the chromosomal count of the fetal chromosomes interrogated in each sample in comparison with the maternal genotype. The distribution of fetal SNPs, compared with maternal, will determine the likelihood that the fetus is monosomic, disomic or trisomic. The SNP-based method also requires a minimum fetal fraction of 3% to 4% to avoid test failure, and its performance is comparable with the NGS counting methods.

208

SE C T I O N 6     Prenatal Screening and Diagnosis

Genotypic data from mother

Data from Human Genome Project (HapMap)

Multiple hypotheses for each chromosome

x

x

x

x

x

x

x

x

y

y

y

y

y

y

y

y

z

z

z

z

z

z

z

z

Subhypotheses with different crossover points

A Sequencing measurements from maternal plasma Compare

B • Fig. 21.4  Single nucleotide polymorphism (SNP)-based analysis for aneuploidy detection from cell-free DNA. A, The maternal genotype is evaluated, and SNPs from the chromosomes of interest are determined. Using this data and data from the human genome project on the frequency and location of crossover events, a subhypothesis of the fetal genotype is determined for monosomy, disomy and trisomy. B, Maximum likelihood estimation determines correct hypothesis. The fetal hypothesis is then compared with the sequencing results from the maternal plasma cell-free DNA. Then using the maximum likelihood estimation of each hypothesis, the most likely correct hypothesis is selected to determine the fetal genotype. The subpicture to the side is an illustration of the readout in which the red upper line is the frequency of aa alleles (a being the more frequent allele) and the blue line being the bb allele (b being the less frequent allele SNP). The green frequency is of the other allele (ab). Note that y-axis or the relative allele frequency of genotype at each allele in the mixture of the determined of the green frequency. (Reprinted with permission from Natera, Inc. Copyright, 2017 Natera, Inc.)

The potential advantages of a SNP-based approach are that additional information is provided concerning the parent of origin of aneuploidy, recombination and inheritance of mutations. It also allows the detection of triploidy, which NGS cannot.83 SNP-based methods can also identify regions of homozygosity in a fetus, indicating consanguinity or uniparental disomy. In certain cases, a sample from the father can be helpful and improve test performance. There is a no-call rate of 5.4%, comparable with other approaches to NIPS.82 On the other hand, the disadvantages include the inadvertent detection of nonpaternity as well as the need for enrichment of fetal DNA, deeper sequencing or higher levels of high-fidelity amplification given that SNPs only account for 1.6% of the human genome.84 

Microarray-Based Technology More recently, an approach has been developed using a microarray-based technology to quantify the relative contributions from specific chromosomes, hence not requiring NGS and thus potentially reducing the cost and turnaround time.85 In this

approach, DNA fragments from the chromosomes of interest are selected using DANSR and hybridised to and analysed using a specially designed array allowing millions of genomic locations to be studied simultaneously with each sample. Juneau and colleagues showed that microarray-based NIPS in combination with DANSR assays and the FORTE algorithm for targeted analysis resulted in high sensitivity (>99.9%, 97.5% and 93.8% for trisomy 21, 18 and 13, respectively) and high specificity (>99.9% for each of the common trisomies) for fetal aneuploidies, screening comparable to NGS.85,86 The advantages of microarray-based NIPS are: decreased variability that will allow testing samples with lower fetal fraction to be analysed, improved turnaround time and reduced cost because of individual hybridisation instead of sample multiplexing.85 

Methylation-Based Technology Another approach to cfDNA analysis focuses on epigenetic markers to differentiate fetal DNA from maternal DNA.87 In 2005, Chim and colleagues showed that the maspin gene, among

CHAPTER 21  Noninvasive Screening for Cytogenetic Disorders (Fetal Aneuploidy Including Microdeletions)

others, is hypomethylated in placental cells but hypermethylated in maternal blood cells.88 A large number of fetomaternal methylation differences have subsequently been detected, primarily on chromosome 21.89,90 This differential pattern of DNA methylation offers a basis for differential analysis of fetal and maternal fragments for NIPS.91-93 However, this approach is limited by the relatively small number of regions that are differentially methylated and have a restriction site suitable for testing with a methylation-sensitive restriction enzyme. The modern use of immunoprecipitation with antibodies specific to 5-methylcytidine has allowed enrichment of hypermethylated DNA sequences specific to chromosome 21 in placental cells.94 In 2009, Papageorgiou and colleagues identified 2000 differentially methylated regions on chromosomes 21, 18, 13, X and Y using this method, the majority in CG-poor nongenic regions.94 Immunoprecipitation allowed the selective enrichment of cffDNA to undergo real-time PCR to measure differences in the amount of chromosome 21-derived DNA from the fetus allowing detection of trisomy 21.90,95 Recent studies continue to define new fetal specific epigenetic markers. Hatt and colleagues presented a comprehensive microarray-based analysis of the methylation status of more than 450,000 CpG sites (regions of DNA where a cytosine nucleotide is followed by a guanine nucleotide in the linear sequence of bases) in maternal blood and CVS samples.96 They defined a list of markers on chromosomes 21, 18, 13 and other autosomes that contain restriction sites for one of 16 different methylation-sensitive restriction enzymes and could potentially be used for NIPS. However, this can only be applied to CpG islands and promoter regions, which cover only a small fraction of the genome. This approach is promising but still has to undergo further clinical trials. If successful, such methodology could reduce the cost of screening dramatically compared to sequencing-based methods.

Methods for Detection of Subchromosomal Abnormalities (Microdeletions and Microduplications) Even before clinical validation, a number of companies offering NIPS have launched and have been offering the option to include a set of five to seven microdeletions: 22 q deletion syndrome (DiGeorge), 5 p (cri-du-chat syndrome), 15 q (Prader–Willi/ Angelman syndromes), 1 p36 deletion syndrome, 4 p (WolfHirschhorn syndrome), 8 q (Langer–Giedion syndrome) and 11 q (Jacobsen syndrome). The frequencies of these microdeletion syndromes are not affected by maternal age and often have no associated fetal anomalies on ultrasound. Although their individual prevalence is overall low ranging from 1 in 4000 (22 q 11.2) to 1 in 50,000 (cri-du-chat), the combined prevalence of all microdeletion and microduplication syndromes is significant. Microdeletions and microduplications are now identifiable by NIPT, but with present technologies, there are still limitations.97,98 Companies quote detection rates ranging from 60% to greater than 99%,98 but there is no doubt that the specificity and positive predictive value (PVV) are significantly lower than NIPS for common aneuploidies because of a lower prevalence in addition to a higher FPR for genomewide deletion detection.99 Data are still lacking to determine accurate sensitivity and specificity and more important, the positive predictive value (PPV) and negative predictive value (NPV). At present, because of the limited clinical testing to

209

evaluate noninvasive detection of deletions, no major national obstetric or genetic organisation recommends screening for microdeletions.100 Depth of sequencing to detect microdeletions and duplications. The statistical power of MPS largely depends on the read

depth and the size of the fetal copy-number variants (CNVs) analysed. To identify increasingly small alterations, deeper sequencing is required to assure sufficient fragments are analysed from the areas of interest. In one approach, sequencing is performed at the level of 1 billion tags as opposed to 10 to 20 million tags with current MPS for fetal whole-chromosome aneuploidy screening.101 Only at this level can fragments from smaller regions of the genome be differentiated. However, although theoretically possible, attaining noninvasive detection of CNVs with the resolution of a chromosomal microarray on amniocytes or villi (CNVs 99.9

49/87

1 in 8506 / 1 in 4265

Monosomy X

90.3

0.23

—

—

—

--

Other sex chromosomes

93.0

0.14

—

—

—

--

Trisomy 21 in TWINS

93.7

0.23

—

—

—

--

(Low/High Risk Pop)

cffDNA, Cell-free fetal DNA.

  

region. The NATUS algorithm would then predict the copy number for the fetus for target regions based on the allele distribution pattern. Wapner and colleagues demonstrated that the SNP-based NIPS for screening a set of 5 microdeletions on 469 samples was highly accurate with detection rates of 97.8% for a 22q11.2 deletion and 100% for Prader-Willi, Angelman, 1p36 deletion and cri-du-chat syndromes.98 FPRs were less than 1%, and no false positives occurred. More recently, Gross and colleagues published their experience using SNP-based NIPS for 22q11.2 deletion syndrome.108 The method is exactly as described earlier with 672 SNPs targeting this specific region. Ninety-five of 21,948 samples were reported as high risk for fetal 22q11.2 deletion; 61 cases had subsequent diagnostic testing. The true positive rate was 18.0%, and the FPR 82.0% giving an overall PPV of 18.0% and a lower PPV of 4.9% for cases with no ultrasound abnormalities. The sensitivity in this study was not available since all negative cases were not followed. 

Clinical Performances of Noninvasive Screening Using Cell-Free Fetal DNA Initial studies of the performance of cfDNA screening were performed on women undergoing diagnostic testing for either advanced maternal age or a positive sequential or combined screening test thus having an exceptionally high risk for the common fetal aneuploidies. These studies demonstrated sensitivities for trisomy 21 of between 97% and 99% and for trisomies 18 and 13 of approximately 85%. The FPR was approximately 1 to 3 per 1000. For sex chromosome detection, the sensitivity was approximately 90%. Two recent metaanalyses of up to 41 studies have recently evaluated this performance and demonstrated even higher sensitivities and reduced FPRs in both low- and high-risk populations109,110 (Table 21.3). To evaluate the performance of NIPS in the general risk population, the Noninvasive Examination of Trisomy (NEXT) study completed in 2015 evaluated almost 16,000 sequential cases regardless of maternal age or a priori risk.111 The mean maternal age was 31 years, and the overall trisomy 21 risk was 1 in 417. All cases of trisomy 21 were detected as were 90% (9 of 10 cases) of trisomy 18 and 100% (2 2 cases) of trisomy 13. The screen-positive rate for each of the common aneuploidies was 3 in 10,000.

In a subgroup analysis of women younger than 35 years of age, the detection rate for trisomy 21 was 100%, and for the lowest risk group of woman having negative biochemical and NT screening and an a priori risk of less than 1 in 270, the performance was equally good. All patients in this study also had conventional first trimester screening performed. In the overall group and the subgroups, cfDNA performed better than NT and biochemistry having both a higher sensitivity and a significantly lower FPR.111

Importance of Fetal Fraction The ability to accurately screen for cytogenetic abnormalities depends on the relative proportion of fetal to maternal cfDNA because lower fetal fractions require deeper sequencing to assure adequate fetal fragment representation.112 If the fetal fraction is below 3% to 4%, many laboratories will not perform NIPS because a reliable result cannot be assured.51-52,112 As explained earlier, one of the more common causes of low fetal fraction is maternal obesity, most likely caused by an excess of DNA fragments from the mother, although this mechanism is still poorly understood.59-60,112,113 Specific fetal aneuploidies are associated with smaller placentas and hence lower levels of fetal cfDNA.65,67 One example is trisomy 18, known to be associated with a smaller placental volume by three-dimensional ultrasound imaging114 and thus having a lower fetal fraction of cfDNA. Rava and colleagues showed the mean fetal fraction for trisomy 18 and trisomy 13 to be 29.7% and 28.3% lower than in euploid pregnancies.67 Several series have shown that failed cffDNA screens increase aneuploidy risk with an odds ratio of 2.5 to 6.2.111,115 Because of this, when a low fetal fraction (A (C)ty et al. 201110). A, Restriction digest of PCR products for two thanatophoric dysplasia (TD) mutations. (A) The c.742C>T type I TD mutation was detected using PCR followed by digestion with AfeI, BsiHKAI and DraIII. cffDNA from a woman carrying an unaffected fetus is digested with AfeI (lane 3) but remains uncut using BsiHKAI (lane 4) and DraIII (lane 5); conversely, in an affected fetus, the AfeI site is destroyed, leaving some of the cffDNA undigested (lane 8), and a BbsI site (lane 9) and a DraIII site (lane 10) are created. B, The c.1948A>G mutation was detected using digestion with the BbsI enzyme and two different primer sets. In the presence of an unaffected fetus, all cffDNA is digested by BbsI (lanes 3 and 8), whereas with an affected fetus, the BbsI restriction site is destroyed, leaving some of the cffDNA uncut (lanes 5 and 10). C, Polymerase chain reaction (PCR) showing amplification of the 132 base pair (bp) region of exon 8 containing the mutation causative for achondroplasia using 5, 10 or 20 μL of deoxyribonucleic acid (DNA) extracted from 400 μL or 800 μL of plasma, as well as on genomic DNA from a normal and a positive control. In the unaffected DNA sample, restriction digest of the PCR product with BsrG1 does not cut the DNA, giving rise to a single 132 bp fragment, whereas if the mutation is present a BsrG1 restriction site is created, and digestion produces fragments of 132, 112 and 20 bp, which results in two bands, rather than the single band seen in the control sample. In all six dilutions of the maternal plasma sample the faint second band can be seen, indicating the presence of the mutation in the maternal plasma sample. (A and  B from  Chitty LS, Khalil A, Barrett AN, et al. Safe, accurate, prenatal diagnosis of thanatophoric dysplasia using ultrasound and free fetal DNA.

The relatively simple technique of PCR restriction enzyme digest (PCR-RED) can be applied to determine presence or absence of some mutations. This method ideally requires knowledge of the mutation before testing and is limited to those in which a restriction enzyme exists which can recognise the mutant or WT allele. This method has been applied by many researchers (see Table 22.2), and it was our initial approach to NIPD which we used for the diagnosis FGFR3-related skeletal dysplasias.10,23 Achondroplasia was one of the early conditions diagnosed using NIPD (see Table 22.2) and is an ideal starting point for development and implementation of NIPD into the routine diagnostic laboratory repertoire as 98% of cases are caused by a single mutation (FGFR3 c. 1138G>A), most cases arise sporadically (resulting from a de novo paternal mutation) and presentation is usually late in pregnancy, when levels of cffDNA are higher, after detection of short limbs on ultrasonography.10 The same technique was also applied for the development of NIPD for thanatophoric dysplasia, also caused by mutations in the FGFR3 gene.23 However, in this condition, there are about 12 causative mutations, some of which are not amenable to the PCR-RED approach. Furthermore, because this is a lethal dominant condition, there is no prior knowledge of the causative mutation when offering diagnosis in cases detected by fetal ultrasound arising de novo. The results delivered by PCR-RED require subjective interpretation of an electrophoresis image (Fig. 22.2), limiting utility of the approach, but still yielding high sensitivity and specificity.16 In a study of 75 cases at risk for achondroplasia or thanatophoric dysplasia, there were five (7%) inconclusive and one false-negative (c.742C>T) result, the latter caused by low fetal fraction despite being tested after 20 weeks’ gestation. For thanatophoric dysplasia, there was a further three cases with other thanatophoric dysplasia-causing mutations not covered by the PCR-RED assays, further demonstrating the limited utility of tests based on individual mutation detection.16 Digital PCR is an alternative approach to PCR-RED which does not rely on subjective interpretation because it gives a digital readout and is more

CHAPTER 22  Noninvasive Prenatal Diagnosis for Single-Gene Disorders

TABLE Estimated Target Molecules for Wild-Type (WT) 22.4 and Mutant Alleles for Fraser Syndrome and

ARPKD Cases Sample

Fraser syndromea

ARPKD

WT Targets

Mutant Targets

Maternal gDNA

1363

1.5

Paternal gDNA

4328

4116

First pregnancy

120

17

Second pregnancy (1)

90

0

Second pregnancy (2)

206

1

Maternal gDNA

2878

0

Paternal gDNA

2698

0

First pregnancy

935

86

Second pregnancy

361

0

aTwo

samples were tested for the second Fraser syndrome pregnancy because the first sample was such an early gestation. Adapted from Lench N, Barrett A, Fielding S, et al. The clinical implementation of non-invasive prenatal diagnosis for single gene disorders: challenges and progress made. Prenat Diagn 33:555–562, 2013. ARPKD, autosomal recessive polycystic kidney disease.

  

sensitive, detecting mutant alleles in maternal plasma that were not detected by PCR-RED.9 Examples are given in Table 22.4 and Figure 22.3, which show results in pregnancies with Fraser syndrome and autosomal recessive polycystic kidney disease (ARPFD). Both PCR-RED and digital PCR have a number of limitations, including low throughput, the need to know (or be able to easily surmise) the mutation in question and a separate assay being required to confirm the presence of cffDNA in the absence of fetal-specific mutation detection. MPS has become increasingly feasible over recent years as sequencing costs and processing time reduce, along with increased output. It is more sensitive, the presence of cffDNA can be confirmed in the same assay as mutation detection, and because it is scalable in terms of sample numbers and regions of interest that can be analysed simultaneously, it is more appropriate for use in a busy service laboratory. Other technologies may offer more potential in the future, but at the moment, MPS offers the best flexibility as illustrated by the paternal mutation exclusion assays now in routine clinical practice in the United Kingdom for a number of diseases using the highly targeted approach of sequencing of specific PCR products (amplicon sequencing).11,16 Primers have been designed to create an FGFR3skeletal dysplasia panel to screen pregnancies with sonographic findings suggestive of achondroplasia or thanatophoric dysplasia and to exclude a recurrence for couples with a previously affected pregnancy.16 As part of the validation process before clinical implementation, 47 samples were tested using the MPS panel, 27 having also been tested by PCR-RED. MPS was 96.2% sensitive (81%–99.3%) and 100% specific (85%–100%), with no inconclusive results, including one positive which was inconclusive by PCR-RED. The MPS panel also detected thanatophoric dysplasia mutations in three cases where no PCR-RED assay was available. There was one false-negative result caused by a rare FGFR3 mutation not covered by the panel, but assay redesign is straightforward, allowing additional mutations to be readily incorporated as required.16 Similar assays have been developed for Apert syndrome9 and to cover a panel of the most common cystic fibrosis (CF) mutations for paternal exclusion in families in which parents carry different mutations.11

221

Extension of the sequencing approach to include families at risk for a wider range of much rarer conditions has recently been reported, including tuberous sclerosis, neurofibromatosis, Rhabdoid tumour predisposition, early infantile epileptic encephalopathy, osteogenesis imperfecta and Fraser syndrome.75 Development of these assays includes sequencing of parental gDNA and, because MPS is a very sensitive technology, in the course of working up tests for these families low level mosaicism has been identified in three mothers and two fathers. This not only increases recurrence risks but, in the case of maternal mosaicism, also renders NIPD impractical because of the background of maternal mutation. Overall, more than 30% of all prenatal molecular genetic tests for monogenic disorders performed in our public-sector Regional Genetics Laboratory in North East Thames in 2015 were delivered via NIPD. Considering all results, 4.3% were inconclusive, one (0.4%) was false negative and others (confirmed after delivery) included 31% mutation positive, and in three families (1.2%), NIPD was not offered because of low-level maternal mosaicism. The relatively high throughput and use of MPS has allowed sample multiplexing to reduce costs and optimise turnaround times to within 5 days. However, because the sensitivity of sequencing can identify unexpected mosaicism in parents, analysis of maternal gDNA should be done in parallel with cfDNA testing to avoid false-positive results caused by low-level maternal mosaicism. 

Noninvasive Prenatal Diagnosis for Autosomal Recessive and Sex-Linked Disorders The paternal exclusion approach to NIPD for recessive conditions in which parents carry different mutations has been described by a number of groups for conditions, including CAH, CF and β-thalassaemia (see Table 22.2), but an invasive test is still required if the paternal mutation is present to determine inheritance of the maternal mutation. Approaches that enable very sensitive estimation of mutation load are required if the paternal allele has been inherited, when parents carry the same mutation and for diagnosis in X-linked conditions because the high background level of the maternal mutation needs to be taken into account. Here RMD can be applied to determine the significance of any allelic imbalance caused by the fetal contribution to cfDNA. Calculation of fetal fraction is required because fetal mutation load will vary with fetal fraction, and thus an additional assay targeting a fetal specific marker is required. Digital PCR and MPS are both very sensitive techniques which allow for single-molecule counting and have been used with RMD for NIPD in these circumstances. Digital PCR has been used for NIPD for β-thalassaemia, haemophilia and sickle cell disorder (see Table 22.2), but it has the same drawbacks discussed earlier – the mutation must be known, a separate assay is required for the fetal-specific marker and multiplexing is not possible. Thus MPS will offer a more practical approach, particularly as some of the more commonly requested prenatal tests for monogenic disorders are for conditions such as β-thalassaemia and CF in which there are multiple causative mutations. Some conditions and mutations require more complex technical approaches. Pathogenic genes with known pseudogenes or regions of high homology are challenging to assess using cfDNA because the methods such as long-range PCR which can be used on gDNA to specifically amplify the gene of interest cannot be applied to the short fragments of cfDNA. As discussed earlier, for recessive or X-linked disorders, RMD needs to be performed to determine the fetal contribution. Although digital PCR has shown some application to definitive diagnosis of recessive disease, standard PCR amplicon methods do not resolve down to the

222

SE C T I O N 6     Prenatal Screening and Diagnosis

Panel02 - Maternal gDNA-c.10261C (wild type)

Panel01 - Maternal gDNA-c.10261T (mutant)

Panel03 - Paternal gDNA-c.10261C (wild type)

Panel04 - Paternal gDNA-c.10261C (wild type)

Panel03 - Paternal gDNA-c.10261T (mutant)

Panel04 - Paternal gDNA-c.10261T (mutant)

Panel05 - 1st Pregnancy cfDNAc.10261C (wild type)

Panel06 - 1st Pregnancy cfDNAc.10261C (wild type)

Panel05 - 1st Pregnancy cfDNAc.10261T (mutant)

Panel06 - 1st Pregnancy cfDNAc.10261T (mutant)

Panel01 - Maternal gDNA-c.10261C (wild type)

Panel02 - Maternal gDNA-c.10261T (mutant)

Panel07 - 2nd Pregnancy cfDNAc.10261C (wild type)

Panel08 - 2nd Pregnancy cfDNAc.10261C (wild type)

Panel07 - 2nd Pregnancy cfDNAc.10261T (mutant)

Panel08 - 2nd Pregnancy cfDNAc.10261T (mutant)

Panel09 -Unaffected cfDNA-c.10261C (wild type)

Panel10 -Unaffected cfDNA-c.10261C (wild type)

Panel09 -Unaffected cfDNA-c.10261T (mutant)

Panel10 -Unaffected ffDNA-c.10261T (mutant)

Panel11 -NTC -c.10261C (wild type)

Panel12 -NTC -c.10261C (wild type)

Panel11 -NTC -c.10261T (mutant)

Panel12 -NTC -c.10261T (mutant)

A Panel01 - Maternal gDNA-c.9374C (wild type)

Panel02 - Maternal gDNA-c.9374C (wild type)

Panel01 - Maternal gDNA-c.9374T (mutant)

Panel02 - Maternal gDNA-c.9374T (mutant)

Panel03 - Paternal gDNA-c.9374C (wild type)

Panel04 - Paternal gDNA-c.9374C (wild type)

Panel03 - Paternal gDNA-c.9374T (mutant)

Panel04 - Paternal gDNA-c.9374T (mutant)

Panel05 - 1st Pregnancy cfDNAc.9374T (mutant)

Panel06 - 1st Pregnancy cfDNAc.9374T (mutant)

Panel05 - 1st Pregnancy cfDNAc.9374C (wild type)

Panel06 - 1st Pregnancy cfDNAc.9374C (wild type)

Panel07 - 2nd Pregnancy cfDNAc.9374C (wild type)

Panel08 - 2nd Pregnancy cfDNAc.9374C (wild type)

Panel07 - 2nd Pregnancy cfDNAc.9374T (mutant)

Panel09 -Unaffected Control cfDNAc.9374C (wild type)

Panel10 -Unaffected Control cfDNAc.9374C (wild type)

Panel09 -Unaffected Control cfDNAc.9374T (mutant)

Panel11 -NTC -c.9374C (wild type)

Panel12 -NTC -c.9374C (wild type)

Panel11 -NTC -c.9374T (mutant)

Panel08 - 2nd Pregnancy cfDNAc.9374T (mutant)

Panel10 -Unaffected Control cfDNAc.9374T (mutant)

Panel12 -NTC -c.9374T (mutant)

B • Fig. 22.3  Heat map images showing digital polymerase chain reaction for Fraser syndrome and autosomal recessive polycystic kidneydisease (ARPKD). Samples were run in duplicate, with wild-type (WT) alleles labelled red and mutant alleles blue. A, For a family with pregnancies at risk for Fraser syndrome (FRAS1 c. 10261C>T), whereas maternal genomic DNA (gDNA) contained only the WT sequence, the paternal had approximately equal numbers of WT and mutant, clearly indicating that he was a carrier of the recessive condition. In the first pregnancy, the fetus had inherited the paternal mutant allele (17 mutant allele counts and 120 WT) ,and the second pregnancy had no paternal mutant allele (see Table 22.4) and thus could not be affected. B, ARPKD samples with WT signal present in all samples but the nonmaternal causative mutation (c.9374C>CT on PKHD1) only found in the affected pregnancy, not in the maternal or paternal gDNA or the cfDNA from the second (unaffected) pregnancy, indicating that this mutation arose de novo. ARPKD, autosomal recessive polycystic kidney disease (With permission from Lench N, Barrett A, Fielding S, et al. The clinical implementation of non-invasive prenatal diagnosis for single gene disorders: challenges and progress made. Prenat Diagn 33:555–562, 2013.)

CHAPTER 22  Noninvasive Prenatal Diagnosis for Single-Gene Disorders

single molecule level, and PCR bias can be introduced. In addition, the mutation must be known and be a single base change or a small indel. RMD is not a useful tool for pathogenic large deletions or rearrangements. However, the haplotyping method for NIPD initially described by the Hong Kong group,2,51 who linked SNPs to the mutation to determine phase and deliver NIPD for β-thalassaemia, has potential for broad application, although it requires sequencing of parental and affected proband DNA. This approach has been reported for CAH,40 maple syrup urine disease43 and DMD56 and uses the affected proband and parental samples to construct mutant and normal haplotypes by linking SNPs to the mutation in question (Fig. 22.4). This has the benefit of not needing to sequence the specific mutation and can therefore be applied in cases of pseudogenes, deletion or conversions and means that a generic assay can be applied to multiple families with different mutations in the same gene. In our laboratory, we have designed, validated, gained UKGTN approvals and implemented definitive MPS haplotyping assays for CF (including cases in which parents carry the same mutation) and for CAH. The workflow for these assays varies.75 For CAH, the first stage is to determine fetal sex. If the fetus is male, no further investigation is required unless the family require definitive diagnosis regardless of fetal sex. For both conditions, if the paternal allele is not inherited, then further investigation is not required. To establish inheritance of the maternal allele, relative haplotype dosage analysis (RHDO) analysis is required. This is based on the principles of RMD described earlier, except that instead of using the single mutation point for RMD, an accumulation of counts from multiple SNPs linked to the maternal mutation are used for a robust assessment of inheritance of maternal alleles. To date, we have found this a very accurate means of prenatal diagnosis, but it can only be applied when both parents and an offspring (affected or unaffected) are available to determine phase. 

Paternal

Proband

Step 3.

Ethical and Social Issues The clinical benefits of introducing NIPD for fetal sex determination and direct diagnosis of single-gene disorders are compelling because these tests are safe, available early in pregnancy and easy to access. It is, however, important that implementation addresses stakeholder views and ethical concerns. Several studies have been undertaken to assess stakeholder attitudes to NIPD, with viewpoints sought from parents who have had NIPD for fetal sex determination72 or NIPD for skeletal dysplasias,76 as well as with carriers of single-gene disorders77-79 and health professionals.70,80 Attitudes to NIPD are generally positive, and stakeholders highlight the many practical and psychological benefits NIPD affords through opportunities for safe and early testing.76,77,79 One key benefit is that decision making may be emotionally easier because parents do not have to consider the risk for miscarriage.77,79,80 Notably, easier decision making was highlighted by parents with a low risk for recurrence after a pregnancy with a skeletal dysplasia because NIPD in subsequent pregnancies allows early reassurance without putting the pregnancy at risk.76 The most common concerns held by stakeholders were the potential for NIPD to be viewed as routine or expected, which may undermine informed choice and increased pressure to have prenatal testing as NIPD because the test is safe and simple to perform. For most, the benefits of NIPD were thought to balance out these concerns, which stakeholders thought could be addressed with formal regulation of services and appropriate pretest counselling in which the consent process is highlighted and

Step 2.

Step 1.

Maternal

223

Proband Step 4.

Maternal mutant allele

Paternal wild-type allele

Maternal wild-type allele

SNP linked maternal mutant allele

Paternal mutant allele

SNP linked paternal mutant allele

•

Fig. 22.4 Haplotyping approach for noninvasive prenatal diagnosis of recessive conditions. Step 1: DNA from couple at risk for affected pregnancy and their affected proband is obtained. Step 2: DNA enriched for heterozygous single nucleotide polymorphisms (SNPs), and these SNPs are linked to affected allele. Step 3: In subsequent at-risk pregnancy, cellfree DNA (cfDNA) is extracted from maternal plasma and cfDNA enriched for SNPs linked to affected allele. Step 4: To determine if paternal mutant allele inherited by fetus, the presence or absence of SNPs linked to paternal mutation (blue stars) is established. Inheritance of maternal mutant allele is established if there is an overrepresentation of SNPs linked to maternal mutation (red stars).

discussion includes the benefits and limitations of NIPD, alternatives, encouragement to reflect on the reasons for choosing NIPD and the impact of results.76-78 For service delivery, women and health professionals highlighted the need for NIPD to be provided through specialised genetics services so that counselling would be undertaken by health professionals with specialist knowledge of the condition and trained in counselling for prenatal testing. Implementation must be accompanied, but a health educational

224

SE C T I O N 6     Prenatal Screening and Diagnosis

programme to ensure that those offering NIPD highlight all aspects of testing to encourage informed parental choice.72,76,77 In a number of studies, carriers of recessive conditions, including those who had previously declined invasive prenatal diagnosis, said they would choose to have NIPD in subsequent pregnancies, not necessarily to inform decisions regarding termination of pregnancy but because they would value the information to prepare for the birth of an affected child.77,79 There were ethical concerns over the appropriateness of directing resources to testing that would not change pregnancy management, particularly in state-funded health systems. 

Economic Issues Comparison of total costs of prenatal diagnosis in the United Kingdom using NIPD or invasive testing for a representative set of single-gene disorders has shown that for autosomal dominant conditions with straightforward molecular techniques, NIPD was cheaper than invasive testing (£314 less), but the more complex and, as a result, more expensive NIPD approaches needed for autosomal recessive and X-linked conditions increased costs above those of invasive testing (£141–£1090 more).81 However, as discussed earlier, research with stakeholders strongly suggests that uptake of NIPD is likely to be high, and many of the couples that would not consider invasive testing because of the risk for miscarriage will want NIPD. This increased uptake is expected to result in the overall costs of the NIPD care pathways being considerably more expensive than current invasive testing pathways.11,81 However, as sequencing costs continue to fall, the extent of these discrepancies will be reduced. The cost of delivering a NIPD service can be reduced by multiplexing testing for conditions, such as CF or sickle cell disorder, or testing for several different conditions or multiple causative mutations in a single assay. As we have moved most of our NIPD for monogenic disorders to MPS platforms, we have seen a reduction in cost but also a reduction in turnaround times because the volume of testing done is such that we have several sequencing runs a week. This then raises the question as to how many laboratories should develop these services. Furthermore, for the very rare single-gene disorders, there is

a question as to the cost and benefits of developing NIPD when tests are performed so infrequently, an issue that raises potential ethical concerns regarding restricting access to safer testing. This is particularly the case for bespoke testing because costs are high because of the cost of consumables and staff time involved in working up a test.75 

Conclusions Noninvasive prenatal diagnosis based on cfDNA is dramatically changing prenatal care. Fetal sex determination is well established as a clinical service in many countries and enables accurate determination of fetal sex from 7 to 9 weeks’ gestation. NIPD is now available for some single-gene disorders which arise de novo, such as achondroplasia, and new technologies such as digital PCR and MPS are allowing NIPD to be offered through some accredited public-sector laboratories for a range of recessive conditions, including CF and CAH. The availability of NIPD for monogenic disorders still seems largely limited to laboratories in the United Kingdom, and we have a long way to go to offer equity of access to families at risk for monogenic disorders worldwide. There is significant scope for the future of NIPD for single-gene disorders as proof-of-principle studies have shown that is possible to map the entire fetal genome using cffDNA. Although currently restricted by the cost of the large amount of sequencing involved, this technology will ultimately allow testing for mutations when there is a known family history as well as de novo mutations. However, any advance must be accompanied by rigorous and largescale evaluations before clinical implementation, and continued research considering ethical issues, stakeholder views and implementation strategies is essential to ensure cfDNA testing is being offered appropriately. Finally, after tests are introduced into clinical practice, ongoing audit and monitoring of both test accuracy and service delivery through recognised quality assurance schemes is important. Access the complete reference list online at ExpertConsult.com. Self-assessment questions available at ExpertConsult.com.

References 1. Lo YM, Zhang J, Leung TN, et  al. Rapid clearance of fetal DNA from maternal plasma. Am J Hum Genet. 1999;64:218–224. 2. Lo Y, Chan K, Sun H, et al. Maternal plasma DNA sequencing reveals the genome-wide genetic and mutational profile of the fetus. Sci Transl Med. 2010;2:61ra91. 3. Alberry M, Maddocks D, Jones M, et  al. Free fetal DNA in maternal plasma in anembryonic pregnancies: confirmation that the origin is the trophoblast. Prenat Diagn. 2007;27:415–418. 4. Hill M, Finning K, Martin P, et  al. Noninvasive prenatal determination of fetal sex: translating research into clinical practice. Clin Genet. 2011;80:68–75. 5. Norton ME, Brar H, Weiss J, et  al. NonInvasive Chromosomal Evaluation (NICE) study: results of a multicenter prospective cohort study for detection of fetal trisomy 21 and trisomy 18. Am J Obstet Gynecol. 2012;207: 137.e1–e8. 6. Leung TY, Qu JZ, Liao GJ, et al. Noninvasive twin zygosity assessment and aneuploidy detection by maternal plasma DNA sequencing. Prenat Diagn. 2013;33:675–681. 7. Hyett JA, Gardener G, Stojilkovic-Mikic T, et al. Reduction in diagnostic and therapeutic interventions by non-invasive determination of fetal sex in early pregnancy. Prenat Diagn. 2005;25:1111–1116. 8. Finning K, Martin P, Daniels G. A clinical service in the UK to predict fetal Rh (Rhesus) D blood group using free fetal DNA in maternal plasma. Ann N Y Acad Sci. 2004;1022:119–123. 9. Lench N, Barrett A, Fielding S, et  al. The clinical implementation of non-invasive prenatal diagnosis for single gene disorders: challenges and progress made. Prenat Diagn. 2013;33:555–562. 10. Chitty LS, Griffin DR, Meaney C, et al. New aids for the non-invasive prenatal diagnosis of achondroplasia: dysmorphic features, charts of fetal size and molecular confirmation using cell-free fetal DNA in maternal plasma. Ultrasound Obstet Gynecol. 2011;37:283–289. 11. Hill M, Twiss P, Verhoef TI, et  al. Noninvasive prenatal diagnosis for cystic fibrosis: detection of paternal mutations, exploration of patient preferences and cost analysis. Prenat Diagn. 2015;35:950–958. 12. Saito H, Sekizawa A, Morimoto T, et  al. Prenatal DNA diagnosis of a single-gene disorder from maternal plasma. Lancet. 2000;356:1170. 13. Li Y, Holzgreve W, Page-Christiaens GC, et  al. Improved prenatal detection of a fetal point mutation for achondroplasia by the use of size-fractionated circulatory DNA in maternal plasma - case report. Prenat Diagn. 2004;24:896–898. 14. Li Y, Page-Christiaens GC, Gille JJ, et  al. Non-invasive prenatal detection of achondroplasia in size-fractionated cell-free DNA by MALDI-TOF MS assay. Prenat Diagn. 2007;27:11–17. 15. Lim JH, Kim MJ, Kim SY, et al. Non-invasive prenatal detection of achondroplasia using circulating fetal DNA in maternal plasma. J Assist Reprod Genet. 2011;28:167–172. 16. Chitty LS, Mason S, Barrett AN, et al. Noninvasive prenatal diagnosis of achondroplasia

and thanatophoric dysplasia: next-generation sequencing allows for a safer, more accurate, and comprehensive approach. Prenat Diagn. 2015;35:656–662. 17. Au PK, Kwok YK, Leung KY, Tang LY, Tang MH, Lau ET. Detection of the S252W mutation in fibroblast growth factor receptor 2 (FGFR2) in fetal DNA from maternal plasma in a pregnancy affected by Apert syndrome. Prenat Diagn. 2011;31:218–220. 18. Raymond FL, Whittaker J, Jenkins L, et  al. Molecular prenatal diagnosis: the impact of modern technologies. Prenat Diagn. 2010;30:674–681. 19. González-González MC, Trujillo MJ, Rodríguez de Alba M, et  al. Huntington diseaseunaffected fetus diagnosed from maternal plasma using QF-PCR. Prenat Diagn. 2003;23:232–234. 20. Bustamante-Aragones A, Trujillo-Tiebas MJ, Gallego-Merlo J, et  al. Prenatal diagnosis of Huntington disease in maternal plasma: direct and indirect study. Eur J Neurol. 2008;15:1338–1344. 21. González-González MC, Garcia-Hoyos M, Trujillo-Tiebas MJ, et  al. Improvement in strategies for the non-invasive prenatal diagnosis of Huntington disease. J Assist Reprod Genet. 2008;25:477–481. 22. Amicucci P, Gennarelli M, Novelli G, Dallapiccola B. Prenatal diagnosis of myotonic dystrophy using fetal DNA obtained from maternal plasma. Clin Chem. 2000;46:301–302. 23. Chitty LS, Khalil A, Barrett AN, et al. Safe, accurate, prenatal diagnosis of thanatophoric dysplasia using ultrasound and free fetal DNA. Prenat Diagn. 2013;33:416–423. 24. Meaney C, Norbury G. Non-invasive prenatal diagnosis of early onset primary dystonia I in maternal plasma. Prenat Diagn. 2009;29:1218–1221. 25. Chiu RW, Lau TK, Cheung PT, et al. Noninvasive prenatal exclusion of congenital adrenal hyperplasia by maternal plasma analysis: a feasibility study. Clin Chem. 2002;48:778– 780. 26. Galbiati S, Stenirri S, Sbaiz L, et al. Identification of an 18 bp deletion in the TWIST1 gene by CO-amplification at lower denaturation temperature-PCR (COLD-PCR) for non-invasive prenatal diagnosis of craniosynostosis: first case report. Clin Chem Lab Med. 2014;52:505–509. 27. González-González MC, García-Hoyos M, Trujillo MJ, et al. Prenatal detection of a cystic fibrosis mutation in fetal DNA from maternal plasma. Prenat Diagn. 2002;22:946–948. 28. Bustamante-Aragones A, Gallego-Merlo J, Trujillo-Tiebas MJ, et al. New strategy for the prenatal detection/exclusion of paternal cystic fibrosis mutations in maternal plasma. J Cyst Fibros. 2008;7:505–510. 29. Fucharoen G, Tungwiwat W, Ratanasiri T, Sanchaisuriya K, Fucharoen S. Prenatal detection of fetal hemoglobin E gene from maternal plasma. Prenat Diagn. 2003;23(5):393–396. 30. Tungwiwat W, Fucharoen G, Fucharoen S, et  al. Application of maternal plasma DNA analysis for non-invasive prenatal diagnosis of Hb E-beta-thalassemia. Transl Res. 2007;150:319–325. 31. Lazaros L, Hatzi E, Bouba I, et  al. Noninvasive prenatal detection of paternal origin Hb lepore in a male fetus at the 7th week of gestation. Fet Diagn Ther. 2006;21:506–509.

32. Bustamante-Aragones A, Vallespin E, Rodriguez de Alba M, et al. Early non-invasive prenatal detection of a fetal CRB1 mutation causing Leber congenital amaurosis. Mol Vis. 2008;14:1388–1394. 33. Bustamante-Aragones A, Pérez-Cerdá C, Pérez B, et al. Prenatal diagnosis in maternal plasma of a fetal mutation causing propionic acidemia. Mol Genet Metab. 2008;95:101– 103. 34. Tungwiwat W, Fucharoen S, Fucharoen G, et al. Development and application of a realtime quantitative PCR for prenatal detection of fetal alpha(0)-thalassemia from maternal plasma. Ann N Y Acad Sci. 2006;1075:103– 107. 35. Ho SS, Chong SS, Koay ES, et al. Noninvasive prenatal exclusion of haemoglobin Bart’s using foetal DNA from maternal plasma. Prenat Diagn. 2010;30(1):65–73. 36. Galbiati S, Brisci A, Lalatta F, et  al. Full COLD-PCR protocol for non-invasive prenatal diagnosis of genetic diseases. Clin Chem. 2011;57:136–138. 37. Papasavva T, Kalakoutis G, Kalikas I, et  al. Non-invasive prenatal diagnostic assay for the detection of beta-thalassemia. Ann N Y Acad Sci. 2006;1075:148–153. 38. Papasavva T, Kalikas I, Kyrri A, Kleanthous M. Arrayed primer extension for the noninvasive prenatal diagnosis of beta-thalassemia based on detection of single nucleotide polymorphisms. Ann N Y Acad Sci. 2008;1137: 302–308. 39. Ramezanzadeh M, Salehi M, Farajzadegan Z, Kamali S, Salehi R. Detection of paternally inherited fetal point mutations for betathalassemia in maternal plasma using simple fetal DNA enrichment protocol with or without whole genome amplification: an accuracy assessment. J Matern Fetal Neonatal Med. 2016;29:2645–2649. 40. New MI, Tong YK, Yuen T, et al. Noninvasive prenatal diagnosis of congenital adrenal hyperplasia using cell-free fetal DNA in maternal plasma. J Clin Endocrinol Metab. 2014;99:E1022–E1030. 41. Ma D, Ge H, Li X, et  al. Haplotype-based approach for noninvasive prenatal diagnosis of congenital adrenal hyperplasia by maternal plasma DNA sequencing. Gene. 2014;544:252–258. 42. Mouawia H, Saker A, Jais JP, et al. Circulating trophoblastic cells provide genetic diagnosis in 63 fetuses at risk for cystic fibrosis or spinal muscular atrophy. Reprod Biomed Online. 2012;25:508–520. 43. You Y, Sun Y, Li X, et al. Integration of targeted sequencing and NIPT into clinical practice in a Chinese family with maple syrup urine disease. Genet Med. 2014;16:594–600. 44. Gu W, Koh W, Blumenfeld YJ, et  al. Noninvasive prenatal diagnosis in a fetus at risk for methylmalonic acidemia. Genet Med. 2014;16:564–567. 45. Barrett AN, McDonnell TCR, Allen Chan KC, Chitty LS. Digital PCR analysis of maternal plasma for non-invasive detection of sickle cell anemia. Clin Chem. 2012;58:1026–1032. 46. Phylipsen M, Yamsri S, Treffers EE, et  al. Non-invasive prenatal diagnosis of betathalassemia and sickle-cell disease using pyrophosphorolysis-activated polymerization and melting curve analysis. Prenat Diagn. 2012;32:578–587.

224.e1

224.e2

References

47. Chen M, Lu S, Lai Z, et al. Targeted sequencing of maternal plasma for haplotype-based noninvasive prenatal testing of spinal muscular atrophy. Ultrasound Obstet Gynecol. 2017; 49:799–802. 48. Sirichotiyakul S, Charoenkwan P, Sanguansermsri T. Prenatal diagnosis of homozygous alpha-thalassemia-1 by cell-free fetal DNA in maternal plasma. Prenat Diagn. 2012;32(1):45–49. 49. Yan TZ, Mo QH, Cai R, et al. Reliable detection of paternal SNPs within deletion breakpoints for non-invasive prenatal exclusion of homozygous α-thalassemia in maternal plasma. PLoS One. 2011;6(9):e24779. 50. Lun FM, Tsui NB, Chan KC, et al. Noninvasive prenatal diagnosis of monogenic diseases by digital size selection and relative mutation dosage on DNA in maternal plasma. Proc Natl Acad Sci U S A. 2008;105:19920–19925. 51. Lam KW, Jiang P, Liao GJ, et  al. Noninvasive prenatal diagnosis of monogenic diseases by targeted massively parallel sequencing of maternal plasma: application to beta-thalassemia. Clin Chem. 2012;58:1467–1475. 52. Papasavva T, van Ijcken WF, Kockx CE, et al. Next generation sequencing of SNPs for non-invasive prenatal diagnosis: challenges and feasibility as illustrated by an application to β-thalassaemia. Eur J Hum Genet. 2013;21(12):1403–1410. 53. Zafari M, Gill P, Kowsaryan M, Alipour A, Banihashemi A. High-resolution melting analysis for noninvasive prenatal diagnosis of IVS-II-I (G-A) fetal DNA in minor betathalassemia mothers. J Matern Fetal Neonatal Med. 2016;29:3323–3328. 54. Tsui NB, Kadir RA, Chan KC, et  al. Noninvasive prenatal diagnosis of hemophilia by microfluidics digital PCR analysis of maternal plasma DNA. Blood. 2011;117:3684–3691. 55. Bustamante-Aragones A, Garcia-Hoyos M, Rodriguez De Alba M, et  al. Detection of a paternally inherited fetal mutation in maternal plasma by the use of automated sequencing. Ann N Y Acad Sci. 2006;1075:108–117. 56. Parks M, Court S, Cleary S, et al. Non-invasive prenatal diagnosis of Duchenne and Becker muscular dystrophies by relative haplotype dosage. Prenat Diagn. 2016;36:312–320. 57. Pertl B, Sekizawa A, Samura O, et al. Detection of male and female fetal DNA in maternal plasma by multiplex fluorescent polymerase chain reaction amplification of short tandem repeats. Hum Genet. 2000;106:45–49. 58. Page-Christiaens GC, Bossers B, VAN DER Schoot CE, DE Haas M. Use of bi-allelic insertion/deletion polymorphisms as a positive control for fetal genotyping in maternal

blood: first clinical experience. Ann N Y Acad Sci. 2006;1075:123–129. 59. White HE, Dent CL, Hall VJ, Crolla JA, Chitty LS. Evaluation of a novel assay for detection of the fetal marker RASSF1A: facilitating improved diagnostic reliability of non-invasive prenatal diagnosis. PLoS One. 2012;7:e45073. 60. Wang E, Batey A, Struble C, et al. Gestational age and maternal weight effects on fetal cellfree DNA in maternal plasma. Prenat Diagn. 2013;33:662–666. 61. Barrett AN, Zimmermann BG, Wang D, Holloway A, Chitty LS. Implementing prenatal diagnosis based on cell-free fetal DNA: accurate identification of factors affecting fetal DNA yield. PLoS One. 2011;6:e25202. 62. Minear MA, Lewis C, Pradhan S, Chandrasekharan S. Global perspectives on clinical adoption of NIPT. Prenat Diagn. 2015;35:959–967. 63. Bustamante-Aragones A, Rodriguez de Alba M, Gonzalez-Gonzalez C, et  al. Foetal sex determination in maternal blood from the seventh week of gestation and its role in diagnosing haemophilia in the foetuses of female carriers. Haemophilia. 2008;14:593–598. 64. Tardy-Guidollet V, Menassa R, Costa JM, et  al. New management strategy of pregnancies at risk of congenital adrenal hyperplasia using fetal sex determination in maternal serum: French cohort of 258 cases (2002-2011). J Clin Endocrinol Metab. 2014;99:1180–1188. 65. Devaney SA, Palomaki GE, Scott JA, Bianchi DW. Noninvasive fetal sex determination using cell-free fetal DNA: a systematic review and meta-analysis. JAMA. 2011;306:627–636. 66. Heland S, Hewitt JK, McGillivray G, Walker SP. Preventing female virilisation in congenital adrenal hyperplasia: the controversial role of antenatal dexamethasone. Aust N Z J Obstet Gynaecol. 2016;56:225–232. 67. Everett TR, Chitty LS. Cell-free fetal DNA: the new tool in fetal medicine. Ultrasound Obstet Gynecol. 2015;45:499–507. 68. Sillence KA, Roberts LA, Hollands HJ, et al. Fetal sex and RHD genotyping with digital PCR demonstrates greater sensitivity than real-time PCR. Clin Chem. 2015;61:1399– 1407. 69. Jacob RR, Saxena R, Verma IC. Noninvasive diagnosis of fetal gender: utility of combining DYS14 and SRY. Genet Test Mol Biomarkers. 2015;19:505–511. 70. Hill M, Compton C, Lewis C, Skirton H, Chitty LS. Determination of foetal sex in pregnancies at risk of haemophilia: a qualitative study exploring the clinical practices and

attitudes of health professionals in the United Kingdom. Haemophilia. 2011;18:575–583. 71. Hill M, Taffinder S, Chitty LS, Morris S. Incremental cost of non-invasive prenatal diagnosis versus invasive prenatal diagnosis of fetal sex in England. Prenat Diagn. 2011;31:267–273. 72. Lewis C, Hill M, Skirton H, Chitty LS. Non-invasive prenatal diagnosis for fetal sex determination: benefits and disadvantages from the service users’ perspective. Eur J Hum Genet. 2012;20:1127–1133. 73.  Association of Clinical Genetics Science. Association of Clinical Genetics Science Activity Audit 2014 -15; 2016. Available from: http: //www.acgs.uk.com/media/982691/acgsaudit 14_15_reviewedv2a.pdf. 74. Bustamante-Aragones A, Rodriguez de Alba M, Perlado S, et  al. Non-invasive prenatal diagnosis of single-gene disorders from maternal blood. Gene. 2012;504:144–149. 75. Jenkins LA, Deans ZC, Lewis C, Allen S. Delivering an accredited non-invasive prenatal diagnosis service for monogenic disorders and recommendations for best practice. Prenat Diagn. 2018;38(1):44–51. 76. Lewis C, Hill M, Chitty LS. Non-invasive prenatal diagnosis for single gene disorders: experience of patients. Clin Genet. 2014;85:336–342. 77. Hill M, Compton C, Karunaratna M, Lewis C, Chitty L. Client views and attitudes to non-invasive prenatal diagnosis for sickle cell disease, thalassaemia and cystic fibrosis. J Genet Couns. 2014;23:1012–1021. 78. Skirton H, Goldsmith L, Chitty LS. An easy test but a hard decision: ethical issues concerning non-invasive prenatal testing for autosomal recessive disorders. Eur J Hum Genet. 2015;23:1004–1009. 79. Hill M, Suri R, Nash E, Morris S, Chitty LS. Preferences for prenatal tests for cystic fibrosis: a discrete choice experiment to compare the views of adult patients, carriers of cystic fibrosis and health professionals. J Clin Med. 2014;3:176–190. 80. Hill M, Karunaratna M, Lewis C, Forya F, Chitty L. Views and preferences for the implementation of non-invasive prenatal diagnosis for single gene disorders from health professionals in the United Kingdom. Am J Med Genet A. 2013;161A:1612–1618. 81. Verhoef TI, Hill M, Drury S, et  al. Noninvasive prenatal diagnosis (NIPD) for single gene disorders: cost analysis of NIPD and invasive testing pathways. Prenat Diagn. 2016;36:636–642.

23

Invasive Diagnostic Procedures ANTHONY O. ODIBO AND GANESH ACHARYA

KEY POINTS • Amniocentesis is used from 15 weeks of gestation onwards for prenatal diagnosis of chromosomal abnormalities, single-gene disorders, fetal lung maturity, fetal infections and inflammation. • Chorionic villus sampling (CVS) is used from 10 weeks of gestation onwards for prenatal diagnosis of single-gene defects and chromosomal abnormalities. • Early amniocentesis (30% live beyond 1 yr • >50% of semi- and lobar HPE >1 yr

Neurologic impairment

• No individuals with alobar HPE can sit or speak • 50% of individuals with lobar HPE able to walk, have mildly impaired hand function and speak single words • Mild interhemispheric variant: walking with assistance, speak, function with mild impairment

Morbidity

• Ventriculoperitoneal shunts: 17% • Anticonvulsant therapy: 40% • Cerebral palsy • Swallowing problems, chronic lung disease caused by aspiration • Poor gastric emptying, reflux, constipation • Hypothalamic dysfunction: sleeping problem, temperature regulation, endocrine disorders

Adapted from Stashinko EE, Clegg NJ, Kammann HA, et al. A retrospective survey of perinatal risk factors of 104 living children with holoprosencephaly. Am J Med Genet A 128A(2): 114–119, 2004.

  

to develop from the lamina terminalis as a bundle of fibres that connects the two hemispheres. The development of the CC is closely related to the normal appearance of the septa pellucida.68 Anterior to the foramina of Monroe, the space between the septa is called the cavum cave septi pellucidi (CSP); posterior to this structure, it is referred to as the cavum vergae (CV) The CC, which is composed of four segments, starts developing from the 11th week of gestation with the genu, and subsequently the body, isthmus and splenium form. The rostrum, the most anterior part, develops later. Completion of the CC is achieved by 18 to 20 weeks of gestation. At the median surface of the cerebrum, the gyrus cinguli creates a partial girdle around the CC and follows the callosal curve. Normal sonographic development of the anterior and posterior complex in the axial plane indicate adequate midline development; however, morphologic abnormality in both complexes is a strong indicator for midline abnormalities and cortical malformations.69 The midsagittal and the coronal views are the best planes to directly visualise the CC. In a midsagittal two-dimensional view, the CC appears as a thin anechoic space, delineated superiorly and inferiorly by two smooth echogenic lines. The complete visualisation and measurement of the CC is feasible from 18 weeks’ gestation onwards. Normative charts can be used to evaluate the length and thickness of the CC. Transvaginal ultrasound, multiplanar 3D and three-dimensional volume contrast imaging in the C-plane (VCI-C) imaging facilitate the proper identification.70,71 The midsagittal plane reveals the CC with all its neighbouring structures; the CSP, the CV, the cavum veli interpositi and the cingulate gyrus. Using colour-flow Doppler, the anterior cerebral and pericallosal arteries with their branches and the vein of Galen are easily displayed in the early second trimester. The terminology to describe CC dysgenesis includes complete and partial agenesis, hypoplasia (thinning of the CC), hyperplasia (thickening of the CC) and morphologically oddly shaped CC.72 A prevalence of 1.4 and 0.4 per 10,000 live births for ACC and hypoplasia of the CC has been suggested, respectively. These

287

figures may be an underestimate as a proportion of asymptomatic ACC patients escape detection.73 Absence of the Corpus Callosum. Complete absence of the CC (ACC) results in an abnormal induction of medial cerebral convolutions determining a radiate arrangement of the cerebral sulci around the roof of the third ventricle. With ACC, the semicircular loop of the pericallosal artery is lost, and branches of the anterior cerebral artery ascend linearly with a radial arrangement. ACC has been associated with other cranial anomalies, such as abnormalities of the posterior fossa and interhemispheric cyst and neuronal migration disorders. Because the development of the CC coincides with cortical development, lissencephaly, heterotopia, polymicrogyria (PMG) and schizencephaly are often present. The rate of chromosomal anomalies is estimated at about 17.8%, including microdeletions for which array comparative genomic hybridisation might be considered. In addition, ACC-linked syndromes show an autosomal dominant, recessive or X-linked mode of inheritance.74 An OMIM (Online Mendelian Inheritance in Man) search results in more than 200 entries, of which Aicardi and Andermann syndromes are the more common. Occasionally, a metabolic aetiology is found, such as hyperglycemia without ketose, a pyruvate dehydrogenase deficiency or phenylketonuria.75 Indirect sonographic features are often helpful when screening for the absence of the CC. The absent CSP should raise suspicion,68,76 and dilation of the atria and occipital horns (colpocephaly) shaping the lateral ventricles like teardrops is very suggestive for CCA (Fig. 28.23A). There is, however, no progressive VM.77 On a coronal view, the lateral ventricles are displaced laterally because of the failure of the bundles of Probst to cross the midline, making the anterior parts of the lateral ventricles look like bull’s horns. Often the third ventricle is enlarged and extends posterior and cranially. Because of the lack of communicating fibres, there is an increased distance between both hemispheres (Fig. 28.23B), which shows in the axial plane as three parallel lines: the falx cerebri and the medial borders of the two hemispheres. However, at the time of midtrimester ultrasound screening, indirect signs may be either absent or barely visible but may appear more clearly later in gestation. In a sagittal view, ACC results in an abnormal induction of the medial cerebral convolutions, leading to a radial arrangement of the sulci, strengthened by the hypoplasia or absence of the cingulate gyrus. By colour-flow Doppler, the pericallosal arteries display an irregular radiant vascular pattern (Figs. 28.24 and 28.25).  Partial Agenesis of the Corpus Callosum. In partial ACC, the CSP is usually present but is abnormally shaped.78,79 Only the midsagittal views of the fetal brain allows for the differentiation among complete agenesis, hypoplasia or partial formation of the CC80,81 (Fig. 28.26). In hypoplasia of the CC, often the posterior portion is affected. Only a handful of cases have been detected prenatally.82 The pericallosal artery follows the anterior part of the CC but loses its normal course posteriorly.  Corpus Callosum Dysgenesis. A thick CC is identified in 5% of CC abnormalities. It can be associated with macrocephaly, macrocephaly-capillary syndrome83 and Cohen syndrome.84 Hyperechogenicity of the CC is characteristic of pericallosal lipoma which is often isolated. Sometimes it is part of a frontonasal dysplasia, Goldenhar syndrome or Pai syndrome.85 Because of the association with chromosomal abnormalities of various kinds, array CGH is highly recommended. In addition to the fetal

SE C T I O N 7     Diagnosis and Management of Fetal Malformations

288

A

B • Fig. 28.23  Indirect sonographic signs suggesting complete absence of the corpus callosum. A, Colpocephaly with a teardrop-shaped lateral ventricle. B, Three-layer sign.

•

•

neurosonogram, a detailed examination of all fetal organ systems, especially the fetal heart, the genitourinary system and the skeleton, should be performed. The identification by means of fetal MRI of more discrete CNS lesion (≤22.5%) such as abnormal gyration, heterotopia and migration anomalies in association with CCA enables refinement of the diagnosis and prognosis.86,87 MR is less accurate in measuring thickness of the CC because of the low spatial resolution of MRI.88 Subjective callosal thickening should alert the specialist, and additional abnormalities should be looked for.88,89 In addition, new MRI techniques such as fibre tracking and functional MRI may help to differentiate isolated cases with usually a good prognosis from those with additional cerebral lesions and an adverse outcome.90  Neurologic Outcome. Significant neurodevelopmental delay may occur in 15% to 36% of the cases of isolated CCA.80,91 The presence of other cerebral and extracerebral malformations worsens the neurodevelopmental outcome.92 A systematic review assessing the of neurodevelopmental outcome

in 132 fetuses revealed a normal outcome in 74.3% with complete CCA and in 65.5% of partial CCA. Moderate and severe disability was reported in 14.3% and 11.4% of CCA and in 6.9% and 27.6% of partial CCA, respectively.86 Subtle perceptual, neuropsychological and motor defects may arise later in life. Of major importance, an additional 15% of prenatally isolated ACC cases were found to have associated problems after birth.75 At the age of 10 years, 75% of the children have a normal intelligence but frequently with mild learning difficulties.93,94  Absence of the Cavum Septi Pellucidi. Absence of the CSP occurs in about 0.2 to 0.3 per 10,000 pregnancies.95,96 The CSP appears as a fluid-filled box on an axial plane between the frontal horns, the CC and the thalami. In a sagittal plane, it is localised under the anterior part of the CC. The CSP is square in 73% and triangular in 27%; however, in cases with CV, the appearance is rectangular.69 The mean width at midtrimester is 3.4 mm. The CSP progressively decreases in size from 26 weeks of gestation. By term, the closure of the CSP is

Fig. 28.24  Abnormal irregular radial vascularisation pattern of the pericallosal artery in complete absence of the corpus callosum.

Fig. 28.25 Sagittal T2 HASTE image of the brain in a fetus at 31 weeks. Absence of the corpus callosum with visualisation of the fornices (circle) and typical sunburst radiation of the gyri.

CHAPTER 28  Sonography of the Fetal Central Nervous System

seen in 97% of fetuses, although occasionally, this cavity remains open until adulthood.68 Failure to visualise the CSP between 18 and 37 weeks is highly indicative of cerebral anomalies. Although it may occur as a normal variant if isolated,76 absence may be associated with the HPE spectrum, septo-optic dysplasia (SOD), callosal dysgenesis and hypogenesis, chronic severe hydrocephalus resulting from aqueductal stenosis or CM, schizencephaly, porencephaly or hydranencephaly and basilar encephaloceles. Genetic causes are rare. The persistence of an enlarged CSP (>1cm) beyond infancy has been associated with cerebral dysgenesis. 

289

Septo-Optic Dysplasia. Septo-optic dysplasia can be suspected when the CSP is absent in an otherwise normal brain (Fig. 28.27). The frontal horns are fused at the midline, and the CC is frequently thin. VM can be present. In experienced hands, sonographic 3D visualisation and measurement of the chiasma and optic nerves may confirm the diagnosis.97 Fetal MRI is generally more suited to exclude optic tract hypoplasia. Additional endocrinologic evaluation and visual assessment are mandatory to differentiate from isolated absent CSP. The prognosis of an isolated absent CSP is usually good.98 In nonisolated cases, the prognosis is related to the associated conditions. The recurrence risk for isolated absent CSP is low, but in a few cases, Mendelian inheritance is suggested. 

Posterior Fossa Anomalies Introduction. By the fifth or sixth gestational week, the pontine

•

Fig. 28.26 Partial agenesis of the corpus callosum: the rostrum and genu are absent (arrowhead). Note the presence of a well-developed cavum septi pellucidi (asterisk).

flexure creates the plica choroidea and divides the rhombencephalon into the metencephalon and the myelencephalon. Invagination divides the roof of the fourth ventricle into an anterior and a posterior membranous area. The anterior membranous area gives rise to the cerebellar vermis. Cerebellar vermian growth forces the posterior membranous area of the roof of the fourth ventricle to invaginate posteriorly below the vermis, resulting in the Blake pouch. The median fenestration of the Blake pouch by the foramen of Magendie leads to its subsequent disappearance. The foramina of Luschka open later, around the fourth month. In 1% to 2% of healthy subjects, the foramen of Magendie is absent; the communication between the fourth ventricle and the subarachnoid space is established when the foramina of Luschka open.99 The cerebellum results from the development of two lateral primordia that fuse subsequently across the midline to form the vermis, starting at the end of the sixth week. At the 11th week, the cerebellum covers the fourth ventricle and subsequently

• Fig. 28.27  Fusion of the anterior horns and absence of the septum pellucidum has been observed as an isolated finding in association with optic atrophy in septo-optic dysplasia and in the presence of lobar holoprosencephaly.

290

SE C T I O N 7     Diagnosis and Management of Fetal Malformations

starts subdividing into fissures directed perpendicularly to the longitudinal axis of the brainstem. The development of the cerebellar cortex results in the formation of folia.100 Routine sonographic evaluation of the posterior fossa occurs in the axial suboccipital bregmatic view, displaying the two cerebellar hemispheres connected with the vermis and the cisterna magna as the space between the vermis and the inner layer of the occipital bone. The cerebellum appears as a butterfly-shaped structure formed by the round cerebellar hemispheres joined in the middle by the slightly more echogenic cerebellar vermis. The cerebellar hemispheres are well depicted on transverse and coronal images and are hypoechoic with a more echogenic lining. By the end of the second trimester, the increased development of the folia leads to an increased echogenicity characterising the striped appearance. Two retrocerebellar septa, perpendicular to the cerebellum, can be visualised in a transverse view in the cisterna magna in the second and third trimesters in 84% to 92% of fetuses, respectively, and are considered to be remnants of the walls of the Blake pouch.101 A more detailed examination of the vermis can be performed using the midsagittal plane depicting the triangular fourth ventricle (fastigium), the pons, the posterior fossa fluid space and the tentorium cerebelli. The vermis appears markedly hyperechoic, and the primary fissure is constantly observed on the midsagittal image from 24 weeks’ gestation as a transverse more echoic line. Reliable interpretation of the normal development and growth of the cerebellar hemispheres and vermis is possible from 18 weeks onwards.102,103  Dandy-Walker Complex. Posterior fossa malformations have recently been grouped as the Dandy-Walker continuum because of new insights in the embryologic development of this region. This continuum includes isolated enlarged cisterna magna, the Blake pouch cyst, vermian hypoplasia and the Dandy-Walker malformation (DWM). The sonographic categorisation of posterior fossa fluid collections depends on the assessment of the position of the torcular and the integrity of the cerebellar vermis. Pitfalls in the diagnosis of posterior fossa anomalies have been attributed to confusion in terminology describing vermian pathology, the gestational age at diagnosis, the incorrect assessment of the midsagittal plane of the cerebellum and the late development of some pathological conditions.104 More detailed examination by transvaginal ultrasound includes the proper identification of the fastigium and its relation to the vermis by assessment of the angle between vermis or brainstem and tentorium and the appearance of the primary fissure.70,103,105,106 Multiplanar imaging of a standard 3D volume and the use of tomographic ultrasound imaging or VCI (volume contrast imaging) facilitates the visualisation and biometry of the midsagittal structures of the posterior fossa, especially the vermis and the fastigium.70,105 Fetal MRI offers a clearer view of the torcular, and the assessment of the integrity of the vermis remains difficult particularly in midgestation.107 Current fetal MRI, particularly in early gestation before 24 gestational weeks, has limitations in accurately predicting postnatal MRI abnormalities. In 41%, fetal and postnatal MRI diagnoses disagree; postnatal MRI reversed fetal MRI diagnoses in 15% and revealed additional anomalies in 26%.108,109 MRI in early gestation (before 18 weeks), although presumed safe, results in a higher false-positive rate because of fetal motion, small anatomic structures (limited spatial and tissue resolution) and rapid growth of the cerebellum later in pregnancy (second and third trimesters).110 T2-weighted images provide structural information and can be complemented with T1-weighted and echo planar imaging to detect haemorrhage.

Normative charts of the posterior fossa and its structures are available.  Isolated Mega Cisterna Magna. The cisterna magna is a fluidfilled space posterior to the cerebellum. In the second half of gestation, the anteroposterior diameter of the cisterna magna is stable and measures between 2 and 10 mm. Early in gestation as the cerebellar vermis does not completely cover the fourth ventricle, it may give the false impression of a defect of the vermis. A CM of more than 10 mm without associated anomalies suggests the diagnosis of isolated mega cisterna magna (MCM). Isolated MCM is defined as a distance between the vermis of the cerebellum and the inner border of the occipital bone of more than 10 mm on an axial plane through the CSP and the vermis. The vermis is intact, the fourth ventricle is normal and there is no VM nor displacement of the torcular. The prevalence is estimated at 2%. Isolated MCM is usually an incidental finding and may be secondary to a temporary distention of the Blake pouch without displacement of the vermis. The condition should be differentiated from the Dandy-Walker complex, cerebellar hypoplasia and posterior fossa arachnoid cyst.111 Adults with isolated MCM have normal cognitive function but with reduced memory and verbal fluency. Children with an enlarged cisterna magna are at risk for mild developmental delay.112 Most studies report a good prognosis.113 The nonisolated cases of MCM have abnormal developmental function in 11% to 29% of cases. In syndromic conditions, the prognosis depends on the underlying condition.114  Blake Pouch Cyst. If the Blake pouch fails to perforate to form the midline aperture of the foramen of Magendie, the accumulation of CSF results in a cystlike structure projecting into the cisterna magna. The Blake pouch cyst remains in communication with the fourth ventricle. The position of the tentorium cerebelli is intact, but an upward and posterior rotation ( atrial rate/ complete A-V-dissociation

Atrial flutter

Ventricular tachycardia

Treat with sotalol if >50% of time

LQTS: magnesium, lidocaine, propanolol AB mediated: dexamethasone

•

Fig. 29.14  Flowchart for diagnosis of fetal arrhythmia. A suggested clinically oriented approach for the diagnosis of fetal abnormal fetal rhythm. AA interval, Time interval between two consecutive atrial contractions; AB mediated, antibody-mediated; AET, atrial ectopic tachycardia; AV, atrioventricular; AVNRT, atrioventricular nodal reentry tachycardia; AVRT, atrioventricular reentry tachycardia; CHB II°, second-degree congenital heart block; CHB III°, third-degree congenital heart block; ES, extrasystole (i.e., premature contraction); FHR, fetal heart rate; LQTS, long-QT syndrome; PJRT, permanent junctional reciprocating tachycardia; SVT, supraventricular tachycardia.

large defects is warranted either surgically or by a percutaneous approach in childhood. 

Arrhythmia Overview. Abnormal cardiac rhythm is common during development and is commonly detected by routine prenatal ultrasound, cardiotocography or during auscultation of the fetal heart at a routine antenatal appointment. Broadly, arrhythmia can be classified into one of three categories: (i) irregular rhythm (110–180 beats/ min), (ii) bradyarrhythmia (180 beats/min). Ectopic beats (i.e., extrasystole) are the most common type of fetal arrhythmia and are usually benign. Sustained brady- or tachyarrhythmia warrants referral to an experienced fetal cardiologist to assess cardiac morphology and perform rhythm analysis.  Ultrasound findings. Because fetal ECG is not readily available, the prenatal classification and diagnosis rely primarily

on the mechanical interaction of the atria and ventricles using ultrasound. M-mode tracings across atrium and ventricle and simultaneous pulsed-wave Doppler of LV inflow and outflow,90,91 SVC and aorta91 or pulmonary artery and vein92 can be used interchangeably, but interpretation of recordings are sometimes difficult, and cases should be referred to an expert. A suggested diagnostic flowchart for fetal arrhythmia is shown in Fig. 29.14.  Perinatal management and treatment. Skin lotions containing cocoa butter and ingestion of cocoa products provoke ectopic beats, and a reduction in their use is usually helpful therapeutically. No intervention is necessary in the majority of cases with ectopic beats; however, monitoring is warranted if they are frequent or blocked because there is an increased risk for the development of supraventricular tachycardia.93 Prenatal treatment for supraventricular tachycardia is indicated if it is sustained (>50% of the time) or in the presence of hydrops. Digoxin, sotalol and flecainide have been widely used as first-line

CHAPTER 29  The Heart

therapy. To date, no randomised controlled trial has been performed to demonstrate the superiority of any of these drugs. No treatment is indicated for blocked atrial bigeminy, which resolves spontaneously. Anti-SSA or -SSB–positive pregnant women have a 3% risk for developing congenital heart block (CHB) in their fetuses, with 15% in pregnancies following an affected case. Management of these pregnancies by serial echocardiography in the office or hospital has not been successful in preventing the development of CHB. Studies are underway using home monitoring with a portable device to determine whether this will be more successful in preventing CHB.94 The decision to treat bradycardia or tachycardia requires expert evaluation, and there is great variety in treatment strategies because the evidence base is poor. Management of bradyarrhythmia depends on the underlying cause: The use of transplacental steroids to treat and prevent

323

complete heart block has been extensively debated. It may reduce hydrops or inflammation and prevent progression to CHB; conclusive evidence, however, is lacking, and side effects must be weighed against possible benefits.95 

Conclusion Prenatal detection of most CHD is feasible, and screening of the entire pregnant population is recommended using the five transverse view fetal heart examination protocol. Pregnancies complicated by CHD should be referred to a tertiary care centre to make a full diagnosis, provide interdisciplinary fetal and maternal monitoring and planning of delivery and (immediate) postnatal interventions depending on the type of CHD and any associated defects. Access the complete reference list online at ExpertConsult.com. Self-assessment questions available at ExpertConsult.com.

References 1. Hoffman JIE, Kaplan S. The incidence of congenital heart disease. J Am Coll Cardiol. 2002;39(12):1890–1900. 2.  Van Der Linde D, Konings EEM, Slager MA, et  al. Birth prevalence of congenital heart disease worldwide: a systematic review and meta-analysis. J Am Coll Cardiol. 2011;58(21):2241–2247. 3. Carvalho JS, Allan LD, Chaoui R, Copel JA, et  al. ISUOG Practice Guidelines (updated): sonographic screening examination of the fetal heart. Ultrasound Obstet Gynecol. 2013;41(3):348–359. 4. The American Institute of Ultrasound in Medicine. AIUM Practice Guideline for the Performance of Fetal Echocardiography. J Ultrasound Med. 2013;32(6):1067–1082. 5. National Health Service. NHS Fetal Anomaly Screening Programme: Standards [Internet]. https://www.gov.uk/topic/population-screening-programmes/fetal-anomaly. 6. Yagel S, Cohen SM, Achiron R. Examination of the fetal heart by five short-axis views: a proposed screening method for comprehensive cardiac evaluation. Ultrasound Obstet Gynecol. 2001;17(5):367–369. 7. Mistry H, Gardiner HM. The cost-effectiveness of prenatal detection for congenital heart disease using telemedicine screening. J Telemed Telecare. 2013;19(4):190–196. 8.  National Institute for Cardiovascular Outcomes Research [Internet]. https://nicor4.nic or.org.uk/chd/an_paeds.nsf/vwContent/Anten atal Diagnosis?Opendocument. 9. Allan LD, Crawford DC, Chita SK, et  al. Familial recurrence of congenital heart disease in a prospective series of mothers referred for fetal echocardiography. Am J Cardiol. 1986;58(3):334–337. 10. Arya B, Glickstein JS, Levasseur SM, Williams IA. Parents of children with congenital heart disease prefer more information than cardiologists provide. Congenit Heart Dis. 2013;8(1):78–85. 11. Allan LD, Cook A, Sullivan I, Sharland GK. Hypoplastic left heart syndrome: effects of fetal echocardiography on birth prevalence. Lancet. 1991;337(8747):959–961. 12. Ashburn DA, McCrindle BW, Tchervenkov CI, et al. Outcomes after the Norwood operation in neonates with critical aortic stenosis or aortic valve atresia. J Thorac Cardiovasc Surg. 2003;125(5):1070–1082. 13. Copel JA, Pilu G, Kleinman CS. Congenital heart disease and extracardiac anomalies: associations and indications for fetal echocardiography. Am J Obstet Gynecol. 1986;154(5):1121–1132. 14. Tennstedt C, Chaoui R, Körner H, Dietel M. Spectrum of congenital heart defects and extracardiac malformations associated with chromosomal abnormalities: results of a seven year necropsy study. Heart. 1999;82(1):34–39. 15. Tulzer G, Arzt W, Franklin RCG, et al. Fetal pulmonary valvuloplasty for critical pulmonary stenosis or atresia with intact septum. Lancet. 2002;360(9345):1567–1568. 16. Chaturvedi RR, Ryan G, Seed M, et al. Fetal stenting of the atrial septum: technique and initial results in cardiac lesions with left atrial hypertension. Int J Cardiol. 2013;168(3):2029– 2036.

17. Arzt W, Wertaschnigg D, Veit I, et  al. Intrauterine aortic valvuloplasty in fetuses with critical aortic stenosis: experience and results of 24 procedures. Ultrasound Obstet Gynecol. 2011;37(6):689–695. 18. Freud LR, McElhinney DB, Marshall AC, et  al. Fetal aortic valvuloplasty for evolving hypoplastic left heart syndrome: postnatal outcomes of the first 100 patients. Circulation. 2014;130(8):638–645. 19. Peake LK, Draper ES, Budd JL, Field D. Outcomes when congenital heart disease is diagnosed antenatally versus postnatally in the UK: a retrospective population-based study. BMC Pediatr. 2015;15(1):1–8. 20. Markkanen HK, Pihkala JI, Salminen JT, et al. Prenatal diagnosis improves the postnatal cardiac function in a population-based cohort of infants with hypoplastic left heart syndrome. J Am Soc Echocardiogr. 2013;26(9):1073–1079. 21. Morris SA, Ethen MK, Penny DJ, et  al. Prenatal diagnosis, birth location, surgical centre, and neonatal mortality in infants with hypoplastic left heart syndrome. Circulation. 2014;129(3):285–292. 22. Peyvandi S, De Santiago V, Chakkarapani E, et  al. Association of prenatal diagnosis of critical congenital heart disease with postnatal brain development and the risk of brain injury. JAMA Pediatr. 2016;170(4):e154450. 23. Freud LR, Escobar-Diaz MC, Kalish BT, et al. Outcomes and predictors of perinatal mortality in fetuses with Ebstein anomaly or tricuspid valve dysplasia in the current era: a multicentre study. Circulation. 2015;132(6):481–489. 24. Calderon J, Angeard N, Moutier S, et  al. Impact of prenatal diagnosis on neurocognitive outcomes in children with transposition of the great arteries. J Pediatr. 2012;161(1):94–99. 25. Li Y, Yin S, Fang J, et al. Neurodevelopmental delay with critical congenital heart disease is mainly from prenatal injury not infant cardiac surgery: current evidence based on a meta-analysis of functional magnetic resonance imaging. Ultrasound Obstet Gynecol. 2015;45(6):639– 648. 26. Paladini D, Alfirevic Z, Carvalho JS, et  al. ISUOG consensus statement on current understanding of the association of neurodevelopmental delay and congenital heart disease: impact on prenatal counseling. Ultrasound Obstet Gynecol. 2017;49(2):287–288. 27. Arzt W, Tulzer G. Fetal surgery for cardiac lesions. Prenat Diagn. 2011;31(7):695–698. 28. Wohlmuth C, Wertaschnigg D, Wieser I, et  al. Tissue Doppler imaging in fetuses with aortic stenosis and evolving hypoplastic left heart syndrome before and after fetal aortic valvuloplasty. Ultrasound Obstet Gynecol. 2016;47(5):608–615. 29. Arzt W, Tulzer G, Aigner M, et al. Invasive intrauterine treatment of pulmonary atresia/intact ventricular septum with heart failure. Ultrasound Obstet Gynecol. 2003;21(2):186–188. 30. Tworetzky W, McElhinney DB, Marx GR, et al. In utero valvuloplasty for pulmonary atresia with hypoplastic right ventricle: techniques and outcomes. Pediatrics. 2009;124(3):e510– e518. 31. Gardiner HM, Kovacevic A, Tulzer G, et  al. Natural history of 107 cases of fetal aortic stenosis from a European multicentre retrospective study. Ultrasound Obstet Gynecol. 2016;48(3):373–381.

32. McElhinney DB, Marshall AC, WilkinsHaug LE, et  al. Predictors of technical success and postnatal biventricular outcome after in utero aortic valvuloplasty for aortic stenosis with evolving hypoplastic left heart syndrome. Circulation. 2009;120(15):1482– 1490. 33. Samanek M, Voriskova M. Congenital heart disease among 815,569 children born between 1980 and 1990 and their 15-year survival: a prospective Bohemia survival study. Pediatr Cardiol. 1999;20:411–417. 34. Moerman PL, Van Dijck H, Lauweryns JM, et  al. Premature closure of the foramen ovale and congenital pulmonary cystic lymphangiectasis in aortic valve atresia or in severe aortic valve stenosis. Am J Cardiol. 1986;57(8):703– 705. 35. Graziano JN, Heidelberger KP, Ensing GJ, et al. The influence of a restrictive atrial septal defect on pulmonary vascular morphology in patients with hypoplastic left heart syndrome. Pediatr Cardiol. 2002;23(2):146–151. 36. Song MS, Hu A, Dyamenahalli U, et al. Extracardiac lesions and chromosomal abnormalities associated with major fetal heart defects: comparison of intrauterine, postnatal and postmortem diagnoses. Ultrasound Obstet Gynecol. 2009;33(5):552–559. 37. Galindo A, Nieto O, Villagrá S, et  al. Hypoplastic left heart syndrome diagnosed in fetal life: associated findings, pregnancy outcome and results of palliative surgery. Ultrasound Obstet Gynecol. 2009;33(5):560–566. 38. Glatz JA, Tabbutt S, Gaynor JW, et al. Hypoplastic left heart syndrome with atrial level restriction in the era of prenatal diagnosis. Ann Thorac Surg. 2007;84(5):1633–1638. 39. Schneider C, McCrindle BW, Carvalho JS, et al. Development of Z-scores for fetal cardiac dimensions from echocardiography. Ultrasound Obstet Gynecol. 2005;26(6):599–605. 40. Pasquini L, Mellander M, Seale A, et  al. Z-scores of the fetal aortic isthmus and duct: an aid to assessing arch hypoplasia. Ultrasound Obstet Gynecol. 2007;29(6):628–633. 41. Rychik J, Szwast A, Natarajan S, et al. Perinatal and early surgical outcome for the fetus with hypoplastic left heart syndrome: a 5-year single institutional experience. Ultrasound Obstet Gynecol. 2010;36(4):465–470. 42. Rychik J, Rome JJ, Collins MH, et  al. The hypoplastic left heart syndrome with intact atrial septum: atrial morphology, pulmonary vascular histopathology and outcome. J Am Coll Cardiol. 1999;34(2):554–560. 43. Donofrio MT, Moon-Grady AJ, Hornberger LK, et  al. Diagnosis and treatment of fetal cardiac disease: a scientific statement from the American Heart Association. Circulation. 2014;129(21):2183–2242. 44. Wald RM, Tham EB, McCrindle BW, et  al. Outcome after prenatal diagnosis of tricuspid atresia: a multicentre experience. Am Heart J. 2007;153(5):772–778. 45. Beroukhim RS, Gauvreau K, Benavidez OJ, et al. Perinatal outcome after prenatal diagnosis of single-ventricle cardiac defects. Ultrasound Obstet Gynecol. 2015;45(6):657–663. 46. Giglia TM, Mandell VS, Connor AR, et  al. Diagnosis and management of right ventricledependent coronary circulation in pulmonary atresia with intact ventricular septum. Circulation. 1992;86(5):1516–1528.

323.e1

323.e2

References

47. Daubeney PE, Sharland GK, Cook AC, et al. Pulmonary atresia with intact ventricular septum: impact of fetal echocardiography on incidence at birth and postnatal outcome. UK and Eire Collaborative Study of Pulmonary Atresia with Intact Ventricular Septum. Circulation. 1998;98(6):562–566. 48. Gardiner HM, Belmar C, Tulzer G, et al. Morphologic and functional predictors of eventual circulation in the fetus with pulmonary atresia or critical pulmonary stenosis with intact septum. J Am Coll Cardiol. 2008;51(13):1299–1308. 49. Daubeney PEF, Wang D, Delany DJ, et  al. Pulmonary atresia with intact ventricular septum: predictors of early and medium-term outcome in a population-based study. J Thorac Cardiovasc Surg. 2005;130(4):1–9. 50. Watson H. Natural history of Ebstein’s anomaly of tricuspid valve in childhood and adolescence. An international cooperative study of 505 cases. Br Heart J. 1974;36(5):417–427. 51. Baek JS, Yu JJ, Im YM, Yun T-J. Outcomes of neonatal Ebstein’s anomaly without right ventricular forward flow. J Thorac Cardiovasc Surg. 2016;152(2):516–521. 52. Fesslova V, Villa L, Nava S, et al. Spectrum and outcome of atrioventricular septal defect in fetal life. Cardiol Young. 2002;12(1):18–26. 53. Delisle MF, Sandor GG, Tessier F, Farquharson DF. Outcome of fetuses diagnosed with atrioventricular septal defect. Obstet Gynecol. 1999;94(5 Pt 1):763–767. 54. Craig B. Atrioventricular septal defect: from fetus to adult. Heart. 2006;92(12):1879–1885. 55. Taketazu M, Lougheed J, Yoo S-J, et al. Spectrum of cardiovascular disease, accuracy of diagnosis, and outcome in fetal heterotaxy syndrome. Am J Cardiol. 2006;97(5):720–724. 56. Jowett V, Paramasivam G, Seale A, et  al. Diaphragmatic hernia: a postnatal complication of anomalous drainage of the umbilical vein. Ultrasound Obstet Gynecol. 2013;41(5):589–591. 57. Berg C, Knüppel M, Geipel A, et al. Prenatal diagnosis of persistent left superior vena cava and its associated congenital anomalies. Ultrasound Obstet Gynecol. 2006;27(3):274–280. 58. Szwast A, Tian Z, McCann M, et al. Vasoreactive response to maternal hyperoxygenation in the fetus with hypoplastic left heart syndrome. Circ Cardiovasc Imaging. 2010;3(2):172–178. 59. Hiramatsu T, Matsumura G, Konuma T, et al. Long-term prognosis of double-switch operation for congenitally corrected transposition of the great arteries. Eur J Cardio-Thoracic Surg. 2012;42(6):1004–1008. 60. Poon LCY, Huggon IC, Zidere V, Allan LD. Tetralogy of Fallot in the fetus in the current era. Ultrasound Obstet Gynecol. 2007;29(6):625–627. 61. Zhao Y, Abuhamad A, Fleenor J, et al. Prenatal and postnatal survival of fetal Tetralogy of Fallot: a meta-analysis of perinatal outcomes and associated genetic disorders. J Ultrasound Med. 2016;35(5):905–915. 62. Kim N, Friedberg MK, Silverman NH. Diagnosis and prognosis of fetuses with double outlet right ventricle. Prenat Diagn. 2006;26(8):740–745. 63. Hartge DR, Niemeyer L, Axt-Fliedner R, et al. Prenatal detection and postnatal management

of double outlet right ventricle (DORV) in 21 singleton pregnancies. J Matern Fetal Neonatal Med. 2012;25(1):58–63. 64. Smith RS, Comstock CH, Kirk JS, et  al. Double-­ outlet right ventricle: an antenatal diagnostic dilemma. Ultrasound Obstet Gynecol. 1999;14(5):315–319. 65. Collett RW, Edwards JE. Persistent truncus arteriosus; a classification according to anatomic types. Surg Clin North Am. 1949;29(4):1245–1270. 66. Volpe P, Paladini D, Marasini M, et al. Common arterial trunk in the fetus: characteristics, associations, and outcome in a multicentre series of 23 cases. Heart. 2003;89(12):1437– 1441. 67. Swanson TM, Selamet Tierney ES, Tworetzky W, et  al. Truncus arteriosus: diagnostic accuracy, outcomes, and impact of prenatal diagnosis. Pediatr Cardiol. 2009;30(3):256–261. 68. Paladini D. How to identify the thymus in the fetus: the thy-box. Ultrasound Obstet Gynecol. 2011;37(4):488–492. 69. Matsui H, Adachi I, Uemura H, et  al. Anatomy of coarctation, hypoplastic and interrupted aortic arch: relevance to interventional/ surgical treatment. Expert Rev Cardiovasc Ther. 2007;5(5):871–880. 70. Becker AE, Becker MJ, Edwards JE. Anomalies associated with coarctation of aorta: particular reference to infancy. Circulation. 1970;41(6):1067–1075. 71. Paladini D, Volpe P, Russo MG, et al. Aortic coarctation: prognostic indicators of survival in the fetus. Heart. 2004;90(11):1348–1349. 72. Brown DL, Durfee SM, Hornberger LK. Ventricular discrepancy as a sonographic sign of coarctation of the fetal aorta: how reliable is it? J Ultrasound Med. 1997;16(2):95–99. 73. Brown ML, Burkhart HM, Connolly HM, et al. Coarctation of the aorta: lifelong surveillance is mandatory following surgical repair. J Am Coll Cardiol. 2013;62(11):1020–1025. 74. Canniffe C, Ou P, Walsh K, Bonnet D, Celermajer D. Hypertension after repair of aortic coarctation—a systematic review. Int J Cardiol. 2012;167(6):2456–2461. 75. Mogra R, Saaid R, Kesby G, et al. Early fetal echocardiography: experience of a tertiary diagnostic service. Aust N Z J Obstet Gynaecol. 2015;55(6):552–558. 76. Edwards JE. Vascular rings related to anomalies of the aortic arches. Mod Concepts Cardiovasc Dis. 1948;17(8):1. 77. Yoo S-J, Min J-Y, Lee Y-H, et al. Fetal sonographic diagnosis of aortic arch anomalies. Ultrasound Obstet Gynecol. 2003;22(5):535– 546. 78. Naidu DP, Wohlmuth C, Gardiner HM. Prenatal diagnosis of double aortic arch: can we predict airway obstruction (pseudo-CHAOS) and need for airway EXIT? Ultrasound Obstet Gynecol. 2017;49(5):660–661. 79. Perolo A, De Robertis V, Cataneo I, et al. Risk of 22q11.2 deletion in fetuses with right aortic arch and without intracardiac anomalies. Ultrasound Obstet Gynecol. 2016;48(2):200–203. 80. Wohlmuth C, Gardiner HM. The biplane mode: it takes two to tango. Ultrasound Obstet Gynecol. 2017;49(4):540.

81. Zielinsky P, Piccoli AL, Manica JLL, et  al. Reversal of fetal ductal constriction after maternal restriction of polyphenol-rich foods: an open clinical trial. J Perinatol. 2012;32(8):574– 579. 82. Bubols GB, Zielinsky P, Piccoli AL, et  al. Nitric oxide and reactive species are modulated in the polyphenol-induced ductus arteriosus constriction in pregnant sheep. Prenat Diagn. 2014;34(13):1268–1276. 83. Kiserud T. Physiology of the fetal circulation. Semin Fetal Neonatal Med. 2005;10(6):493– 503. 84. Pérez-Jiménez J, Neveu V, Vos F, Scalbert a. Identification of the 100 richest dietary sources of polyphenols: an application of the Phenol-Explorer database. Eur J Clin Nutr. 2010;64(suppl 3):S112–S120. 85. Gewillig M, Brown SC, De Catte L, et al. Premature foetal closure of the arterial duct: clinical presentations and outcome. Eur Heart J. 2009;30(12):1530–1536. 86. Seale AN, Uemura H, Webber SA, et al. Total anomalous pulmonary venous connection: morphology and outcome from an international population-based study. Circulation. 2010;122(25):2718–2726. 87. Seale AN, Carvalho JS, Gardiner HM, et  al. Total anomalous pulmonary venous connection: impact of prenatal diagnosis. Ultrasound Obstet Gynecol. 2012;40(3):310–318. 88. Berg C, Georgiadis M, Geipel A, Gembruch U. The area behind the heart in the four-chamber view and the quest for congenital heart defects. Ultrasound Obstet Gynecol. 2007;30(5):721– 727. 89. Kawazu Y, Inamura N, Shiono N, et al. ‘PostLA space index’ as a potential novel marker for the prenatal diagnosis of isolated total anomalous pulmonary venous connection. Ultrasound Obstet Gynecol. 2014;44(6):682–687. 90. Strasburger JF, Huhta JC, Carpenter RJ, et al. Doppler echocardiography in the diagnosis and management of persistent fetal arrhythmias. J Am Coll Cardiol. 1986;7(6):1386–1391. 91. Nii M, Hamilton RM, Fenwick L, et al. Assessment of fetal atrioventricular time intervals by tissue Doppler and pulse Doppler echocardiography: normal values and correlation with fetal electrocardiography. Heart. 2006;92(12):1831–1837. 92. Carvalho JS, Prefumo F, Ciardelli V, et al. Evaluation of fetal arrhythmias from simultaneous pulsed wave Doppler in pulmonary artery and vein. Heart. 2007;93(11):1448–1453. 93. Donofrio MT, Moon-Grady AJ, Hornberger LK, et  al. Diagnosis and treatment of fetal cardiac disease: a scientific statement from the American Heart Association. Circulation. 2014;129(21):2183–2242. 94.  Prospective Maternal Surveillance of SSA (Sjögren Syndrome A) Positive Pregnancies Using a Hand-held Fetal Heart Rate Monitor. NCT02920346. http://www.clinicaltrials.gov. 95. Eliasson H, Sonesson S-E, Sharland G, et  al. Isolated atrioventricular block in the fetus: a retrospective, multinational, multicentre study of 175 patients. Circulation. 2011;124(18):1919– 1926.

30

Fetal Lung Lesions JAMES COOK, ANGELA YULIA, COLIN WALLIS AND PRANAV P. PANDYA

KEY POINTS • A limited number of congenital malformations of the respiratory tract can be identified directly by prenatal sonography. • These malformations should be described systematically because definitive diagnosis requires histologic examination. • The identification of subtle lesions that have no detrimental effect on a fetus or postnatal respiratory function is increasingly common. • A lack of evidence surrounding the natural history of asymptomatic cystic lung lesions has resulted in highly divergent postnatal management strategies. • A conservative approach to postnatal management of asymptomatic cases is a reasonable option. • Careful prenatal counselling is recommended.

Congenital Malformations of the Respiratory Tract Embryologic development of the respiratory tract requires the appropriate growth of the upper airway and the six ‘trees’ that make up the lower respiratory tract – bronchial, arterial (systemic and pulmonary), venous (systemic and pulmonary) and lymphatic – together with a normal thoracic volume, a normal thoracic skeletal structure and normal neuromuscular function. Defects in any of these elements (except the systemic venous system because there are none known) can affect anatomical organisation resulting in a variety of congenital abnormalities. Whereas some lesions can be detected by direct prenatal sonographic visualisation, other lesions are suspected because of the presence of nonspecific findings (e.g., mediastinal shift) or form part of a more generalised genetic syndrome, for example, the pulmonary hypoplasia seen in skeletal dysplasias such asphyxiating thoracic dystrophy. Other congenital malformations of the lungs only become apparent in the postnatal period when they cause symptoms. Not all congenital malformations have a detrimental impact on a fetus or postnatal respiratory function. Advances in sonographic technology allow prenatal detection of subtle lesions which may have no immediate clinical impact. A lack of evidence surrounding the natural history of asymptomatic cystic lung lesions has resulted in divergent postnatal management strategies and difficult prenatal counseling. In this chapter, we will identify thoracic malformations, excluding congenital diaphragmatic hernia (covered in Chapter 31), that 324

can be detected directly on prenatal sonography, describe a system to classify these lesions sonographically, define their salient pathological features and discuss the merits of prenatal and postnatal management options. 

Thoracic Malformations Detected on Prenatal Ultrasound Thoracic malformations detectable on prenatal sonography are detailed in Table 30.1; however, a definitive diagnosis of these lesions (excluding congenital diaphragmatic hernia (CDH)) requires histologic confirmation. A prenatal or postnatal diagnosis is not possible by radiologic means alone. The imaging appearance of different types of lesions can be identical, rendering specific pathological diagnoses redundant and risking confusion in communication between medical professionals and families. Moreover, it is now apparent that there can be considerable overlap in histologic features within lesions, further highlighting the increasing complexity of diagnosis and the limitations of diagnosis based on imaging modalities alone. In light of these difficulties, a system whereby malformations detected by prenatal sonography are described meticulously in simple language, based on their appearance, and without the presumption of a single pathological diagnosis has been recommended.1 Within this system, all thoracic malformations are described under the umbrella term congenital thoracic malformation (CTM). Malformations are then defined further using descriptive terms, including the presence and size of cysts, the presence of a feeding vessel, the degree of echogenicity, the presence or absence of mediastinal shift, polyhydramnios and the presence of anomalies in other systems. In practical terms, lesions have been most usefully classified sonographically as either macrocystic or microcystic (see Table 30.1).2 Detection of CTMs, often at the routine 20-week anomaly scan, allows detailed planning of further prenatal management, including serial sonographic monitoring, delineation of the lesion and intrauterine therapeutic interventions if required. Planning for appropriate neonatal support on delivery can be prepared in advance. The exception to this pattern are pleural effusions, which often present later when scanning is undertaken because of a suspicion of increased amniotic fluid or as an incidental finding on ultrasound (USS) undertaken during the third trimester. The appeal of enhanced prenatal radiologic definition of lung CTMs has led to the exploration of magnetic resonance imaging (MRI) as an additional modality. MRI has been used in the delineation of fetal lung lesions3 and the identification of feeding vessels,4 although this is readily done using Doppler ultrasound.

CHAPTER 30  Fetal Lung Lesions

Whether this enhanced imaging provides any additional information of practical value over ultrasound alone has yet to be determined, and in most centres at present, MRI is not part of routine practice. 

Macrocystic Lung Lesions Macrocytic lung lesions include congenital pulmonary airway malformations (CPAMs), bronchogenic cysts, enteric cysts, bronchial atresia and congenital lobar emphysema (see Table 30.1). The sonographic appearance includes a cystic lesion(s) of varying size in the thorax with or without mediastinal shift (Fig. 30.1). An essential differential diagnosis to consider on identification of a macrocystic lung lesion is a left-sided diaphragmatic hernia, in which the stomach or bowel herniated into the thorax can be TABLE Differential Diagnosis of Congenital Thoracic 30.1 Malformation

Macrocystic Lesions Congenital pulmonary airway malformation Bronchogenic cyst or enteric cyst Congenital diaphragmatic hernia Bronchial atresia Congenital lobar emphysema Pleuropulmonary blastoma

Microcystic Lesions Congenital pulmonary airway malformation Pulmonary sequestration Pleural effusion Tracheal or laryngeal atresia Pulmonary hypoplasia or agenesis Mediastinal teratoma Rhabdomyoma Ectopia

  

A

325

mistaken for a cystic structure. The crucial importance of this differentiation is the subsequent management strategy for CDH, particularly the place of delivery, associations with chromosomal abnormalities and genetic syndromes and general prognosis. Additional sonographic features that assist in the correct identification of a CDH include the absence of a stomach within the abdomen, the visualisation of peristalsis within the thorax, the paradoxical movement of abdominal viscera within the thorax during fetal breathing movements and absence of a diaphragm. However, differentiation can remain difficult on occasion. In a case series of 110 fetuses diagnosed with a CTM, two were later identified as having a diaphragmatic hernia, both after serial prenatal scanning.2

Congenital Pulmonary Airway Malformations Congenital pulmonary airway malformations were previously known as congenital cystic adenomatoid malformations (CCAM). In 2002, Stocker recommended the term CPAM as being preferable to the term congenital cystic adenomatoid malformation because not all types of CPAM are cystic and adenomatoid.5 The new terminology enables a better description of the entity’s alterations. For example, type 0 is not a cystic lesion, and types 0, 1 and 4 are not adenomatoid lesions. Congenital pulmonary airway malformations represent the most common cystic lesions diagnosed on prenatal ultrasound. Currently, the best estimate of incidence reported by the European Surveillance of Congenital Anomalies (EUROCAT) is 0.94 in 10 000 live births.6 Although CPAMs can be defined as macrocystic or microcystic, both types are described in this section. Opinions vary as to the aetiology of these lesions. Genetic abnormalities that may influence normal lung development or external insults disrupting lung growth have been postulated. The subclassification of CPAM types remains contentious. Various systems of classification have been proposed with the most widely accepted that of Stocker.5 Within this system, CPAMs are classified into five types according to the level of the bronchial tree at which the defect is thought to have occurred. The strength of this classification is that specific neoplasms are associated with specific CPAM subgroups; however, these are not distinguishable with prenatal ultrasound, and postnatal histologic examination is required. There can be significant histologic overlap of lesions previously considered distinct (e.g., hybrid forms of CPAM, pulmonary sequestration (PS) and bronchial atresia).

B • Fig. 30.1  A, Axial view through the chest of a fetus at 22 weeks with multiple cystic lesions in the chest. Note the shift and compression of the heart. B, A single cyst is seen in the axial view with some mediastinal shift.

326

SE C T I O N 7     Diagnosis and Management of Fetal Malformations

Type 0 or acinar dysplasia. This is a rare type of CPAM and

is thought to develop at the level of the bronchus. On histologic examination, bronchial airways are present, but the distal parenchyma is highly unusual and consists mainly of mesenchymal tissue. Macroscopically, the lungs are small, and the condition is not compatible with life. The condition is also termed acinar dysplasia.5  Type 1. This is the most common type of CPAM, accounting for approximately 60% to 70% of cases.5 and thought to develop at the bronchial/bronchiolar level. The cysts range in diameter up to 10 cm, with at least one cyst more than 2 cm in diameter required for diagnosis. They are lined with pseudostratified ciliated columnar epithelium, with mucous cell proliferation also present on occasion.5  Type 2. These are less common than type 1 CPAMs, accounting for 15% to 20% of cases,5 and are thought to arise at the bronchiolar level. Lesions typically consist of multiple small cysts which range in size but must be less than 2 cm in diameter for diagnosis. The cysts are related to dilated bronchiole-like structures and are surrounded by simplified alveolar tissue.5 These lesions can be associated with other congenital abnormalities such as renal agenesis or dysplasia7 as well as cardiovascular and neurologic abnormalities.  Type 3 or lung hyperplasia. These are a rare type of CPAM, accounting for 5% to 10% of cases5 thought to arise at the bronchiolar/alveolar duct level. Their inclusion as a CPAM is controversial because the microscopic features of excess bronchiolar ducts and parenchyma typical of fetal lung is considered by some pathologists to represent lung hyperplasia.8 The lesions can be large and affect an entire lobe with consequent compression of surrounding lung tissue.  Type 4 or regressed pleuropulmonary blastoma. These are very rare lesions and the most controversial of the CPAM diagnoses. The cysts can be large and are impossible to distinguish radiologically from type 1 CPAMs. On histologic examination, however, the cysts are lined with alveolar or bronchiolar epithelial cells upon mesenchymal tissue.5 The only difference between this lesion and a pleuropulmonary blastoma (PPB) is the absence of blastema. Some argue that these lesions in fact represent a regressed neoplasm rather than a form of CPAM.9 

of cystic structures broadly termed foregut cysts.11 Subdivision of these cysts can be made on the basis of their histology. Bronchogenic cysts have histologic features in keeping with the primitive airway and are commonly identified as single cysts within the mediastinum but may be situated anywhere along the bronchial tree or even in extrathoracic locations. They typically present as a single cyst and are lined with respiratory-type epithelium and contain cartilage in the wall on histologic examination. Clinical manifestations are most commonly attributable to airway compression but cysts may also act as a nidus of infection and bleeding. In contrast to bronchogenic cysts, enteric cysts have histologic features differentiated towards the gut rather than the bronchus.11 They can be subdivided according to where they occur along the gastrointestinal (GI) tract. Cysts arising from the oesophagus are termed oesophageal cysts, and cysts arising at a distal point along the GI tract are termed gastroenteric. Malignant change has been described in enteric cysts. 

Congenital Lobar Emphysema This lesion is characterised by hyperinflation of a lobe or segment of the lung and ordinarily presents in a neonate or infant with respiratory distress. Occasionally, it is detected prenatally as an apparent macrocystic lesion on ultrasound or the cause of a profound mediastinal shift. Partial airway obstruction caused by a mucosal flap, twisting of the lobe on its pedicle or a defect in bronchial cartilage results in air trapping. Presumably, the prenatal features, when present, are also a result of partial bronchial obstruction and the accumulation of lung fluid. Histologically, a normal number of distended and sometimes ruptured alveoli are demonstrated. Rarely, there are increased alveolar counts, which has been termed polyalveolar lobe.12 

Microcystic Lung Lesions Congenital Pulmonary Airway Malformation Microcystic CPAMs appear as a uniformly hyperechogenic lesion in the chest on prenatal USS (Fig. 30.2). As with macrocystic lesions, they can be associated with mediastinal shift and hydrops. 

Bronchial Atresia

Pulmonary Sequestration

Bronchial atresia describes interruption of a lobar, segmental or subsegmental bronchus either caused by discontinuity or membranous interruption. It results in the cystic degeneration of the distal lung parenchyma likely caused by accumulation of obstructed fetal lung fluid. As previously stated, CPAMs are also thought to originate from a spectrum of bronchial defects, and bronchial atresia may also lie along this spectrum. Indeed, evidence of bronchial atresia is often identified in hybrid association with CPAMs and PS.10 Despite complete interruption of the bronchus, the distal lung can fill with air in the postnatal period and even become hyperinflated, although the mechanism is not well understood. Prenatally, there may be a cystic appearance or just mediastinal shift. 

Pulmonary sequestrations are defined as isolated areas of lung tissue that do not communicate with the bronchial tree of the normal lung and receive a blood supply from a systemic vessel. They are subdivided into two groups: intralobar, in which the lesion lies within the visceral pleura, and extralobar, in which the sequestration is invested in its own pleura. There can be considerable histologic overlap with features of type 2 CPAMs and bronchial atresia,13 and in common with these other cystic lesions, the aetiology of PS is not understood. Many sequestrations present as an echogenic mass in the fetal chest or abdomen at the 20-week anomaly scan. Intralobar sequestrations are typically identified in the left lower lobe, and extralobar lesions are identified beneath the left lower lobe or within the abdomen. Doppler ultrasound can be used to identify the blood supply (Fig. 30.3). Some sequestrations communicate directly with the GI tract, most commonly the lower oesophagus and stomach.14 Careful monitoring prenatally is required, particularly if there are signs of hydrops as the anomalous blood supply can result in cardiac failure. 

Bronchogenic Cysts and Enteric Cysts (Duplication Cysts) The embryonic lung develops from an outgrowth of the primitive foregut. Disruption of this division can result in the formation

CHAPTER 30  Fetal Lung Lesions

327

Laryngeal or Tracheal Atresia

• Fig. 30.2  Axial view through the chest of a fetus at 21 weeks with microcystic congenital pulmonary airway malformation.

During embryogenesis, the epithelial lining of both the larynx and trachea is derived from the endoderm. As the endoderm proliferates, occlusion of the lumen of the larynx and trachea occurs followed by recanalization at approximately 10 weeks’ gestation. Failure of recanalization can be partial or complete, with complete failure associated with laryngeal or tracheal atresia and partial failure associated with laryngeal webs. Laryngeal and tracheal atresia are rare malformations but should be considered if bilateral enlarged and hyperechogenic lungs are present on ultrasound of the fetus. Careful ultrasound examination may reveal a dilated trachea below the level of the obstruction (Fig. 30.4). The mass effect of the distended lungs results in the other typical radiologic features of mediastinal compression and convexity of the diaphragms. Nonspecific features such as polyhydramnios and fetal hydrops may also be evident. Although these lesions can be isolated, they may also be part of a genetic syndrome (e.g., Fraser syndrome), mandating detailed fetal anomaly scanning and genetic investigations if deemed appropriate. 

Pulmonary Hypoplasia and Agenesis

•

Fig. 30.3 Axial view through the chest of a fetus with a pulmonary sequestration which is echogenic; the extra feeding vessel is seen.

A

B

Pulmonary hypoplasia and pulmonary agenesis are related malformations characterised by a spectrum of lung parenchymal underdevelopment. Whereas pulmonary agenesis lies at the extreme end of the spectrum with complete absence of lung parenchymal tissue, pulmonary hypoplasia is defined by diminished numbers of airways and alveoli resulting in a reduced lung size. The features for both malformations may be unilateral or bilateral. The lesions are thought to result from insults during lung embryogenesis, the timing of which relates to the point on the spectrum of the clinical features. Complete agenesis of one or both lungs is exceptionally rare and is often associated with other congenital abnormalities (e.g., scimitar syndrome associated with anomalous pulmonary venous return). The remaining lung in unilateral agenesis is hypertrophied and causes mediastinal shift. Pulmonary hypoplasia is a more frequent finding and is characterised by reduced lung volume on prenatal ultrasound with the appearance of a small chest or increased heart:lung ratio. Associated congenital abnormalities are present in at least 50% of cases. The pathogenesis might include: • A deficiency in the thoracic volume inhibiting normal lung growth, such as may be seen in skeletal dysplasias associated with short ribs, in which the rib size restricts pulmonary growth

C

• Fig. 30.4  A, Axial view through the chest of a fetus at 23 weeks with laryngeal atresia. Note the appearance of bilateral enlarged and hyperechogenic lungs. B, Parasagittal view of enlarged bilateral hyperechogenic lungs with flattening of the diaphragm. C, Parasagittal view of laryngeal atresia showing a dilated trachea.

328

SE C T I O N 7     Diagnosis and Management of Fetal Malformations

A

B

• Fig. 30.5  A, Parasagittal view of chest of a fetus at 21 weeks with fetal akinesia deformation sequence. Note the appearance of the small chest and short ribs. B, Midsagittal view of the small chest.

• A deficiency in fetal breathing movements inhibiting normal lung development, as in the fetal akinesia deformation sequence (e.g., Pena Shokeir syndrome type 1) in which a general lack of movement and minimal or no breathing or swallowing movements inhibits normal lung development (Fig. 30.5) • Severe oligohydramnios from early in pregnancy is the commonest cause of pulmonary hypoplasia, as may be seen in bilateral renal agenesis, early-onset bilateral cystic renal disease or severe preterm prelabour rupture of membranes (PPROM), where a lack of amniotic fluid prevents the normal passage of liquid in and out of the lungs which then cannot develop to their full potential. • A deficient vascular supply inhibiting normal lung growth, which may result from congenital heart disease 

Prenatal Management General Considerations The detection of a CTM on prenatal ultrasound should prompt a detailed and expert scan to exclude CDH and detect other congenital anomalies that may aid definition of the underlying pathology. Identification of a systemic blood supply to the lesion by Doppler USS is useful in defining a likely sequestration (see Fig. 30.3). 

Cystic Lung Lesions A definitive diagnosis of a congenital cystic lung lesion is not possible using ultrasound, and even histologic examination often reveals hybrid features of different entities. As such, there is also considerable overlap in the clinical management of these malformations. In general, the prognosis for a fetus with a cystic lung lesion is good with the risk for intrauterine or perinatal death less than 1% in the University College Hospital case series.2 Serial ultrasound scans should be undertaken at least every 4 weeks and sometimes more frequently if there is significant mediastinal shift because these lesions can change during the course of gestation with most reducing in size over the course of the pregnancy.15,16 About half17 may increase in size until the middle of the second trimester after which a reduction in size or even apparent sonographic resolution is possible in up to 76% of cases.18 Features such as fetal hydrops or mediastinal shift, usually associated with poor prognosis,19 may also resolve spontaneously. Attempts have been made to identify prognostic features for use within predictive outcome models such as various cyst volume ratios.20 Crombleholme and colleagues reported a useful tool to

classify the risk for hydrops, the need for fetal intervention and the perinatal survival rate.18 The CPAM volume ratio (CVR) is calculated by dividing the volume of the CPAM (length × height × width × 0.52) by the head circumference. A CVR greater than 1.6 predicts an increased risk for fetal hydrops (≤75%), and a CVR less than or equal to 1.6 is associated with a less than 3% risk for hydrops.18 CVR has proven on retrospective and prospective assessment to be the most useful predictor for the development of fetal hydrops. In addition, CVR can also be very useful in counselling parents, guiding the frequency of subsequent scans and determining which patients to preemptively treat with antenatal interventions described later. Other parameters such as a mass– thorax ratio, cystic predominance of the lesion and eventration of the diaphragm, do not add independent predictive value to the CVR.20,21 However, the unpredictability of the natural history of these lesions in utero means that regular sonographic assessment remains the most powerful tool in the shaping of individual management strategies. A variety of fetal interventions have been attempted to reduce the mass effect of a lesion, prevent the progression of complications and improve the outcome for these fetuses. However, these prenatal interventions are infrequently used and usually only in cases of persistent preterm hydrops when the prognosis is poor. Interventions include: • Maternal administration of betamethasone21,22 • Thoracocentesis23 • Shunt insertion23 • Resection of the cysts by open fetal surgery23 • Sclerotherapy by percutaneous injection24 • Radiofrequency ablation20 • Laser ablation25 Over the years, maternal betamethasone treatment has been suggested to have beneficial effects on large microcystic CPAMs.26-28 In 1998, Higby and colleagues first described resolution of a large CPAM when steroids therapy was initially given for fetal lung maturation.29 Since then, several others have reported similar effect with two standard doses of 12 mg of betamethasone intramuscularly, 24 hours apart, in which they showed a decrease in CPAM growth lesions with CVR of 1.4 or greater at 19 to 26 weeks of gestation27,30 Betamethasone seems to be a good choice because it does not cause decreased alveolarisation compared with dexamethasone.31 The exact mechanism by which steroids induce CPAM regression is not well understood. Curran and associates hypothesised that steroids promote the maturation of the lung cells,28 and San Feliciano and colleagues postulated that antenatal corticosteroids produce effects on vascular endothelial growth factor, which plays a crucial role in pulmonary development.31

CHAPTER 30  Fetal Lung Lesions

Others have speculated that steroids affect cell proliferation and apoptosis and downregulate several genes related to abnormal lung development, consequently reducing CPAM growth.22 Current evidence suggests that in large CPAMs with hydrops, a course of steroids appears to be a reasonable first-line therapy. However, a variable response on maternal betamethasone treatment has also been reported. In a study by Morris and colleagues 15 of 20 patients (13 were hydropic and 2 were nonhydropic) with highrisk fetal CPAM (defined as a CPAM associated with hydrops or a CVR >1.6) received at least one course of steroids.22 Seven of the 13 hydropic fetuses (54%) showed an initial response to steroid administration, but the 2 nonhydropic high-risk fetuses progressed to birth without developing hydrops. Seven of the 15 patients, however, resulted in fetal demise or early postnatal death, giving a survival rate of 53%.22 For a subset of high-risk CPAMs with CVR greater than 1.6 that do not adequately respond to a single course of steroids, multiple courses of antenatal betamethasone may facilitate the stabilisation or regression of CPAM and result in favourable short-term outcomes without the need for open fetal resection.32 Concerns regarding the long-term effects of maternal betamethasone on fetal development have been raised, but there are no studies so far to support deleterious effects occurring after one or two courses of maternal betamethasone.33,34 Whether steroids should also be used in low risk CPAMs without hydrops is more debatable, as the prognosis of low risk CPAMs without intervention is generally good and spontaneous regression may occur.35 One of the most commonly applied invasive intervention for the management of cystic lung lesion is thoracocentesis. In the majority of cystic lung lesions described by Cavoretto and colleagues, treatment was aimed primarily at drainage of the effusion rather than surgery of the lesions.36,37 However, treatment by thoracocentesis alone lead to the subsequent reaccumulation of the effusion in most of the cases, thus necessitating the placement of a thoracoamniotic shunt. Schrey and colleagues studied 11 fetuses with macrocystic CPAM who underwent thoracoamniotic shunting.37 Shunts were inserted at a mean gestational age of 24.6 (range, 17–32) weeks. Marked mediastinal shift was present in all cases. Six fetuses were hydropic, and of the remaining five, one had severe polyhydramnios, three had lesions that were rapidly increasing in size and one had a very large lesion at initial presentation. One hydropic fetus that underwent the procedure at 17 weeks died 1 day later. The mean gestational age of delivery of the 10 fetuses who survived was 38.2 weeks. The group concluded that fetal thoracoamniotic shunting for large macrocystic CPAM is associated with favourable outcome in most cases and should be considered in severe cases even before hydrops develops.37 Open surgery to resect lesions has serious maternal side effects and has only been practiced in one or two units in the United States. Growing evidence supports a potential benefit of fetal laser ablation of the systemic feeding artery (FLAFA) in improving survival and avoiding the need for postnatal surgery in fetuses with bronchopulmonary sequestration (BPS).25,38,39 In one case report, a hydropic fetus with a microcystic lung lesion and associated systemic arterial supply underwent successful laser coagulation of the feeding vessel and resolution of the hydrops.38 Cruz-Martinez and colleagues assessed the effectiveness of FLAFA in five fetuses with hybrid lung lesion associated with hydrops at risk for perinatal death.25,40 FLAFA was performed successfully in all cases at a median gestational age of 24.9 (range, 24.4–31.7) weeks. After the intervention, the dimensions of both lungs increased and fluid effusions resolved in all cases. All cases were delivered liveborn at term, without respiratory

329

morbidity, resulting in perinatal survival of 100%. During postnatal follow-up, three (60%) cases showed progressive regression of the entire lung mass and did not require postnatal surgery; in two (40%) cases, a progressive decrease in size of the mass was observed, but a cystic portion of the lung mass persisted and postnatal lobectomy was required. They concluded that in fetuses with large hybrid lung lesions at risk for perinatal death, FLAFA is feasible and could improve survival and decrease the need for postnatal surgery.25 However, it is important to be aware that these interventions are not commonly applied techniques and are only reserved in cases with a very poor prognosis, in which without any interventions, the fetuses are unlikely to survive. Because of the rarity of these cases, it is difficult to collect good quality controlled data to support the use of these more aggressive interventions. Management should be in a specialist centre with appropriate experience in fetal therapy. The majority of lung lesions are small, not associated with mediastinal shift, or the mediastinal shift resolves as pregnancy progresses. In these cases, delivery can be planned without the need for neonatal intensive care support. In contrast, if mediastinal shift remains into the third trimester, neonatal intensive care support with access to surgical backup is recommended. 

Laryngeal and Tracheal Atresia (see Fig. 30.4) Upper airway atresia can occur in isolation but may also be associated with other congenital abnormalities (e.g., Fraser syndrome).41 In cases associated with multiple abnormalities termination of the pregnancy may be discussed. When atresia is an isolated finding, then a prenatal intervention involving the placement of a tracheostomy or tracheoplasty below the obstruction to assist in lung development has been attempted with variable results.42,43 An intervention designed to secure a definitive airway during delivery, before the onset of respiration, is termed ex utero intrapartum treatment (‘EXIT procedure’). The technique requires the positioning of a definitive airway by tracheostomy or intubation during delivery, whilst fetal oxygenation is still maintained via the placenta.44 When stable, delivery of the fetus is completed and surgery to correct the obstruction can be planned (see Chapter 31). Similar procedures may also be considered in cases of complete high airway obstruction from other causes (e.g., cystic hygroma). Malformations that cause complete high airway obstruction, including laryngeal and tracheal atresia, have been classified into a group designated ‘congenital high airway obstruction syndrome’ (CHAOS).45 

Pulmonary Hypoplasia and Agenesis There are multiple causes of lung hypoplasia (described previously). Correction of the underlying cause, if possible, may permit further alveolar development, both prenatally and postnatally. In many of the conditions associated with pulmonary hypoplasia or pulmonary agenesis, such as those described earlier, the underlying abnormality is so severe that survival would not be possible even if the pulmonary hypoplasia could be corrected. Amnioinfusion in women with early preterm rupture of membranes is not proven to improve outcome and therefore is not recommended.46 

Postnatal Management Cystic Lung Lesions In common with prenatal management, there is considerable overlap in management strategies for of all of these lesions. Only a

330

SE C T I O N 7     Diagnosis and Management of Fetal Malformations

minority of neonates with a CTM exhibit respiratory distress due to the mass effect of the lesion. In these cases, however, surgical intervention is warranted, and the management is resection of the lesion. In a case series of 119 neonates cared for at Great Ormond Street Hospital (GOSH) diagnosed prenatally with CPAM or PS, only 8 (6.7%) required emergency surgery during the neonatal period.47 This is in keeping with another large case series from Southampton of 72 neonates with a prenatal diagnosis of congenital lung malformation in which only one required emergency surgery. The vast majority of neonates remain asymptomatic. A CT scan of the chest during the first 3 months of life48 should be undertaken in all cases with a prenatally diagnosed cystic lung lesion to enable an accurate assessment because chest radiographs alone are unreliable and may falsely indicate resolution of the lesion.48 An echocardiogram can delineate the systemic feeding vessel in cases of likely PS. After initial investigations, further management remains the subject of intense debate because the need for surgical intervention is less clear. This has resulted in highly divergent management strategies largely because of a deficiency in our knowledge of the natural history of these lesions. Supporters of a conservative approach suggest that these lesions were largely clinically silent in the population before routine anomaly scanning, the risk for complications appears low49 and postnatal spontaneous resolution may occur.50 However, advocates of a surgical approach cite concerns regarding the potential for malignant transformation and recurrent infection, resulting in approximately 70% of lesions being resected worldwide.51 Divergent management strategies make prenatal counselling of parents challenging and emphasises the need for a multidisciplinary approach involving the obstetrician, neonatologist, paediatrician and paediatric surgeon, all providing a consistent message. In the following section, we will summarise the arguments for and against a surgical approach to asymptomatic cystic lung lesions. There are four arguments used to justify a surgical approach9: • The risk for malignancy • The risk for complications such as infection •  The potential for compensatory lung regrowth after early resection • A low complication rate after elective surgery The risk for malignancy. The two types of malignancy associated with cystic lung lesions are PPB and bronchioloalveolar carcinoma (BAC), both of which are rare in early life.49,50 Pleuropulmonary blastomas are a distinct pathological entity. Crucially, a type 1 PPB cannot be distinguished radiologically from a benign cystic lung lesion such as a type 1 CPAM. The risk for a prenatally diagnosed benign cystic lesion in fact representing a PPB is small because the majority of PBBs are identified postnatally. A total of 350 pathology-confirmed PBB cases have been identified to date by The International PPB Registry,52 of which only 9 were identified on prenatal ultrasound scan. The probability that a benign cystic lung lesion may be a PPB is increased if there is a family history of PPB associated tumours (e.g., ovarian, renal and thyroid) or mutations within the DICER1 gene (demonstrated to be associated with 66% of cases) are detected.54,55 The prognosis of these lesions depends on their histologic staging. Type 1 PBB has a better outcome (91% 5-year survival rate) than types two and three (71% and 53% 5-year survival rates, respectively).52 Bronchioloalveolar carcinomas occur after the transformation of mucinous cells, which are only identified within type 1 CPAMs. These cells have the potential to develop into areas of atypical adenomatous hyperplasia, transform into BAC (noninvasive) and

subsequently adenocarcinoma (AC) (invasive).54,55 The time course of the progression described or if progression is inevitable is not known. Both BAC and AC are exceptionally rare with only 24 cases described in total, usually as an incidental finding in adult patients. Importantly, there is evidence that prophylactic resection of cystic lung lesions does not eliminate the risk for malignancy. Adenocarcinoma has been described after previous resection of CPAM in early life,56 and a PPB has been described after prior resection of a cystic lesion from an anatomically distinct area of the lung.57  The risk for other complications including recurrent infection.

The incidence of respiratory infection in children with CTM is low in early life. Stanton and colleagues51 described 505 conservatively managed infants, of whom 3.2% became symptomatic in the first year. Ng and colleagues49 reported a series of 74 patients of whom 5% of developed symptoms over a median follow-up period of 5 years. The GOSH cohort (described earlier) has been followed up for a median period of 9.9 years.58 We have established that after the second year, the rate of resection for recurrent respiratory infection diminishes, and there were no resections in those over the age of 5 years. In cases of PS, an additional complication is the possibility of heart failure caused by high flow through the feeding vessel. In these cases, embolization of the feeding vessel or resection of the lesion has been performed. In addition, involution of these lesions has been described after embolization.59 Bronchogenic and enteric cysts, if symptomatic, tend to cause symptoms associated with airway compression, but other complications, including bleeding, have been described.  The potential for compensatory lung growth after early resection. Based on the traditional view that alveolar division

only continues for the first 2 years of life, it has been suggested that improved compensatory lung growth may occur if resection is completed during this period. However, new evidence suggests that genuine new alveolar growth is likely to occur into adolescence.60 Furthermore, the majority of lesions identified are small and appear to continue to reduce in size as the lung grows in early life. Indeed, in the GOSH cohort, we identified that 5.9% of lesions had disappeared on CT imaging during the postnatal period, which is a similar proportion to that reported previously.50  The risk of surgery. If surgical intervention is being considered, then the risks associated with surgery must be balanced against the risks of complications and intended benefits described earlier. In their meta-analysis, Stanton and colleagues51 reported a complication rate of 5% in elective procedures carried out on asymptomatic infants, including air leak, infection, effusion and death in one case. More recently, Hall and colleagues61 have reported a complication rate of 23% in a case series of 60 patients with asymptomatic CPAM who underwent surgery. This included 3 cases of major complications: tension pneumothorax, aggressive chest wall fibromatosis and near-fatal haemorrhage. 

Pulmonary Agenesis and Hypoplasia The outcome of babies born with pulmonary hypoplasia or agenesis is generally poor and is associated with high neonatal mortality and significant long-term morbidity rates.62,63 After delivery, these infant need respiratory support, which can range from supplying supplemental oxygen to mechanical ventilation, including highfrequency ventilation and extracorporeal membrane oxygenation. Infants with severe respiratory failure secondary to pulmonary hypoplasia and documented persistent pulmonary hypertension of the newborn may benefit from inhaled nitric oxide (NO), but

CHAPTER 30  Fetal Lung Lesions

the data are limited.64 Aggressive ventilation in these infants can cause overexpansion of lungs with compresses intraalveolar capillaries, which further exacerbates pulmonary hypertension. There is a small population study which demonstrated that NO may be beneficial as adjunct therapy in combination with sildenafil and dopamine infusions to improve neonatal survival; however, larger studies are needed to support the practice.65 

Conclusion There are a wide range of congenital malformations of the respiratory tract, some of which are detected on prenatal sonography. It is not possible to distinguish among these lesions sonographically, and prenatal reports should be based on a purely descriptive system of reporting. There is also considerable overlap in the pathological features of various cystic lung lesions, the aetiology of which is poorly understood.

331

Interventions are usually only undertaken in the prenatal and postnatal period if significant symptoms are present. The vast majority of lesions detected are asymptomatic, with many appearing to resolve spontaneously in both the pre- and postnatal periods. Postnatal management remains controversial because of a lack of knowledge of their natural history. A conservative approach to prenatally diagnosed asymptomatic CTMs is a reasonable option in some asymptomatic cases. Careful explanation to parents and prenatal counselling regarding these lesions is recommended. Unfortunately, there remains no prospect of a randomised trial to assist with decision making in these difficult cases, and clinical judgment is required. Continued long-term follow-up of cases will assist in further definition of their natural history. Access the complete reference list online at ExpertConsult.com. Self-assessment questions available at ExpertConsult.com.

References 1. Bush A. Congenital lung disease. A plea for clear thinking and clear nomenclature. Pediatr Pulmonol. 2001;32:328–337. 2. Bush A, Hogg J, Chitty LS. Cystic lung lesions—prenatal diagnosis and management. Prenat Diagn. 2008;28:604–611. 3. Liu YP, Chen CP, Shih SL, et al. Fetal cystic lung lesions: evaluation with magnetic resonance imaging. Pediatr Pulmonol. 2010;45:592–600. 4. Zeidan S, Hery G, Lacroix F, et  al. Intralobar sequestration associated with cystic adenomatoid malformation: diagnostic and thoracoscopic pitfalls. Surg Endosc. 2009;23:1750–1753. 5. Stocker JT. Congenital pulmonary airway malformation—a new name and an expanded classification of congenital cystic adenomatoid malformations of the lung. Histopathology. 2002;41:424S–431S. 6. EUROCAT: European Surveillance of Congenital Anomalies. EUROCAT Guide 1.4 and Reference Documents 2013. www.eurocat‐network. eu/. 7. Stocker J, Madewell J, Drake R. Congenital cystic adenomatoid malformation of the lung. Classification and morphologic spectrum. Hum Pathol. 1977;8:155–171. 8. Langston C. New concepts in the pathology of congenital lung malformations. Semin Pediatr Surg. 2003;12:17–37. 9. Kotecha S, Barbato A, Bush A, et al. Antenatal and postnatal management of congenital cystic adenomatoid malformation. Paediatr Respir Rev. 2012;13:162–170. 10. Kunisaki SM, Fauza DO, Nemes LP, et al. Bronchial atresia: the hidden pathology within a spectrum of prenatally diagnosed lung masses. J Pediatr Surg. 2006;41(1):61–65. 11. Nobuhara KK, Gorski YC, La Quaglia MP, et al. Bronchogenic cysts and esophageal duplications. Common origins and treatment. J Pediatr Surg. 1997;32:1408–1413. 12. Mani H, Suarez E, Stocker JT. The morphologic spectrum of infantile lobar emphysema. A study of 33 cases. Paediatr Respir Rev. 2004;5(suppl 1):S313–S320. 13. Cass DL, Cromblehome TM, Howell LJ, et al. Cystic lung lesions with systemic blood supply: a hybrid of congenital cystic adenomatoid malformation and bronchopulmonary sequestration. J Pediatr Surg. 1997;32: 986–990. 14. Newman B. Congenital bronchopulmonary foregut malformations: concepts and controversies [review]. Pediatr Radiol. 2006;36(8):773–791. 15. Cavoretto P, Molina F, Poggi S, et  al. Prenatal diagnosis and outcome of echogenic fetal lung lesions. Ultrasound Obstet Gynecol. 2008;32: 769–783. 16. Ierullo AM, Ganapathy R, Crowley S, et al. Neonatal outcome of antenatally diagnosed congenital cystic adenomatoid malformations. Ultrasound Obstet Gynecol. 2005;26:150–153. 17. Kunisaki SM, Barnewalt CE, Estroff JA, et al. Large fetal congenital cystic adenomatoid malformations: growth trends and patient survival. J Pediatr Surg. 2007;42:404–410. 18. Crombleholme TM, Coleman B, Hedrick H, et al. Cystic adenomatoid malformation volume ratio predicts outcome in prenatally diagnosed cystic adenomatoid malformation of the lung. J Pediatr Surg. 2002;37(3):331–338. 19. Schott S, Mackensen-Haen S, Wallwiener M, et al. Cystic adenomatoid malformation of the lung causing hydrops fetalis: case report and review of the literature. Arch Gynecol Obstet. 2009;280:293–296. 20. Vu L, Tsao K, Lee H, et al. Characteristics of congenital cystic adenomatoid malformations associated with nonimmune hydrops and outcome. J Pediatr Surg. 2007;42:1351–1356. 21. Curran PF, Jelin EB, Rand L, et  al. Prenatal steroids for microcystic congenital cystic adenomatoid malformations. J Pediatr Surg. 2010;45: 145–150. 22. Morris LM, Lim FY, Livingston JC, et  al. High–risk fetal congenital pulmonary airway malformations have a variable response to steroids. J Pediatr Surg. 2009;44:60–65. 23. Adzick NS, Harrison MR, Crombleholme TM, et al. Fetal lung lesions. Management and outcome. Am J Obstet Gynecol. 1998;179:884–889. 24. Bermúdez C, Pérez–Wulff J, Arcadipane M, et  al. Percutaneous fetal sclerotherapy for congenital cystic adenomatoid malformation of the lung. Fetal Diagn Ther. 2008;24:237–240. 25. Cruz–Martinez R, Mendez A, Duenas–Riano J, et al. Fetal laser surgery prevents fetal death and avoids the need for neonatal sequestrectomy in cases with bronchopulmonary sequestration. Ultrasound Obstet Gynecol. 2015;46:627–628.

26. Loh KC, Jelin E, Hirose S, et al. Microcystic congenital pulmonary airway malformation with hydrops fetalis: steroids vs open fetal resection. J Pediatr Surg. 2012;47:36–39. 27. Tsao K, Hawgood S, Vu L, et al. Resolution of hydrops fetalis in congenital cystic adenomatoid malformation after prenatal steroid therapy. J Pediatr Surg. 2003;38:508–510. 28. Curran PF, Jelin EB, Rand L, et  al. Prenatal steroids for microcystic congenital cystic adenomatoid malformations. J Pediatr Surg. 2010;45: 145–150. 29. Higby K, Melendez BA, Heiman HS. Spontaneous resolution of nonimmune hydrops in a fetus with a cystic adenomatoid malformation. J Perinatol. 1998;18:308–310. 30. Peranteau WH, Wilson RD, Liechty KW, et al. Effect of maternal betamethasone administration on prenatal congenital cystic adenomatoid malformation growth and fetal survival. Fetal Diagn Ther. 2007;22:365–371. 31. San Feliciano L, Remesal A, Isidoro-García M, Ludeña D. Dexamethasone and betamethasone for prenatal lung maturation: differences in vascular endothelial growth factor expression and alveolarization in rats. Neonatology. 2011;100:105–110. 32. Derderian S, Coleman A, Jeanty C, et al. Favorable outcomes in high– risk congenital pulmonary airway malformations treated with multiple courses of maternal betamethasone. J Pediatr Surg. 2015;50:515–518. 33. Stutchfield PR, Whitaker R, Gliddon AE, et al. Behavioural, educational and respiratory outcomes of antenatal betamethasone for term cesarean section (ASTECS trial). Arch Dis Child Fetal Neonatal Ed. 2013;98: F195–F200. 34. Wapner RJ, Sorokin Y, Thom EA, et al. Single versus weekly courses of antenatal corticosteroids: evaluation of safety and efficacy. Am J Obstet Gynecol. 2006;195:633–642. 35. Witlox RS, Lopriore E, Oepkes D. Prenatal interventions for fetal lung lesions. Prenat Diagn. 2011;31:628–636. 36. Cavoretto P, Molina F, Poggi S, et  al. Prenatal diagnosis and outcome of echogenic fetal lung lesions. Ultrasound Obstet Gynecol. 2008;32:769– 783. 37. Schrey S, Kelly EN, Langer JC, et  al. Fetal thoracoamniotic shunting for large macrocystic congenital cystic adenomatoid malformations of the lung. Ultrasound Obstet Gynecol. 2012;39(5):515–520. 38. Oepkes D, Devlieger R, Lopriore E, et al. Successful ultrasound–guided laser treatment of fetal hydrops caused by pulmonary sequestration. Ultrasound Obstet Gynecol. 2007;29(4):457–459. 39. Mallmann MR, Geipel A, Bludau M, et al. Bronchopulmonary sequestration with massive pleural effusion: pleuroamniotic shunting vs intrafetal vascular laser ablation. Ultrasound Obstet Gynecol. 2014;44:441–446. 40. Cruz-Martinez R, Martínez-Rodríguez M, Bermúdez-Rojas M, et  al. Fetal laser ablation of feeding artery of cystic lung lesions with systemic arterial blood supply. Ultrasound Obstet Gynecol. 2017;49(6):744–750. 41. Lacy DE, Shaw NJ, Pilling DW, et  al. Outcome of congenital lung abnormalities detected antenatally. Acta Paediatr. 1999;88:454–458. 42. Paek BW, Callen PW, Kitterman J, et  al. Successful fetal intervention for congenital high airway obstruction syndrome. Fetal Diagn Ther. 2002;17:272–276. 43. Saadai P, Jelin EB, Nijagal A, et  al. Long–term outcomes after fetal therapy for congenital high airway obstructive syndrome. J Pediatr Surg. 2012;47(6):1095–1100. 44. Wilson RD, Chitty LS. Anomalies of the fetal thorax and abdomen: diagnosis, management and outcome. Prenat Diagn. 2008;28(7):567. 45. Lim FY, Crombleholme TM, Hedrick HL, et  al. Congenital high airway obstruction: natural history and management. J Pediatr Surg. 2003;38:940–945. 46. Roberts D, Vause S, Martin W, et al. Amnioinfusion in very early preterm prelabor rupture of membranes (AMIPROM): pregnancy, neonatal and maternal outcomes in a randomized controlled pilot study. Ultrasound Obstet Gynecol. 2014;43:490–499. 47. Cook J, Chitty LS, De Coppi P, et al. The natural history of prenatally diagnosed congenital cystic lung lesions: long-term follow-up of 119 cases. Arch Dis Child. 2017;102:789–790. 48. Epelman M, Kreiger PA, Servaes S, et  al. Current imaging of prenatally diagnosed congenital lung lesions. Semin Ultrasound CT MR. 2010;31: 141–157. 49. Ng C, Stanwell J, Burge DM, et al. Conservative management of antenatally diagnosed cystic lung malformations. Arch Dis Child. 2014;99:432–437. 50. Butterworth SA, Blair GK. Postnatal spontaneous resolution of congenital cystic adenomatoid malformations. J Pediatr Surg. 2005;40:832–834.

331.e1

331.e2

References

51. Stanton M, Njere I, Ade-Ajayi N, et  al. Systematic review and meta– analysis of the postnatal management of congenital cystic lung lesions. J Pediatr Surg. 2009;44:1027–1033. 52. Messinger YH, Stewart DR, Priest JR, et al. Pleuropulmonary blastoma: a report on 350 central pathology–confirmed pleuropulmonary blastoma cases by the International Pleuropulmonary Blastoma Registry. Cancer. 2015;121:276–285. 53. Schultz KA, Harris A, Williams GM, et al. Judicious DICER1 testing and surveillance imaging facilitates early diagnosis and cure of pleuropulmonary blastoma. Pediatr Blood Cancer. 2014;61:1695–1697. 54. Mani H, Shilo K, Galvin JR, et al. Spectrum of precursor and invasive neoplastic lesions in type 1 congenital pulmonary airway malformation: case report and review of the literature. Histopathology. 2007;51:552–580. 55. Travis WD, Garg K, Franklin WA, et al. Bronchioloalveolar carcinoma and lung adenocarcinoma: the clinical importance and research relevance of the 2004 world health organization pathologic criteria. J Thorac Oncol. 2006;1:S13–S19. 56. Ioachimescu O, Mehta A. From cystic pulmonary airway malformation, to bronchioloalveolar carcinoma and adenocarcinoma of the lung. Eur Respir J. 2005;26:1181–1187. 57. Papagiannnopoulos K, Sheppard M, Bush A, et  al. Pleuropulmonary blastoma: is prophylactic resection of congenital lung cysts effective. Ann Thorac Surg. 2001;72:604–605. 58. Cook J, Chitty LS, De Coppi P, et al. The natural history of prenatally diagnosed congenital cystic lung lesions: long-term follow-up of 119 cases. Arch Dis Child. 2017;102(9):798–803.

59. Cho MJ, Kim DY, Kim SC, et al. Embolization versus surgical resection of pulmonary sequestration: clinical experiences with a thoracoscopic approach. J Pediatr Surg. 2012;47(12):2228–2233. 60. Narayanan M, Owers-Bradley J, Beardsmore CS, et  al. Alveolarization continues during childhood and adolescence: new evidence from helium–3 magnetic resonance. Am J Respir Crit Care Med. 2012;185:186– 191. 61. Hall NJ, Chiu PP, Langer JC. Morbidity after elective resection of prenatally diagnosed asymptomatic congenital pulmonary airway malformations. Pediatr Pulmonol. 2016;51(5):525–530. 62. Williams O, Hutchings G, Debieve F, Debauche C. Contemporary neonatal outcome following rupture of membranes prior to 25 weeks with prolonged oligohydramnios. Early Hum Dev. 2009;85:273– 277. 63. Kilbride HW, Yeast J, Thibeault DW. Defining limits of survival: lethal pulmonary hypoplasia after midtrimester premature rupture of membranes. Am J Obstet Gynecol. 1996;175:675–681. 64. Keszler M. Guidelines for rational and cost–effective use of iNO therapy in term and preterm infants. J Clin Neonatol. 2012;1(2):59–63. 65. Tiryaki S, Ozcan C, Erdener A. Initial oxygenation response to inhaled nitric oxide predicts improved outcomes in congenital diaphragmatic hernia. Drugs D R. 2014;14(4):215–219.

31

Congenital Diaphragmatic Hernia FRANCESCA MARIA RUSSO, LIESBETH LEWI, JUTE RICHTER AND JAN DEPREST

KEY POINTS • Congenital diaphragmatic hernia occurs in 1 to 4 in 10,000 births. The condition is isolated in more than 50% of the cases. • The main causes of mortality and morbidity are respiratory insufficiency and persistent pulmonary hypertension of the newborn. • Prenatal diagnosis should be made by screening ultrasound, after which patients are referred to specialised centres. • In isolated cases, the size of the lungs and the presence of liver herniation are antenatal predictors of outcome. • In cases with anticipated poor outcome, a potential option is fetal treatment in the form of fetoscopic endoluminal tracheal occlusion.

Epidemiology and Background Congenital diaphragmatic hernia (CDH) is a developmental anomaly with a prevalence ranging between 1 and 4 in 10,000 births, which means that in Europe, around 2000 children are born with this condition every year.1 Despite being relatively uncommon, CDH is a major clinical concern inside the realm of neonatology, with important implications for diagnosis, management and prognosis. Although medical and surgical management have improved the outcome of this condition, CDH remains associated with high mortality and significant morbidity.2 A primary characteristic of CDH is that the diaphragm fails to form properly during embryogenesis. Normally, the diaphragm develops to form a continuous sheet that completely separates the thoracic and abdominal cavities before the major period of internal organ growth. In the case of CDH, a significant proportion of the diaphragm is absent.3 The defect is usually on the left side (85%) but can also occur on the right (12%–15%) or bilaterally. In some rare instances, a true agenesis of the hemidiaphragm is present, but in most cases, the defect is limited to the posterolateral region of the diaphragm (referred to as Bochdalek hernia). The anterior (Morgagni hernia; 25%–30%) or central regions (2%–5%) can also be affected.4 Less often, the diaphragm is present but thinned and devoid of muscular fibres (diaphragmatic eventration).5 The diaphragmatic defect allows herniation of abdominal viscera into the thorax, where they compete for the space normally reserved to accommodate the growing lungs. When the defect is located on the left side, the thorax may contain small and large bowel, the spleen, the stomach, the left lobe of the liver and, 332

occasionally, the kidney. Right-sided CDHs virtually always contain part of the right lobe of the liver and sometimes the bowel, kidney, or both.6 The loss of a continuous diaphragmatic muscle also impairs fetal breathing movements that are necessary for proper stretch-induced lung maturation.7 Lungs of fetuses with CDH display variable degrees of lung hypoplasia, with impairment of both airway and vascular maturation. These changes become symptomatic immediately after birth, when neonates have variable degrees of respiratory insufficiency and persistent pulmonary hypertension (PPH), which is often resistant to inhaled nitric oxide (iNO).8 In 50% to 60% of cases, the diaphragmatic defect and lung hypoplasia are the only significant anomalies. In the remaining cases, there are major nonpulmonary congenital anomalies.9 Cardiovascular defects such as ventricular septal defects, cardiac outflow anomalies (tetralogy of Fallot, double outlet right ventricle, transposition of the great vessels and others) and abnormal great vessels (right aortic arch, double aortic arch, truncus arteriosus, abnormal subclavian arteries and others) are the most common associated anomalies, found in about one third of patients with CDH.10 Left ventricular hypoplasia has also been described, yet its occurrence and clinical relevance are debated.11 Musculoskeletal defects such as anomalies of the limbs or of the number and shape of the vertebral bodies or ribs, neural tube defects,9 abdominal wall defects,12 craniofacial defects or urinary tract anomalies have also been reported. Associated malformations are sometimes components of Pallister-Killian and Fryns syndromes; GhersoniBaruch syndrome; Wilms tumour, aniridia, genitourinary anomalies, and mental retardation (WAGR); Denys-Drash; and other syndromes. Some chromosomal anomalies, such as 9p tetrasomy, have CDH as part of their spectrum. For further information we refer to the excellent review by Slavotinek and colleagues.13 Finally, the presence of the intestine in the thorax during late fetal development causes malrotation, malfixation, or both, which can further complicate the disease. 

Aetiology and Pathogenesis The causes of CDH are largely unknown, although exposure to teratogens or pharmacologic agents has been suggested. In particular, phenmetrazine, thalidomide, quinine, nitrofen and vitamin A deficiency have been linked to this disease.14 In the classical view, the defect in CDH occurs first in the muscular part of the diaphragm. However, studies in rats have suggested that CDH is a primary lung pathology, even in humans.15 Keijzer and colleagues proposed the ‘dual-hit hypothesis’, that is, that two independent events cause the major features seen in CDH.16 These hits disturb

CHAPTER 31  Congenital Diaphragmatic Hernia

normal lung development (first hit) and diaphragm formation (second hit). Data from animal studies confirm this hypothesis: in the toxic nitrofen model in rats, abnormalities in the ipsilateral as well as the contralateral lung are present already before the development of the diaphragm.17 Another theory is based on the hypothesis that nonclosure of the pleuroperitoneal canals would be caused by a defect in the pleuroperitoneal folds (PPFs), the source of diaphragmatic cells. Of interest, the diaphragmatic defect in the nitrofen model is located more medial than could be expected from nonclosure of these canals.18 Therefore it has been proposed that the origin of the diaphragmatic defect lies in the amuscular mesenchymal precursor cells of the diaphragm, which are also derived from the PPFs. This theory is based on the observation that although the migration of muscular precursors is not disturbed, a defect occurs in regions of the underlying mesenchymal substratum of the PPF. This would subsequently contribute to the defective region in CDH. Finally, an involvement of the retinoid signalling pathway is likely in CDH. Both animal and clinical studies have shown that retinol and retinol-binding proteins are decreased in newborns with this malformation.19,20 Moreover, some of the genes involved in the pathogenesis of human CDH are tightly related to retinoid signalling.21 

Prognosis Congenital diaphragmatic hernia was described many years ago,22 but survival after repair was not achieved until the 20th century.6 The symptoms of insufficient gas exchange are associated with those of PPH, and unless invasive treatment is undertaken, the respiratory condition deteriorates rapidly until death. Survival rates vary widely amongst various neonatal management centres. When only liveborn children undergoing surgery are included, survival until discharge approaches 70% for isolated CDH.23 If terminations of pregnancy, spontaneous abortions, stillbirths, prehospital or preoperative deaths and surgical mortality are taken into account, the mortality rate is between 50% and 60%.12 The gap between these numbers is usually referred to as the ‘hidden mortality’. However, significant advances in the postnatal management, with the introduction of ‘gentle ventilation and permissive hypercarbia’, have resulted in improved survival rates over the past 2 decades.24 The improved survival of very sick babies is, however, associated with a higher risk for severe morbidity24 persisting beyond the initial hospitalisation, especially in those treated with extracorporeal membrane oxygenation (ECMO). The severity of lung hypoplasia and PPH are the key determinants of both mortality and morbidity and thus of quality of life.25 More than half of survivors are oxygen dependent at 28 days of age,2 and 16% require oxygen at the time of discharge for a mean duration of 14.5 months.26 Restrictive and obstructive lung diseases have also been reported in CDH survivors many years after operation.27 Diaphragmatic rigidity and thoracic deformities can play a minor role in chronic lung disease.28 Bronchodilators may be needed in 40% of patients in the first year of life.26 PPH may persist in up to 30% of patients at 2 months of age and is associated with increased risk for early death and increased morbidity.29 PPH also deeply affects the quality of life in CDH survivors.30 Nonpulmonary diseases are also relatively frequent in CDH survivors. Gastroesophageal reflux is caused by a distorted anatomy of the diaphragmatic sling, both congenital and related to the surgical CDH repair. Also, esophageal motility and gastroesophageal

333

sphincter function are disturbed.31 In addition, malrotation may delay gastric emptying, and the abnormal balance of pressures in the thorax and abdomen may facilitate retrograde passage of gastric contents to the oesophagus. Reflux can complicate the preexisting respiratory disease, and for all these reasons, a considerable proportion of these patients respond poorly to medical treatment and ultimately require antireflux surgery.32 Reflux and the need for antireflux surgery are dependent on the degree of pulmonary hypoplasia.33 Neurodevelopmental deficits are possible in patients in whom brain oxygenation was marginal for long periods of time and particularly when ECMO required major vascular occlusion.34 Neurosensory deafness occurs in a small proportion of children surviving CDH.34 This is progressive, and it is often tied to both prolonged antibiotic treatment and a particular sensitivity or a developmental defect of the inner ear. These sequelae, and the frequent associated malformations, require long-term follow-up for early diagnosis and proactive management.6 Most babies eventually lead a life very close to normal provided when managed in a multidisciplinary follow-up program.35 

Congenital Diaphragmatic Hernia–Related Aberrations in Lung Development In CDH, the lung is hypoplastic not only on the side of the hernia, but the contralateral side is affected as well but to a variable extent. Peripheral airways are less developed, and there are markedly less and smaller alveoli, thickened alveolar walls and an increased amount of interstitial tissue36 so that there is less alveolar airspace and gas exchange surface area. Parallel to airway changes, there is a reduction in arteries, resulting in a hypoplastic vascular bed. Morphologically, a thickening of the vascular wall is determined by an increase in arterial media and adventitia and by neomuscularisation of the small pulmonary arteries, which are normally partially or nonmuscularised.37 The structural remodelling of the small pulmonary arteries reduces their ability to dilate to increase the vascular bed capacity and reduce the pressure in the pulmonary circulation after birth.38 After birth, further muscularisation of this ‘immature’ pulmonary vasculature occurs, with migration of adventitial fibroblasts into the media and smooth muscle cells into the intima. These morphologically abnormal vessels respond abnormally to mechanical and chemical stimuli, including the shear stress accompanying raised blood flow through the narrowed vessels. Increased contractility and impaired relaxation of pulmonary arteries have been demonstrated in animal models39 and could be responsible for the low efficacy of conventional vasodilatory therapy. 

Prenatal Diagnosis and Outcome Prediction Today, high-resolution ultrasound and advances in prenatal diagnosis and genetic testing have made it possible to diagnose CDH relatively early and to rule out a number of associated anomalies. Ideally, antenatal ultrasound screening identifies cases in utero, reportedly in more than 70% of cases, yet lower numbers have been reported as well.40 Intrathoracic abdominal organs are the hallmark of CDH. Left-sided CDH typically presents with a mediastinal shift to the right caused by herniation of the stomach, intestines and in some cases liver (Fig. 31.1A). In right-sided CDH, part of the liver is visible in the chest, with mediastinal

334

SE C T I O N 7     Diagnosis and Management of Fetal Malformations

S L

L

B

A

B

C

D

• Fig. 31.1  Ultrasound appearance of congenital diaphragmatic hernia (CDH) on a cross-section at the level of the four-chamber view. A, Fetus with left-sided CDH. The chest contains bowels (B), stomach (S) and part of the liver (L), and there is a shift of the heart and mediastinum towards the right side. B, Right-sided CDH: the liver (L) is visualised in the thorax, and there is a mediastinal shift towards the left. C, Measurement of the lung area and D, measurement of the head circumference for calculation of the observed to expected lung to head ratio. (Courtesy of UZ Leuven.)

shift to the left1 (Fig. 31.1B). Prenatal diagnosis allows in utero referral to a tertiary care centre used to manage the condition for expert assessment, counselling and perinatal management. Additional genetic and morphologic assessment using ultrasound or magnetic resonance imaging (MRI) can be used to rule out associated malformations. For isolated cases, clinicians should individualise prognosis to counsel parents about prenatal options. Most prediction methods are based on lung size, liver herniation and pulmonary circulation, and more recently stomach position.41-46 Ultrasound measurement of the lung-to-head ratio (LHR) is most widely used. The LHR, first described by Metkus and colleagues,43 provides an indirect estimate of the size of the lung contralateral to the hernia normalised for the head circumference (Fig. 31.1C). It is a twodimensional measure and changes over gestation as the lung area grows more rapidly than the head circumference. The observed to expected (o/e) LHR has subsequently been proposed to eliminate the effect of gestational age at assessment.47 The o/e LHR has been shown to be an independent predictor of postnatal survival47 both in left- and right-sided CDH48 and to some extent of short-term morbidity.49 Other methods to assess lung size, such as the lungto-thorax ratio,50 the quantitative lung index51 and three-dimensional measurements of lung volume,52 have also been proposed, but the predictive value of these parameters has not been validated to the same extent as the o/e LHR. Liver herniation can be determined both for left- as well as right-sided hernia, although in the latter case, the liver is nearly always herniating through the defect. For left-sided CDH, herniation of the liver into the thorax has been recognised as a predictor of poor outcome by Harrison and colleagues53 and seems

to be independent from lung size.54 Although it is theoretically possible to quantify the amount of liver in the thorax, liver position on ultrasound is usually categorised binary as either ‘up’ (in the thorax), or ‘down’ (confined to the abdomen). Because the echogenicity of the liver is very similar to that of the lung, it can be difficult to assess liver position with ultrasound (Fig. 31.2A and Video 31.1). Additional indirect signs suggestive of liver herniation are presence of hepatic vessels above the diaphragmatic edge43 (Fig. 31.2B), abnormal position of the ductus venosus or the gallbladder55 (Fig. 31.2C) or deviation (bowing) of the umbilical vein on towards the left side56 (Fig. 31.2D). Finally, evaluation of stomach position has recently been reintroduced as an indirect method to estimate severity of the disease in left-sided CDH because it has been shown to correlate with the proportion of intrathoracic liver determined by MRI.57 The combination of liver herniation and o/e LHR is now a widely accepted method to stratify fetuses with left- and rightsided CDH into groups with an increasing degree of pulmonary hypoplasia and corresponding mortality rates. It is also used to select patients for fetal therapy trials (Fig. 31.3). Severity assessment by MRI theoretically has several advantages over ultrasound. Visualisation is not limited by maternal habitus, amniotic fluid volume or fetal position. With MRI, the total (left and right) lung volume can be measured, which may better predict postnatal lung function. Volumetry may also accurately quantify liver and stomach herniation.58,59 Although one study has claimed that MRI better predicts outcome than ultrasound,60 the numbers do not allow such a claim nor has this been proven in clinical practice.61

CHAPTER 31  Congenital Diaphragmatic Hernia

335

L

A

B Right

C

CDH

Control

D

Left

•

Fig. 31.2 Ultrasound evidence of liver herniation in left-sided congenital diaphragmatic hernia (CDH) cases. A, Sagittal section of the fetal abdomen and thorax demonstrating liver herniation (L). B, Visualisation of the hepatic vessels above the diaphragmatic edge (arrow). C, As a consequence of the herniation and rotation of the liver, the gallbladder (arrow) is visualised on the left side of the abdomen. D, Bowing of the umbilical vein towards the left side; the same section from a fetus with CDH without liver herniation is provided on the right for comparison. (Courtesy of UZ Leuven.)

Liver in thorax (‘up’)

Liver in abdomen (‘down’)

Total trial for Total trial for severe lung hypoplasia moderate lung hypoplasia

100 90 80 70 60 50 40 30 20 10 0 o/e LHR (%) 45 Mild, 47% of cases

B •

Fig. 31.3 Patient stratification and selection of candidates for intrauterine therapy according to the observed to expected (o/e) lung-to-head ratio (LHR) for left-sided (A) and right-sided (B) congenital diaphragmatic hernia. (Adapted from Jani et al.47 and DeKoninck et al.87)

Lung size and liver herniation also predict neonatal morbidity, such as the duration of assisted ventilation, the need for supplemental oxygen, the need for patch repair and the time it takes to full enteral feeding.46 The literature on prediction of PPH is more limited (systematically reviewed by Russo and colleagues62).

Several candidate parameters have been suggested in single case series, including lung size, presence of visceral herniation and direct assessment of the pulmonary vasculature, which may provide additional information. However, to our knowledge, there is currently no validated antenatal predictor for PPH. 

336

SE C T I O N 7     Diagnosis and Management of Fetal Malformations

Current Neonatal Management In an attempt to improve outcome and permit comparison of outcome data, the CDH EURO consortium has published a standardised neonatal treatment protocol (revised in 201563). Delivery is planned (either via induction or via caesarean section) after 39 weeks in a high-volume tertiary centre, and the newborn is immediately intubated. Historically, high-pressure high oxygen conventional respiratory assistance was used, leading to iatrogenic pneumothorax and acute death. Then it was realised that the hypoplastic and immature CDH lung was severely damaged by excessive oxygen delivery and by excessive airway pressure.64 Since Wung and colleagues introduced the ‘gentle ventilation and permissive hypercarbia strategy’, improved pulmonary outcomes and mortality rates have been reported.65 This policy progressively gained support and it is now the standard in most developed countries. High-frequency oscillatory ventilation was also believed to improve survival and reduce long-term morbidity. However, a recent randomised controlled trial (RCT; the VICI trial: Ventilation in Infants with Congenital diaphragmatic hernia: an International randomised clinical trial.)66 has shown no superiority of this technique versus conventional ventilation. The latter again shows that proper studies have to be done before implementing new technologies. The management of PPH in newborns with CDH remains one of the major concerns in neonatal intensive care units (NICUs). Early cardiac ultrasound (within the first 24 hours of life) is recommended by the international consortia as a noninvasive tool in the diagnosis of PPH. Serial cardiac ultrasonography is then recommended to guide therapeutic choices and to monitor their effect on PPH.25 The cornerstone of postnatal treatment of PPH is the reversal of the vasoconstrictive component to prevent the right ventricle overload and the development of irreversible vascular remodelling. Currently, iNO is the first therapeutic choice in CDH infants with PPH. However, in an RCT, although iNO significantly decreased the need for ECMO in infants with PPH, it did not reduce mortality rate, length of hospitalisation, chronic lung injury or neurodevelopmental impairment.67 Furthermore, although the response rate to iNO in all infants with PPH is around 60%, in infants with CDH, it is estimated to be only 30%.68 Other vasodilatory drugs, such as phosphodiesterase type 5 (PDE5) inhibitors, endothelin antagonists or prostacyclins, have been used alone or in association with nitric oxide in experimental series.69-71 However, their use is highly variable throughout NICUs around the world, which makes it difficult to define their exact role. Again, trials are being designed to clarify this. Apart from pharmacologic therapy, ECMO is in some centres an adjunct in the treatment of CDH-related PPH.25 It is used to unload the right ventricle while putting the lungs at rest, thereby reversing PPH, which would otherwise be lethal. It prevents additional lung injury induced by barotrauma and oxidative stress in case of maximal ventilator support. As for iNO, experience with ECMO achieved the worst results precisely in patients with CDH.72 Therefore there is no evidence that the outcome of CDH is better with ECMO. Together with the limitations of the technique (required weight above 2000 g, need for heparinisation), all this has somewhat tempered the initial enthusiasm followed by its diminished use in many centres.6 Surgical repair should be performed electively after the newborn is stabilised. The optimal surgical technique remains under debate. Minimal access surgery is gaining ground on the open approach (thoracotomy or laparotomy). This approach has

aesthetic advantages but carries a higher risk for recurrence.63 It may also not be applicable in severe cases. For defects that are too large to be closed by primary repair, prosthetic patches may be used to close the gap,73 ranging from first-generation nonabsorbable synthetic materials (Gore-Tex) to biomaterials (xenografts) as well as composite materials. The ideal mesh remains elusive. 

Antenatal Therapeutic Strategies The ability to prenatally identify a future nonsurvivor offers the potential for prenatal interventions that could avoid that outcome. The concept of tracheal occlusion (‘plug the lung until it grows’) was first introduced by Wilson and associates74 and is inspired by clinical observations in fetuses with congenital high airway obstruction, who have a marked increased lung volume and alveolar number.75 Airway obstruction prevents egress of pulmonary fluid, which experimentally has been shown to prompt lung growth by a mechanism of stretch of lung parenchymal cells.76 This leads to increased airway branching morphogenesis, an increase in both alveolar surface area and airspace volume and stimulated alveolisation.77 This concept has been further explored experimentally, demonstrating that tracheal occlusion improves neonatal lung compliance and improves ventilation. The procedure was first clinically attempted by open fetal surgery and clipping of the trachea.78 Progress in minimally invasive fetal surgery made percutaneous fetal endoscopic tracheal occlusion (FETO) with a balloon possible.79 FETO is an investigational minimally invasive, percutaneous procedure that can be done under maternal local anaesthesia (Fig. 31.4A).80 The procedure is usually performed at 27 + 0 to 29 + 6 weeks in severe cases and 30 + 0 to 31 + 6 weeks in moderate cases. The fetus is anesthetised and immobilised with an intramuscular injection of a neuromuscular blocking agent, fentanyl and atropine. A flexible cannula is inserted through the skin and myometrium and targeted to the fetal nose tip under ultrasound guidance. Fetoscopic instruments specifically designed for FETO are then introduced. These consist in a 3.3-mm sheath loaded with a fiberoptic endoscope (1.3 mm; Karl Storz) and a balloon occlusion system (catheter loaded with a detachable inflatable latex balloon with integrated one-way valve Goldbal 2, Balt Extrusion, France). A stylet or forceps can also be inserted through the sheet to remove the balloon if wrongly positioned. Fetoscopic landmarks are the philtrum and upper lip, the tongue and raphe of the palate, uvula, epiglottis, and eventually the vocal cords. The endoscope is advanced into the trachea until identification of the carina, above which the balloon is positioned by inflation and detachment from the catheter (Video 31.2). The median duration of FETO is 10 (range, 3–93) minutes, dependent on both the experience of the operator and the position of the fetus.81 A longer operation time is the main risk factor for membrane rupture. Experimental data suggest benefit of in utero reversal of tracheal occlusion (‘plug-unplug’ sequence). It stimulates lung maturation82 and has the logistic advantage of permitting vaginal delivery and referral to the home institution of the patient. This prompted clinicians to attempt timely in utero reversal as much as possible. Meanwhile, clinical data suggest that prenatal balloon removal increases neonatal survival83 and reduces neonatal morbidity.41 Elective intrauterine occlusion reversal is usually scheduled at 34 weeks in patients with an uneventful postoperative course. This can be performed via ultrasound-guided puncture, fetoscopic removal, tracheoscopic removal on placental circulation or postnatal puncture. Ultrasound-guided in utero balloon puncture is

CHAPTER 31  Congenital Diaphragmatic Hernia

337

A

B •

Fig. 31.4 Fetal therapy for congenital diaphragmatic hernia. A, Schematic drawing of percutaneous fetoscopic endoluminal tracheal occlusion. Inset, A detachable balloon, normally used for endovascular occlusion, is positioned in the trachea. B, Schematic drawing of balloon removal on placental circulation by laryngeotracheoscopy. (Reproduced with permission from UZ Leuven, Leuven, Belgium. Drawing by Myrthe Boymans.)

done after fetal immobilisation and analgesia. After puncture, the balloon is pushed by the lung fluid into the pharynx, from where it is either swallowed or falls into the amniotic cavity (Video 31.3). Fetoscopic removal is done with similar instruments as for insertion. The balloon is first punctured with a stylet and then grasped and retrieved with forceps.84

In 20% of cases, patients present earlier than planned with threatening preterm delivery, with or without ruptured membranes. Whenever clinically possible, in utero balloon removal is still attempted using the same techniques. If in utero retrieval does not seem safe or possible, the balloon is removed by laryngotracheoscopy during a modified caesarean section under locoregional

338

SE C T I O N 7     Diagnosis and Management of Fetal Malformations

TABLE Main Potential Advantages and Risks of Fetal 31.1 Endoscopic Tracheal Occlusion

Pros

Cons

Stimulation of lung growth Minimally invasive procedure

15%–20% risk for pPROM Mean gestational age at birth: 35 weeks Need for a second intervention (unplug) Potential risk for emergency unplug

Potential to increase survival Potential reduction in postnatal morbidity

Effect on PPH not yet demonstrated PPH, Persistent pulmonary hypertension; pPROM, preterm premature rupture of membranes.

  

anaesthesia, with the fetal head and shoulders delivered while the fetus remains on placental circulation85 (Fig. 31.4B and Video 31.4). Postdelivery removal is a last resort, which is done by video laryngotracheoscopy. Blind and ultrasound-guided ex utero puncture have also been reported.86 In a recent study performed in three FETO centres,84 balloon removal was elective in 72% of cases and as an emergency in 28%. The primary method was by fetoscopy in the majority of cases (67%), by ultrasound guidance in 21%, by tracheoscopy on placental circulation in 10% and by postnatal tracheoscopy in 1%. The method of choice for primary removals mainly depended on preference of the surgeon and, surprisingly, was not associated with a difference in the interval to delivery. The main conclusion, however, was that delivery should not take place outside FETO centres because the balloon could not be removed in three cases, leading to iatrogenic death. Observational studies have shown that FETO leads to an apparent increase in survival and reduced early neonatal respiratory morbidity compared with historical controls of similar severity, for both severe left-sided CDH and severe right-sided cases.81,87,88 This potential benefit is now being investigated in two parallel RCTs called Tracheal Occlusion To Accelerate Lung growth (TOTAL; http://www.TOTALtrial.eu), in fetuses with left-sided CDH and severe or moderate lung hypoplasia (NCT01240057 and NCT00763737).89 Right-sided CDH is too uncommon to justify a RCT, and we use a cutoff of o/e LHR of less than45% based on a long single-centre series. 

Experimental Antenatal Treatments The main drawback of FETO is the increased risk for preterm delivery, partly offsetting the beneficial effects of fetal therapy. Historical data on more than 200 cases demonstrated a 25% rate of preterm rupture of membranes at less than 34 weeks and a 30% rate of delivery before 34 weeks, requiring urgent balloon retrieval.81 Apart from being relatively invasive, FETO is also technically challenging and therefore difficult to widely implement. Moreover, the maximum post-FETO survival rate reported in severe cases so far is not higher than 50% to 60%, which in part is caused by insufficient airway growth and, above all, limited vascular development. The problem of PPH seems, at least, to persist despite surgical fetal treatment. The main advantages and disadvantages of FETO are outlined in Table 31.1. The main inclusion and exclusion criteria for participation in the TOTAL trial are outlined in Table 31.2. For the aforementioned reasons, complementary or alternative therapeutic treatments would be welcomed. The ideal fetal therapy should address both the problem of ventilatory insufficiency and

TABLE Main Inclusion and Exclusion Criteria for 31.2 Participation in the TOTAL Trial, for Both the

Severe Arm and the Moderate Arm

Inclusion Criteria

Exclusion Criteria SEVERE ARM

Maternal age 18 years or older

Patient not willing to undergo randomisation

Singleton pregnancy

Multiple pregnancy

Written informed consent

Patient not able to consent in full

Left-sided diaphragmatic hernia

Right-sided or bilateral diaphragmatic hernia

No associated structural anomalies and normal karyotype

Additional major structural or genetic anomalies

Gestational age at surgery, 27.0–29.6 wk

Balloon cannot be placed before 29.6 wk

Severe hypoplasia defined as o/e LHR 15 mm

Cervix 15 mm

Cervix 28 wk

Postnatal (>48 hr)

APRPDa

12

++

Ovoid tibia

AD, gm

>12

+++

+++

Majewski syndrome (SRPS2A; NEK1 SRTD6) Osteogenesis imperfecta types IIA/C and IIC

COL1A1 COL1A2

+/- mild

Skull

Legs

Talipes

Short

Cloverleaf

+/- small

Micrognathia, cardiac defects, sex reversal in males Stippled

Talipes

Micrognathia Narrow

Short

++

Cardiac anomaly Hypo

+/- poly

Narrow

Short

CNS anomaly

Short

Poly

Micrognathia, depressed nasal bridge

Narrow

Short ++

Narrow

Short, beaded

Renal, cardiac, CNS, genital Hypo

SE C T I O N 7     Diagnosis and Management of Fetal Malformations

Diagnosis

Gestational age at presentation (wk)

Osteogenesis imperfecta type IIB

COL1A1 COL1A2

AD, gm

>16

++

+

Osteogenesis imperfecta type III

COL1A1 COL1A2

AD, gm

20

+

Legs

Osteogenesis imperfecta type IV

COL1A1 COL1A2

AD, gm

>20

Rhizomelic CDP (RCDP1,2,3,5) NB Overlapping heterogeneous peroxisomal disorders, Zellweger syndrome

PEX7, GNPAT, AGPS, PEX5

AR

20

Rhizomelic stippled

Saldino-Noonan syndrome (SRPS2B; SRTD3; ATD3)

DYNC2H1

AR

>12

++

SEDC

COL2A1

AD

>12

++

Thanatophoric dysplasia I

FGFR3

AD

C), inherited from the other parent causes hydrops fetalis. α-Thalassemia carrier status is common in Southeast Asian populations, and this autosomal recessive disorder accounts for 28% to 55% of their NIHF. Most other population cohorts report approximately 10% of their cases caused by hematologic disorders. 

Lymphatic Dysplasia Over the past decade, an increased proportion of cases with NIHF have been diagnosed with conditions associated with abnormal lymphatic development (CH and NIHF, chylothorax, or chyloascites and NIHF).2 As discussed in the previous section, CH and NIHF is associated with a high risk of chromosomal abnormalities. When a chromosomal cause has been excluded, a syndromic cause must be considered, with Noonan syndrome being the most common syndrome associated with CH. Noonan syndrome is an autosomal dominant disorder caused by mutations in genes that participate in RAS–mitogen-activated protein kinase signal transduction (RAS-MAPK pathway genes: PTPN11, KRAS, SOS1, RAF1, MAP2K1, BRAF, SHOC2, NRAS, CBL and RIT1) with PTPN11 mutations found in approximately 50% of patients. In 2006, a report of PTPN11 testing of pregnancies presenting with CH with or without hydrops or associated anomalies indicated the detection of a mutation in 16% of cases.23 This finding was replicated in another study that found mutations in both PTPN11 and RAF1 in pregnancies with CH,24 leading to the recommendation that Noonan syndrome gene testing should be considered in all cases of CH with normal karyotypes. Approximately 85% of cases of Noonan syndrome can be diagnosed by of a panel of genes responsible for Noonan syndrome and other RASopathies. Two other groups of genetic disorders should be considered in the context of CH and fetal hydrops: fetal akinesia deformation sequence (FADS) and lethal multiple pterygium syndrome (LMPS). Both disorders have some phenotypic overlap with decreased fetal movements and arthrogryposis. A review of 79 consecutive cases of FADS showed that 15% presented with fetal hydrops.25 Both FADS and LMPS are genetically heterogeneous and have been shown in a proportion of cases to be caused by mutations in the neuromuscular junction genes: recessive mutations in CHRNA1, CHRND and CHRNG have been described in patients with LMPS and recessive mutations in CNTN1, DOK7, RAPSN and SYNE1 in patients with FADS,26 and more recently recessive mutations in RYR1 have been found in 8.3% of patients with FADS/ LMPS phenotypes.27 It has been postulated that the decreased fetal movement in these disorders is associated with decreased lymphatic flow, leading to the CH and fetal hydrops. With exome sequencing studies being performed on cases of FADS and LMPS, it is likely that numerous additional genes causing these phenotypes will be identified. Abnormalities in lymphatic development (chylothorax or chyloascites) can also present in the second and third trimesters. The abnormalities may be reflective of a chromosomal or genetic syndrome or primary lymphatic dysplasia. Congenital chylothorax is the most common type of pleural effusion in fetal life and is associated with fetal hydrops in 60% to 70% of

433

cases.28,29 In one retrospective case series with 10 cases of congenital chylothorax, a diagnosis of trisomy 21 was made in one case and a diagnosis of Noonan syndrome in three cases.29 A larger prospective German nationwide epidemiologic study of chylothorax reported on 27 cases of chylothorax with one case with trisomy 21 and five cases with Noonan syndrome.30 A review of the prenatal features in a series of 47 patients with a molecular diagnosis of Noonan syndrome reported 5 patients with hydrothorax, and all five had associated polyhydramnios. Mutations in PTPN11 and SOS1 were found in four and one patients, respectively.31 Mutations in other genes of the RAS-MAPK pathway have been reported to cause chylothorax and fetal hydrops, including SHOC2 and CBL.32,33 Although Noonan syndrome is the most common syndrome associated with chylothorax and fetal hydrops, other syndromes that are part of the RASopathies, cardio-facio-cutaneous and Costello syndrome, can be the cause of the prenatal findings of lymphatic dysplasia.34 Primary generalised lymphatic dysplasia (GLD), although rare, is a recognised cause of fetal hydrops. GLD results from an inherent developmental abnormality of the lymphatic system involving the viscera. The onset of lymphatic drainage failure can be prenatal or postnatal.35 Hennekam syndrome is an example of an autosomal recessive syndromic form of GLD that is characterised by lymphoedema of the limbs, genitalia and face; secondary facial dysmorphic features; intestinal lymphangiectasia; and varying degrees of intellectual disability.36 In severe cases, it may present prenatally with fetal hydrops.36,37 Recessive mutations in CCBE1 and FAT4 are found in fewer than 50% of Hennekam patients, indicating that the condition is genetically heterogeneous, and some genes remain unknown.38 Another form of recessive GLD with a high incidence of NIHF with either demise or complete resolution of the neonatal oedema followed by childhood onset of lymphoedema with or without systemic involvement has recently been reported in six families harbouring mutations in PIEZO1, a gene coding for a calcium permeable mechanically activated ion channel found in the plasma membrane of various cell types.39 A recent report of hydrops fetalis and pulmonary lymphangiectasia caused by autosomal dominant FOXC2 mutation which presented with lymphoedema of the lower limbs and irregularly implanted eyelashes (lymphoedema-distichiasis syndrome) in the father and numerous family members illustrates that these conditions can have variable presentations and that a thorough analysis of the familial pedigree is important.40 Primary congenital lymphoedema also known as Milroy disease, a dominant condition caused by mutations in FLT4 inherited as autosomal dominant or recessive, typically presents with congenital chronic swelling of the lower limbs. However, it has also been reported to present prenatally with oedema of the lower limbs and transient ascites and pleural effusions, further supporting the concept of variable presentation with the more severe cases presenting with fetal hydrops.41 Given the growing number of genes implicated in lymphatic anomalies,42 it is conceivable that de novo mutations in these genes may account for a significant proportion of the cases currently considered as idiopathic fetal hydrops. Indeed, sequencing analysis of FLT4, FOXC2 and SOX18 of 12 probands who survived in utero generalised oedema or hydrops fetalis of unknown aetiology identified mutations in FLT4 in two patients and a mutation in FOXC2 in one patient, although none of the patients had a positive family history of lymphoedema.43 

434

SE C T I O N 7     Diagnosis and Management of Fetal Malformations

Infection A variety of infectious agents have been associated with NIHF, including parvovirus B19 (most common), cytomegalovirus (CMV), herpes simplex virus, Toxoplasma gondii, rubella, syphilis and chlamydia.44 Infectious agents produce hydrops through effects on fetal bone marrow, myocardium or vascular endothelium. They represent 7% of cases of NIHF. Fetal infections are detailed in Chapter 42. 

Thoracic Masses Thoracic masses account for 5%2 of cases of NIHF and include any thoracic pathology that has a significant mass effect and results in elevated central venous pressure. This includes congenital pulmonary airway malformations (CPAMs; see Chapter 30), diaphragmatic hernia (see Chapter 31), chylothorax (discussed under lymphatic dysplasia earlier) and intrathoracic tumours (see Chapter 37). 

Metabolic Conditions Inborn errors of metabolism are a recognised cause of fetal hydrops, although they are likely to be underdiagnosed given the need for specialised metabolic or genetic testing that may not be readily available. The systematic reviews of hydrops fetalis by Bellini and colleagues (including publications between 1979 and 2007 and 2008 and 2013) found that inborn errors of metabolism accounted for 1.1% and 1.3% of cases, respectively.2,7 However, another systematic review of hydrops fetalis aimed at determining

the contribution of lysosomal storage disease (LSD) (the most common class of inborn error of metabolism causing NIHF) reported that of the 54 retrospective studies and case series with cases of NIHF with its workup described, only 15 (27.8%) reported testing for LSD, and the extent of this workup varied among studies. Amongst the 678 total NIHF cases identified in those 15 publications, 35 cases (5.2%) were diagnosed with LSD.45 In the two studies with the most extensive workup for LSD, 8 of 86 patients (9.3%) had an LSD. This finding strongly suggests that LSD and other disorders of inborn errors of metabolism may account for a significant proportion of cases of idiopathic NIHF. At least 15 different LSDs have been reported in series or case reports as causing NIHF (Table 36.5). The most common conditions reported were mucopolysaccharidosis type VII and type IVa, Gaucher disease, GM1 gangliosidosis, sialidosis, Niemann-Pick types C and A, galactosialidosis, infantile sialic acid storage disorder and mucolipidosis II. The mechanisms contributing to the development of hydrops in storage disorders may include obstruction of venous blood return resulting from organomegaly, anaemia associated with hypersplenism or reduction of erythropoietic bone marrow stem cells caused by infiltrating storage cells, and hypoproteinaemia caused by liver dysfunction.46 A number of inborn errors of metabolism other than LSD have been reported in cases NIHF, and these are listed in Table 36.5. Congenital disorders of glycosylation (CDG) type 1 have been described in a number of cases of NIHF, leading to the suggestion that this class of disorder should be considered in every case of unexplained NIHF and investigations for PMM2-CDG might be pursued given it is the most common form of CDG type 1.47 

TABLE Lysosomal Storage Diseases and Nonlysosomal Inborn Errors of Metabolism Associated With Nonimmune 36.5 Hydrops Fetalis LYSOSOMAL STORAGE DISEASES

ENZYME OR PROTEIN DEFECT

GENE DEFECT

MUCOPOLYSACCHARIDOSIS

MPS I

α-L-iduronidase

IDUA

MPS IVA

N-acetylgalactosamine-6-sulphatase

GALNS

MPS VII

Beta-glucuronidase

GUSB

SPHINGOLIPIDOSIS OR OLIGOSACCHARIDOSES

GM1 gangliosidosis

β-Galactosidase

GLB1

Niemann-Pick A

Sphingomyelin phosphodiesterase

SMPD1

Niemann-Pick C

Niemann-Pick C1 protein Epididymal secretory protein E1

NPC1 NPC2

Sialidosis

Sialidase-1

NEU1

Galactosialidosis

Lysosomal protective protein

CTSA

Gaucher type II

Glucosylceramidase

GBA

Farber disease

Acid ceramidase

ASAH1

Multiple sulfatase deficiency

Sulfatase-modifying factor 1

SUMF1

LYSOSOMAL TRANSPORT DEFECT

Infantile sialic acid storage disease

Sialin

SLC17A5 OTHERS

Wolman disease

Lysosomal acid lipase

LIPA

Mucolipidosis Type II

N-acetylglucosamine-1-phosphotransferase subunits alpha/beta

GNPTAB

CHAPTER 36  Fetal Hydrops

435

TABLE Lysosomal Storage Diseases and Nonlysosomal Inborn Errors of Metabolism Associated With Nonimmune 36.5 Hydrops Fetalis—cont’d NONLYSOSOMAL INBORN ERRORS OF METABOLISM

ENZYME OR PROTEIN DEFECT

GENE

GLYCOGENOSIS

Type IV

1,4-α-Glucan-branching enzyme

GBE1

CONGENITAL DISORDERS OF GLYCOSYLATION

CDG Ia

Phosphomannomutase 2

PMM2

CDG Ik

Chitobiosyldiphosphodolichol β-mannosyltransferase

ALG1

CDG Ih

Probable dolichyl pyrophosphate Glc1Man9GlcNAc2 α-1,3-glucosyltransferase

ALG8

CDG Ig

Dol-P-Man:Man(7)GlcNAc(2)-PP-Dol α-1,6-mannosyltransferase

ALG12

PEROXISOMAL DISORDER

Zellweger syndrome

Peroxisome biogenesis factor 1

PEX1

FATTY ACID OXIDATION DEFECTS

Long-chain-hydroxyacyl CoA dehydrogenase deficiency

Hydroxyacyl-CoA dehydrogenase

HADHA

Primary carnitine deficiency

Solute carrier family 22 member 5

SLC22A5

CHOLESTEROL BIOSYNTHESIS DEFECTS

Smith-Lemli-Opitz syndrome

7-Dehydrocholesterol reductase

DHCR7

Greenberg syndrome: hydrops-ectopic calcification moth-eaten skeletal dysplasia

Lamin-B receptor

LBR

Conradi Hünermann: chondrodysplasia punctata

3-β-Hydroxysteroid-δ(8),δ(7)-isomerase

EBP

OTHERS

Fumarase deficiency

Fumarate hydratase, mitochondrial

FH

Transaldolase deficiency

Transaldolase

TALDO1

S-adenosylhomocysteine hydrolase deficiency

Adenosylhomocysteinase

AHCY

  

Other Genetic Conditions

Idiopathic

A number of genetic conditions and genetic syndromes have been discussed under previous aetiologic categories. Skeletal dysplasias represent another group of genetic disorders that can lead to NIHF. A number of rare skeletal dysplasias include as a consistent feature CH or fetal hydrops in addition to severe micromelia with short ribs and a small chest. Table 36.6 reviews the most common skeletal dysplasias associated with NIHF. Arriving at a specific diagnosis of the type of skeletal dysplasia is important because some have an autosomal recessive mode of inheritance, and others represent new mutations for a dominant gene. (More details on skeletal dysplasias are given in Chapter 34.) 

Approximately 18% of cases of NIHF are classified as idiopathic because no underlying pathology is found to explain the hydrops. As discussed in the section on inborn errors of metabolism, the percentage of idiopathic cases in each series varies and is in part dependent on the extent of the investigations carried out. As whole-exome sequencing becomes a more integral part of the prenatal and postnatal and postmortem workup of NIHF, it is likely that the percentage of idiopathic cases will decrease. 

Other Less Common Categories

In 2015, the Society for Maternal-Fetal Medicine (SMFM) published a clinical guideline for NIHF.49 The recommendations made in this guideline are still valid. However, given progress made in DNA testing for single gene disorders using next generation sequencing (gene panels or whole exome), these modalities

Malformations of the gastrointestinal tract or genitourinary tract and extrathoracic tumours each account for a small percentage of cases of NIHF (see Table 36.2). 

Clinical Evaluation

436

SE C T I O N 7     Diagnosis and Management of Fetal Malformations

TABLE 48 36.6 Skeletal Dysplasia Associated With Nonimmune Hydrops Fetalis

Condition and Incidence

Gestational Age of Presentation

Achondrogenesis types 1A, 1B and 2: 1 in 40,000 for all types of achondrogenesis

Ultrasound Findings

Gene

Inheritance

11–16 wk

Considerable overlap in US findings between the different types Cystic hygroma or hydrops; short long bones; small thorax with slender; short ribs; unossified vertebral bodies; hypomineralisation of the skull in type 1A

1A: TRIP11 1B: DTDST 2: COL2A1

AR AR AD, de novo mutation

Caffey disease, severe lethal variant: very rare

14–20 wk

Hydrops Cortical hyperostosis of long bones and ribs, resulting in limb bowing and shortening, and narrow thorax

Unknown

AR

Desbuquois dysplasia Very rare

20 wk

Hydrops Micrognathia; proptosis; marked rhizomesomelic shortening; short, narrow thorax; cleft palate; large joint dislocations Possible associated anomalies: CHD, omphalocele

CANT1

AR

Greenberg dysplasia (Hydrops-ectopic calcification-motheaten skeletal dysplasia): extremely rare

17 wk

Cystic hygroma, hydrops Severe micromelia, poorly ossified skull vault, abnormal contours of the long bones with irregular hypo- and hyperechoic areas within the bones. Flattened vertebrae and irregular hyperechogenicity of vertebral bodies; small thorax, short ribs Postaxial polydactyly may be present

LBR

AR

Short rib–polydactyly types 1 and 3: very rare

Type 1: 12–14 wk Type 3: milder second to third trimesters

Cystic hygroma and hydrops SRPS1: marked micromelia; polydactyly of hands and sometimes feet; narrow thorax with short, horizontally oriented ribs and a prominent abdomen; occasionally absent fibula Associated anomalies: CHD, renal dysplasia, renal cysts, anal atresia, ambiguous genitalia SRPS3: similar but milder with less frequent associated anomalies

DYNC2H1

AR

Short rib–polydactyly type 2: very rare

12–14 wk

Hydrops Marked micromelia; polysyndactyly of hands and sometimes feet; narrow thorax with short, horizontally oriented ribs and a prominent abdomen Associated anomalies: midline cleft lip, cleft palate, renal dysplasia, renal cysts, ambiguous genitalia, cerebellar and brain anomalies

NEK1 NEKI and DYNC2H1

AR Digenic biallelic mutations

Thanatophoric dysplasia: 1 in 20,000 to 1 in 50,000

12–18 wk

Increased nuchal translucency and occasionally hydrops TD I: short, curved femurs, occasionally clover leaf skull TD II: straight, long femurs with cloverleaf skull Both have narrow thorax with short ribs, trident hands Associated anomalies: ventriculomegaly, CHD, renal anomalies

FGFR3

AD, de novo mutations

AR, Autosomal recessive; AD, autosomal dominant; CHD, congenital heart defect; SRPS, short rib polydactyly syndrome; US, ultrasound.

  

must now be considered in the investigation of fetal hydrops that remain unexplained after all other investigations have been carried out.

Clinical History The assessment of the hydropic fetus should start with a detailed clinical history, including maternal age, current and prior obstetrical history, medication (prescription and nonprescription), infectious exposure, maternal and paternal blood type, single-gene

carrier screening, aneuploidy screening, ethnicity, family history of both patient and partner to assess for consanguinity, inherited conditions, history of stillbirths, recurrent spontaneous losses and infants with birth defects. 

Fetal Assessment Imaging techniques. Real-time fetal ultrasonography, fetal vascular

blood flow Doppler, fetal echocardiography and ultrafast fetal magnetic resonance imaging are the common imaging techniques

CHAPTER 36  Fetal Hydrops

437

TABLE 51 36.7 Distribution of Fluid Collection in HF of Different Aetiologies and Timing of Hydrops

Cause of HF (n = 220) # of Cases by Trimester: First/Second/Third

Ascites (%)

Pleural Effusions (%)

Pericardial Effusions (%)

Skin Oedema (%)

Cystic Hygroma (%)

Aneuploidy (n = 85) 44/31/10

40

35.3

9.4

69.4

51.8

CHD (n = 32) 5/18/9

68.8

31.3

12.5

78.1

12.5

SD (n = 13) 5/5/3

69.2

15.4

30.8

76.9

15.4

PV B19 (n = 9) 2/7/0

66.7

44.5

66.7

55.6

11.2

Immune HF (n = 2) 0/2/0

80% can be diagnosed),6 the antenatal detection rate is improving.7 A detailed first trimester anomaly scan, including cardiac anatomy, should be performed in such cases and further investigations offered. In the event of a normal karyotype or microarray, if the findings do not resolve, and particularly if there is a further anomaly found on ultrasound, genetic counselling should be considered. If antenatal testing is declined, careful assessment of the infant should be performed in the neonatal period. Increased nuchal translucency is also associated with cardiac abnormalities, and in some cases, ultrasound signs of fluid in other fetal compartments such as the thorax, abdomen and generalised skin oedema may be noted; this is known as fetal hydrops or hydrops fetalis. In cases of hydrops, investigations as described earlier should be performed, as well as consideration of other causes such as red cell alloimmunisation or nonimmune causes, including viral infection and metabolic disease (see Chapter 36). In cases of persistent hydrops presenting in the first trimester, the outlook is very poor with few fetuses surviving to delivery.

CHAPTER 37  Fetal Tumours

445

TABLE 37.1 Table of Differential Diagnosis of Fetal Neck Masses

Diagnosis

Ultrasound features

Incidence

Outcome births2

Cervical lymphangiomas

Well-circumscribed or diffuse cystic mass, laterally located, and often arising from cervical area, floor of mouth or the tongue

1 in 1775 live

Can cause complex airway obstruction; referral to ENT and consideration of EXIT procedure recommended; presence in the first trimester is associated with chromosomal abnormalities

Cervical teratomas

Can be massive, usually solid with some cystic areas, and well defined; may have internal calcifications, positioned in the anterior neck; 3:1 female:male ratio

1 in 40,000 live births3

Can cause complex airway obstruction; referral to ENT and consideration of EXIT procedure are recommended

Haemangioma

Typically posterolateral, well-defined, solid masses with slow vascular flow

Rare

May be associated with cortical malformations; brain MRI indicated, can be part of PHACES syndrome

Cervical thymic cyst

Commonly multiloculated but can be uniloculated, most commonly to the left, splaying the carotid artery and jugular vein

Very rare

Congenital goitre

Symmetric thyroid enlargement; associated with maternal propylthiouracil use (for Graves’ disease) and maternal thyroid stimulationblocking antibody

Rare

Unlikely to cause airway obstruction; when present, intubation is usually successful

Brachial cleft cysts

Unilateral, uniloculated anterolateral thin-walled cyst

Rare

Not reported to cause airway obstruction

Vascular malformations

Multiloculated cystic structure often laterally located

Rare

Neuroblastoma

Retropharyngeal, solid mass, with or without calcification, extending into mediastinum or skull

Very rare

ENT, Ear, nose and throat; EXIT, ex utero intrapartum treatment; MRI, magnetic resonance imaging; PHACES, posterior fossa malformation, haemangiomas, arterial anomalies, cardiac defects, eye abnormalities and sternal cleft.

  

In cases of first trimester cystic hygromas that have been appropriately investigated and no genetic abnormality found, particularly in those cases that resolve, the prognosis is very good. The development of a cystic neck mass in the second trimester is likely to have a different aetiology (Table 37.1) from those seen in the first trimester and may carry a better prognosis unless associated with hydrops or generalised oedema, in which cases the prognosis remains poor. 

Teratomas Fetal teratomas are rare tumours with an incidence of 1 in 40,000 live births. Although most commonly found in the region of the lower spine and pelvis (sacrococcygeal teratomas (SCTs); discussed in detail later), 6% are related to the neck. Teratomas are (almost always) formed of tissues derived from the three germ cell layers: endoderm, mesoderm and ectoderm. Although teratomas are predominantly (>80%) benign tumours with well-differentiated tissues, the rapid growth and the mass effect can cause significant complications; primarily by obstruction or deviation of the trachea and oesophagus. In

cases of immature teratoma, with more poorly differentiated tissues, there in increased risk for invasion and metastases; however, the prognosis remains good with a greater than 80% 5-year survival rate.8 The origin of fetal teratomas remains poorly understood. The most widely accepted hypothesis is that aberrant pluripotent cells are sequestered during embryogenesis, in the fourth to fifth week of gestation, that are able to proliferate to form disorganised structures comprising tissue types derived from the three embryonic germ layers.1 

Antenatal Management Tertiary-level ultrasound assessment and multidisciplinary counselling are important in any neck mass. The ultrasound assessment should include details of the site and size of the mass, solid or cystic components, calcification, associated vascularity and assessment of invasion into or deviation of adjacent structures. Attempts should be made to determine the nature of the mass to aid counselling regarding postnatal management, likelihood of complications and long-term outcomes.

446

SE C T I O N 7     Diagnosis and Management of Fetal Malformations

•

Fig. 37.3 Teratoma at 31 weeks’ gestational age. Three- and fourdimensional imaging can help the parents visualise the mass and help with parent counselling.

A routine element of ultrasound assessment of fetal neck masses should be assessment for evidence of tracheal deviation or oesophageal occlusion. Indirect markers of these include polyhydramnios or a small or nonvisible stomach bubble. In some cases, the oropharynx may be fluid filled and readily visible, indicating significant partial or total occlusion. Ultrasound assessment is also important in the determination of fetal well-being, particularly in identifying development of cardiac compromise or hydrops, and to regularly assess the growth and size of the mass. The utility of three-dimensional (3D) US imaging in neck masses is unclear and becomes difficult to achieve in larger tumours at later gestations, particularly in the absence of polyhydramnios. Although its clinical benefit may be limited in such cases, we have found that it is useful to the parents (Fig. 37.3). After 24 weeks’ gestation, ultrasound assessment should be performed every 2 weeks. These assessments should focus on assessing the size of the mass, any changes in characteristics including vascularity, fetal neck extension, amniotic fluid volume and signs of cardiac compromise. To aid delivery planning, fetal presentation and placental site should be carefully mapped. Increasing polyhydramnios and features of cardiac compromise, including hydrops or Doppler abnormalities, are indications for more frequent assessment. In cases of significant polyhydramnios, particularly associated with maternal discomfort, amniodrainage may be indicated, although this is associated with rupture of membranes and preterm labour. In view of the anticipated airway difficulties and the advantages of a planned delivery, amniodrainage should be kept to a minimum and performed in a centre where ex utero intrapartum treatment (EXIT) is possible. Magnetic resonance imaging. Fetal MRI is generally accepted to be safe up to 3 Tesla and is being increasingly exploited. The advantages of the technique are its excellent soft tissue definition and the large field of view it provides, allowing global imaging of the fetal head and neck at any gestation. In addition, it is a useful adjuvant to ultrasound when imaging is limited, for example, in cases of oligohydramnios, poor fetal position or maternal obesity. The difficulty with fetal MRI is in imaging an unpredictably

mobile fetus. To overcome this, fast MRI sequences are used to obtain single-slice acquisitions, producing a motion frozen stack of images, free from artefact. A variety of fast MRI sequences are used to obtain T1- and T2-weighted images (depending on the manufacturer of the hardware), which acquire 15 to 20 3-mm slices in less than 25 seconds. The most common and important indication for fetal MRI in the context of neck masses is to assess airway patency to inform decisions on mode of delivery (Fig. 37.4). Because the fetal airway is fluid filled, it appears bright on T2-weighted sequences. This allows the trachea to be traced through the neck using imaging in three orthogonal planes. This may confirm patency or conversely assist surgeons in planning the best approach for tracheostomy.9 After patency is confirmed, the passage of the trachea can be mapped and displacement assessed. As an aid to determining the degree of airway displacement, the tracheoesophageal displacement index (TEDI) has been developed, which is defined as the sum of the lateral and ventral displacement of the tracheoesophageal complex from its normal anatomical location.10 A TEDI score greater than 12 mm has been reported to be predictive of a complicated airway (100% vs 46%; P = .04)10 (Fig. 37.5). As well as assessing airway patency, fetal MRI can help identifying the underlying cause of neck masses,11 as the improved soft tissue definition and differences in signal from T1- and T2-weighted imaging can help distinguish pathologies.12,13 MRI can also help describe the relationship among the mass, airway and other structures of the neck, head and thorax14 and help assess for neck extension secondary to the tumour, which may necessitate delivery by caesarean section. Additional information on the depth of invasion and structures involved can aid surgical planning.11,14 MRI has been reported to obtain additional findings or make an alternative diagnosis to ultrasound in 83% of cases of fetal neck masses.11 Magnetic resonance imaging is also useful for assessing the fetal lungs, which are clearly visualised in T2-weighted imaging. They can be collapsed, hypoplastic or hyperinflated in cases of fetal neck masses. There is potential for MRI derived total lung volume measurements to help predict lethal pulmonary hypoplasia.15 Determining airway patency remains challenging with MRI because of the relative thickness of the slices compared with the size of the fetal airway structures. There is ongoing research in reconstructing the two-dimensional stacks of slices in three orthogonal planes into anatomically relevant 3D volumes. This would improve spatial resolution and therefore the diagnostic ability of imaging in the future. Reconstructing virtual bronchoscopies as an aid in evaluating airway patency is an exciting future possibility.16  Prognosis. The antenatal prognosis for a fetus with a cystic hygroma and normal karyotype and cardiac structure is good with few cases dying in utero.17 The in utero mortality rate in cases of teratoma is higher because of increased vascularity and associated cardiac demands.18 At present (because of limited worldwide cases), there is no predictive model for adverse in utero outcomes.  In utero therapies. There are a few reports of in utero therapy, including sclerotherapy,19,20 for cystic hygroma and fetal surgery21 for teratoma. The numbers are very limited, and these therapies are not routinely practised, although they may have a role in the case of the previable hydropic fetus, in which the mortality rate is close to 100% if delivered or left untreated. Fetoscopy is used in some centres to assess the patency of the fetal airway; however, there are only currently two reports in the

CHAPTER 37  Fetal Tumours

447

Ventral TEDI = L + V

T

L

V CS Lateral

A A

V

M L

B • Fig. 37.5  A, Measurement of the tracheoesophageal displacement index

B

C •

Fig. 37.4  A, Magnetic resonance images of a lymphangioma at 30 weeks’ gestational age. A right-sided lymphangioma crosses the midline anterior to the airway; the airway can be seen and was thought to be patent. Also note the flow artefact coming from the fetal nose in (thick arrow), similar to Doppler this demonstrates flow of fluid in the fetal airway, and is suggestive but not diagnostic of airway patency (thin arrow). B, Teratoma at 31 weeks’ gestational age. This teratoma originated from the anterior and left side of the neck. The airway could be followed and appeared patent; however, note the displacement of the soft tissue and trachea (thin arrow) to the right of the cervical spine on the axial view (thick arrow). C, A teratoma at 29 weeks’ gestational age. Although the nasal airway and nasopharynx appear patent the remainder of the airway cannot be followed, so airway patency could not be confirmed.

(TEDI). TEDI was defined as the sum of the lateral (L) and ventral (V) displacements of the tracheoesophageal complex (T) from the ventral aspect of the cervical spine (CS) on fetal magnetic resonance imaging (MRI). B, Axial magnetic resonance image of fetal neck demonstrating a large mass (M) and significant displacement of the tracheoesophageal complex (thin arrow) from the ventral aspect of the cervical spine (thick arrow). (Reproduced from Lazar DA, Cassady CI, Olutoye OO, et al. Tracheoesophageal displacement index and predictors of airway obstruction for fetuses with neck masses. J Pediatr Surg 47:46–50, 2012.)

literature of operative intervention for neck masses. One report details the in utero removal of a pedunculated nasopharyngeal tumour using YAG (yttrium aluminum garnet) laser excision.22 A second report details a fetal endoscopic tracheal intubation technique (FETI).23 In this case, a fetus with a giant cervical teratoma was successfully intubated at 35 weeks’ gestation. This allowed a standard elective lower segment caesarean section to be performed with immediate use of the endotracheal tube at birth for ventilation. 

Intrapartum Management: The EXIT Procedure The EXIT procedure is performed to secure the fetal airway before stopping placental circulation. By maintaining the fetus on placental circulation, oxygenation is assured, allowing additional time to establish an airway. The first successful case was described in 199024,25 for the insertion of a tracheostomy in a fetus with an epignathus. However, the term EXIT was coined, and the procedure was first routinely used for management of pregnancies complicated with congenital diaphragmatic hernia treated with fetoscopic endotracheal balloon occlusion26 (FETO) (see Chapter 31). The EXIT procedure is now a widely used technique in

448

SE C T I O N 7     Diagnosis and Management of Fetal Malformations

centres managing the delivery of fetuses with neck masses with a high suspicion of airway complications. Although developed from the routinely performed lower segment caesarean section, an EXIT procedure should not be regarded as such and comes with its unique complexities and risks to both the mother and fetus. The EXIT procedure is used for a variety of fetal anomalies which obstruct the fetal airway, preventing spontaneous respiration and making intubation of the airway either very difficult or impossible. The anomalies can be extrinsic, including teratomas and lymphangiomas, or intrinsic, such as laryngeal atresia, congenital upper airway obstruction, obstructive malformations of the upper airways and intrathoracic lesions such as congenital hydrothorax. It is also sometimes used in cases of FETO in which the balloon has not been electively removed before delivery. Interventions performed with the EXIT procedure include intratracheal intubation, tracheotomy and tracheoplasty. It remains difficult to predict which fetuses will have a complicated airway. Ultrasound and MRI may be unable to demonstrate airway patency throughout its course, and in such cases, it is sensible to assume EXIT is required. A large mass, a suspected diagnosis of teratoma or signs of obstruction such as polyhydramnios or a small or absent stomach bubble all increase the chance of airway obstruction; therefore, if any of these are present, an EXIT should be planned. When the airway is visible but deviates from its normal course, the TEDI (see earlier) provides a guide to when EXIT may be appropriate. In masses that are not teratomas, a TEDI of greater than 12 mm has 100% sensitivity and 86% specificity for a complicated airway.10 However, all cases should be evaluated on a case-by-case basis by an experienced multi-MDT. In our experience, neck masses can change rapidly after delivery, with sudden postpartum increases in vascularity and size, so even in cases in which antenatal imaging suggests a patent airway, careful consideration should be given by an MDT on the possible benefits of an EXIT procedure to safely establish a secure airway in the early neonatal period. Detailed antenatal planning with an MDT, which should include the obstetricians; neonatologists; anaesthetists (both maternal and neonatal); paediatric ear, nose and throat surgeons; radiologists; theatre scrub teams; and critically the parents, is essential for each case. The theatre space itself should be sufficient to accommodate the various teams and ‘zones’ established for each specialty. A detailed brief and ‘talk through’ of the procedure, including plans for foreseeable complications and confirmation that all necessary equipment and drugs are available, should be performed immediately before the procedure. Ideally, delivery should be as close to term gestation as possible; however, because of increasing polyhydramnios, up to 76% of cases are delivered at a late preterm gestation (median, 35weeks).27 The parents of the baby should be involved in the antenatal planning process and a nominated midwife assigned to the case. Discussion should be undertaken antenatally with the parents and plans made for cases in which it is not possible to gain airway access and other worst-case scenarios such as severe hypoxia or prolonged fetal bradycardia. Psychological support for the parents should be offered. To provide adequate fetal perfusion, maternal anaesthesia should aim to provide optimal uterine relaxation and suitable maternal blood pressure. EXIT procedures are usually performed under deep maternal general anaesthesia because volatile anaesthetics assist in uterine relaxation. Regional anaesthesia has been reported but requires intravenous infusion of glyceryl trinitrate (GTN) and remifentanil. Rapid-sequence

induction and general endotracheal anaesthesia with maternal paralysis and epidural for postoperative analgesia is a standard technique.28 After delivery of the fetus, the concentration of volatile agents is reduced and/or converted to intravenous anaesthesia, and oxytocin is given to establish uterine tone. More recently, the SIVA (supplemental intravenous anaesthesia) technique has been used.29 This exploits the tocolytic effects of propofol and allows a reduction in the use of volatile anaesthetic agents. This reduced exposure to volatile agents may reduce fetal myocardial suppression and may also be a more suitable technique in cases in which prolonged maternal exposure to volatile agents is not desirable. Before uterine incision, particularly in cases of extensive anterior placentation, intraoperative sterile ultrasound mapping of the placenta should be performed to avoid inadvertent placenta incision. The head, neck and upper torso of the fetus are delivered along with the fetal right arm (Fig. 37.6). The fetus should have 10 μg/kg fentanyl and 0.1 mg/kg of vecuronium immediately into the deltoid muscle. Monitoring of the fetal saturations and heart rate should be performed with sterile monitoring equipment. Atropine should be available and usage determined by a senior neonatologist. After this, attempts should be made to secure the airway with direct laryngoscopy. If this is not successful, examination of the airway with a rigid bronchoscope, flexible bronchoscopy, or both should be performed and guided intubation attempted. If this is unsuccessful, an attempt at tracheostomy should be considered. After the airway is secured, the baby can be delivered and transferred to the neonatal unit for further assessment. If there are prolonged episodes of desaturation or bradycardia or compromise to the maternal health (bleeding, hypotension), the baby should be delivered immediately even if a definitive airway has not been secured. The EXIT procedure carries increased risks to the mother, predominantly the risk for haemorrhage and risks associated with longer operative time. Approximately 10% of cases need blood transfusions, with haemorrhage caused by placental abruption, uterine atony and bleeding at the incision site.28 With prior planning, operative risks can be minimised, and the EXIT procedure should be considered a safe and effective procedure. In experienced centres, a secured airway can be obtained in close to 100% of planned EXIT procedures. Without the EXIT procedure, the neonatal mortality rate for patients with cervical neck masses has been reported at greater than 50%; however, with the EXIT procedure, this is now less than 10%.17,28,30,31 A review of our own cases in the past 5 years includes seven EXIT procedures (one case of presumed congenital high airways obstruction (CHAOS); six delineate neck masses). In two cases, the fetus died: one baby had a very large anterior teratoma with bronchoscopic evidence of invasion into the upper airway. Endotracheal intubation was not possible via direct laryngoscopy, and a tracheostomy was successfully performed; however, there was early neonatal death secondary to presumed severe hypoxic brain injury; postmortem examination was declined. The other was a case of presumed CHAOS, which was in fact a rare lethal congenital bronchopulmonary disorder (congenital alveolar dysplasia). In the five patients with giant cervical masses who survived delivery, two patients had teratomas which were fully resectable with excellent outcomes. In the three lymphatic malformations, one patient was treated by sclerotherapy alone, and one patient required an initial surgical debulk of cervical compressive disease and sclerotherapy for a compressive mediastinal comment. The latter patient was treated with sclerotherapy and has required surgical

CHAPTER 37  Fetal Tumours

A

449

endoscopy if not already done during the EXIT procedure. If the tracheostomy was performed in an expedited fashion, formalisation of the tracheostomy tract is required to ensure the tract remains established and secure in the postnatal period. Otherwise, further management is largely dependent on the cause of the airway obstruction (i.e., whether the pathology is intrinsic or extrinsic) and identifying the extent of disease. Additional factors have to be considered, including other congenital malformations, particularly cardiac, which may need urgent correction at the same time as the airway. The combined factors may have implications towards strategy of timing of surgery, and input from a dedicated aerodigestive MDT is usually required. 

Postnatal Investigations

B

After delivery of the infant and subsequent airway stabilisation, the initial priority is to evaluate the extent of airway disease. Essential investigations include direct endoscopic evaluation with microlaryngoscopy and tracheobronchoscopy; bronchography; and imaging with ultrasound, computed tomography (CT) and/ or MRI. Adjunct investigations may be necessary and include oesophagoscopy, optical coherence tomography (OCT) imaging of the airway, echocardiography and cardiac catheterisation, and genetic analysis. Direct evaluation of the airway may be performed as part of the EXIT procedure itself or immediately after delivery. Full endoscopic evaluation of the airway may not always be possible because of the pathology itself, and imaging with CT or MRI will further delineate the location and extent of the lesion. Further management focuses on determining if the airway obstruction is amenable to surgical correction. In the case of neck masses, the key question to managing the lesion is whether it is resectable. 

Postnatal Management

C •

Fig. 37.6  Pictures demonstrating the processes involved in an ex utero intrapartum treatment (EXIT) procedure. A, The head, neck and upper torso of the baby are delivered along with the right arm. Fentanyl and vecuronium (with or without atropine) are injected into the deltoid muscle. B, The fetal airway is secured with direct laryngoscopy, visualising the vocal cords under direct vision to ensure correct placement of the endotracheal tube. C, The baby is then delivered, and the umbilical is cord cut.

debulk of the tongue for an invasive component. The latter two children with lymphatic malformations have ongoing tracheostomy because of extensive disease into the upper airway (mediastinal and tongue base invasion) and related tracheomalacia. Otherwise the children do not have any neurologic or swallowing deficits. 

Postnatal Management and Outcomes The EXIT procedure will secure an airway either by an endotracheal route or tracheostomy. Immediate postnatal management includes a brief evaluation of the airway through direct

In general, MRI will determine the resectability of a lesion. Cervical teratomas are often amenable to full excision, and other lesions, including cervical thymic cysts, neuroblastomas and haemangioendotheliomas, can also be considered for surgical removal. However, lymphovascular malformations have to be considered more carefully because the borders are less well defined, and the disease can readily invade neighbouring structures.18,25 Lymphatic malformations are either macro- or microcystic disease or heterogeneous, consisting of both forms. Options include immediate decompression via aspiration (for large interconnecting cysts), sclerotherapy, surgical debulking and excision, or a combination of these.32-34 Sclerotherapy aims to deliver an agent into the lesion, thereby inducing a cascade of thrombitis and fibrosis. The agents used for this include ethanol, bleomycin, doxycycline and sodium tetradecyl sulphate.35 Multiple applications may be required to induce resolution of the lesion. Because the sclerosant induces an inflammatory reaction, there is often a temporary increase in the size of lesion followed by a resolution phase. This has implications for airway compromise if adjacent to the airway. Although sclerotherapy is generally safe with minimal side effects, it is worth noting that posttreatment infections can occur. Sclerosant therapy is normally confined to the lesion but can occasionally leak into surrounding tissues, leading to cutaneous necrosis, damage to muscle fibres and injury to peripheral nerves. In cases in which there is resistance to sclerotherapy or the disease is predominantly microcystic, surgery may be warranted.36

450

SE C T I O N 7     Diagnosis and Management of Fetal Malformations

Surgery aims to remove the compressive pathology, but the extent of surgery often needs to be balanced against the potential morbidity that may occur. In most cases with significant airway compression, surgery is the best modality. It particularly offers the advantage of being immediate; sclerotherapy may require repeated administration of sclerosants and is often complicated by temporary oedema and increased size of the lesion. Conversely, when the pathology infiltrates the tongue base and encases major nerves and vessels, surgery is more challenging and not always indicated. The risks from surgery include permanent cranial nerve injury, dysphagia and problems associated with disturbed lymphatic drainage. In the literature, these have been reported between 10% and 30%.37 Despite surgery and sclerotherapy, recurrence rates can be 15% to 50%.38 Much debate has centred around the optimal treatment, particularly sclerotherapy versus surgery. It is generally accepted that given the heterogeneity of disease, individual cases are preferentially discussed within a dedicated MDT and treatment tailored to the individual child.32 The timing of such surgery is also dependent on concomitant pathology. Congenital cardiac disease may need to be addressed at the same time, if not before corrective airway surgery. If the airway is considered established and uncompromised, repair can be delayed to allow for growth in the child. More often, airway surgery is required immediately to relieve the obstruction and is typically performed as soon as practically possible after EXIT delivery. In general, most lesions can be reduced or fully removed, and outcomes are favourable. Outcome reports from the EXIT procedure for 12 infants with giant neck masses found a mortality rate of 8% (n = 1), and 8% (n = 1) had evidence of mild developmental delay. The remaining patients had no functional deficits.39 

Sacrococcygeal Teratomas Sacrococcygeal teratomas are the commonest form of congenital tumours. However, SCTs are rare with an incidence of 1 in 35,000 to 1 in40,000 in pregnancies and an incidence of 1 in 27,000 at birth. There is an unexplained fourfold greater incidence in females than males. There are data to suggest that antenatally diagnosed SCTs have a poorer prognosis than those diagnosed postnatally, for which the prognosis is good.40 Traditionally, SCTs have been classified into four subtypes according to the Altman classification system41 (Table 37.2). Although useful as a descriptive term, it is doubtful whether the Altman classification has an impact on in utero outcome or postnatal surgical outcomes. A national registry study from the Netherlands did not find an association among Altman class, fetal sex or tumour pathology and functional outcome,42 although a recent study showed that there were no urologic or anorectal complications in type 1 SCTs.43

Antenatal Imaging The initial diagnosis of SCT is most commonly made by ultrasound after 20 weeks’ gestation. It is of interest that neonates with a poor prognosis are reported to be diagnosed with SCT at an earlier median gestational age than those with good prognosis (21.1 weeks (18.50–29.86 weeks) vs 23.9 weeks (16.60–34.14 weeks); P = .030),44 although the overlap in gestations limits it clinical utility in counselling parents. At midtrimester gestations, good assessment of the tumour size can be made (Fig. 37.7). The appearance of the internal component should be described as well as any deviation or obstruction of normal structures. Assessment of the degree to which

TABLE Sacrococcygeal Teratoma Subtypes (Altman 37.2 Classification)

Subtype

Description

Frequency (%)

I

Predominantly external tumours with only a small presacral component

46

II

External tumour mass but with a significant intrapelvic extension

34

III

External tumour mass with a predominantly pelvic or intraabdominal mass

9

IV

Presacral tumour with no external component

10

   the tumour consists of solid and cystic components should be performed. The size of the tumour should be measured, and 3D imaging techniques in both US and MRI can be used for volume calculations, which allow for individual assessment of the proportions of cystic and solid components. Vascularity should be assessed, and particular attention should be taken to identify any large feeding vessels. Magnetic resonance imaging should be seen as a complimentary imaging modality to US. MRI has benefits over US in imaging internal components, particularly in the third trimester. MRI assessment for signs of invasion, obstruction or deviation of internal structures may aid counselling regarding postnatal surgery and may have some benefits in prediction of long term functional urinary or bowel complications.43 

Antenatal Management Prediction of outcome. Several models have been proposed to predict

outcomes of fetuses with SCT. It was already known that features such as high levels of vascularity and fetal hydrops predict adverse outcomes because of cardiac overload. Rodriguez and colleagues45 further hypothesised that the tumour volume to fetal weight ratio (TFR) in the second trimester may be predictive of outcome, with poor outcomes being defined as development of hydrops, fetal demise or neonatal death. Tumour volume was calculated as a prolate ellipsoid using the greatest dimensions of length, height and depth as measured by ultrasound or MRI; estimated weight was calculated with the Hadlock formula. Measured before 24 weeks’ gestation, all fetuses with a TFR of 0.12 or less had a good outcome. Further studies,44,46 including a multi-institutional review including 50 fetuses with SCT, have validated that a TFR greater than 0.12 before 24 weeks’ gestation is predictive of poor prognosis (area under the curve, 0.913; sensitivity, 91.7%; specificity, 76.2%; positive predictive value (PPV), 86.8%; negative predictive value (NPV), 84.2%). The component elements of the tumour may also be predictive. Tumours that had a 50% or greater solid component were seven times more likely to have a poor outcome compared with those with a predominantly cystic mass (70.7% vs 9.1%44). A predictor of adverse outcome that accounts for the cystic:solid nature of SCTs, the solid tumour volume index (STVI), has been proposed.47 Using MRI, the volume of the tumour was assessed, and the maximum dimensions of the solid component of the tumour were used to calculate solid tumour volume and then divided by total tumour volume. Fetuses with an STVI greater than 0.09 were significantly more likely to develop

CHAPTER 37  Fetal Tumours

451

Voluson E10

Voluson E10

1

2

A

B Voluson E10

C

D

• Fig. 37.7  A, Ultrasound of a mostly external sacrococcygeal tumour (SCT) at 26 weeks’ gestational age, invading into the pelvis. Note the mixed cystic and solid component. B, A different case at 28 weeks’ gestational age, clearly showing the extent of pelvic invasion, and the separate bladder (thin arrow), spine (thick arrow) and cord insertion. C, Doppler image demonstrating the vascularity of the lesion in A. Notice the two large feeding vessels (thin arrows). This pregnancy had no evidence of cardiac failure, so in utero treatment was not thought to be appropriate. D, MRI of a different case at 35 + 6 weeks’ gestation of an Altman type 2 SCT with cystic and solid components. The tumour is mostly external but with a significant intrapelvic mass (thin arrow). Normal bladder (thick arrow) and bowel (star) can be seen separate from the tumour.

hydrops or high-output cardiac compromise (PPV, 81.25%; NPV, 100%). It should be noted that this is a complex index to calculate and requires further validation. The tumour growth rate has also been associated with adverse outcomes.48,49 Further investigation of tumour growth rate has shown that an increase tumour volume of more than 61 cm3 per week is associated with adverse outcomes, including hydrops, high-output cardiac failure and in utero death (likelihood ratio (LR), 4.52). Tumour growth rates of more than 165 cm3 per week are highly associated with in utero demise (LR, 18.4).50 A recent review of a UK-based registry showed increased growth rate to be associated with a poor prognosis.51 However, as commented on in this study, it remains unclear as to whether outcomes are associated with growth rate of the whole tumour or growth rate of the solid component, which is likely to have increased metabolic demands and higher vascularity contributing the phenomenon of vascular steal and its subsequent adverse effects on the fetal cardiac function.50 After 24 weeks’ gestation, ultrasound assessment should be performed every 2 weeks. These assessments should focus on assessing the size of the mass and any changes in characteristics, including increasing vascularity or signs of cardiac compromise, which may require in utero intervention or prompt delivery. 

In utero therapies. Several strategies for in utero therapy, including radiofrequency ablation (RFA), laser (both vascular and interstitial), sclerosing therapies and coiling of the main vessels have been attempted with a view to improving fetal outcomes, particularly in hydropic fetuses and fetuses with high output cardiac failure. Because of the heterogeneity of interventions, the risk for selection bias in small cohorts or case reports and incomplete descriptions, drawing conclusions as to the success of each individual intervention is not possible. A case series and systematic review by Van Meigham and colleagues52 described results from 34 cases of SCT treated with minimally invasive procedure. The survival rate was 30% (6 of 20) in patients with evidence of cardiac failure and 67% (8 of 12) in those without. In the subset of hydropic fetuses treated with RFA or interstitial laser, the survival rate was 45% (5 of 11). Because previability cardiac compromise is usually fatal, this result is encouraging, suggesting that in utero intervention, particularly RFA and laser treatment, may improve outcomes; however, the data are very limited and of mixed quality. Prematurity was a common occurrence after intervention with a mean gestational age at delivery of 29.7 ± 4.0 weeks in this series. More concerning, perhaps, are the complications arising from the interventions, mostly reported in RFA (although the reporting of complications and follow-up was poor in many studies). These included a fetus

452

SE C T I O N 7     Diagnosis and Management of Fetal Malformations

with perineal skin necrosis; another with gluteal necrosis, ischial and femoral head hypoplasia and sciatic nerve trauma; and a third fetus with a leg palsy (although the association with intervention was not clear in the latter case). Any in utero treatment should therefore be considered experimental and should only be performed by experienced operators in fetal therapy centres after adequate patient counselling. Comparison has also been made between the use of ‘interstitial’ techniques, which aim to directly ablate the tumour and ‘vasculartargeted’ techniques that aim to ablate the major feeding vessel.53 The data analysed are a reassessment of many of those included in the previous paper, plus a further five cases from two expert centres (totalling 33 cases). There is a tendency towards improved survival in the vascular-targeted group (64%, 7 of 11) compared with interstitial ablation (41%, 9 of 22). In hydropic fetuses, resolution of hydrops was seen more often in the vascular targeted group than in those that underwent interstitial intervention (75% (6 of 8) vs 33% (2 of 6)). Again, the authors express the same limitations of data and the need for multicentre prospective data collection. Historically, open fetal surgery has been reported. This is not discussed in detail here because of improvements in minimally invasive techniques, and no data have been published for more than a decade. In view of the complexity and uncertainty regarding in utero therapy and the improvements in neonatal care, early delivery from 28 weeks’ gestation should be considered in rapidly growing tumours or deteriorating fetuses that are not yet hydropic.54 

Planned delivery should take place in a unit with neonatal and paediatric surgical experience in teratomas. In cases of SCT with a predominantly internal component or with only a small external component, a vaginal delivery can be anticipated. In SCTs with a large external component (>5 cm) or in cases of fetal compromise or prematurity, a caesarean section should be planned. It should be anticipated that the SCT may be vascular and friable. Care should be taken to minimise trauma to the SCT, and a classical caesarean section may be required in larger cases. In larger vascular cases, neonatal blood should be available in case of bleeding and urgent need for transfusion. A consultant paediatric surgeon should be present in the delivery suite in cases of SCT with a large external component for the risk for bleeding. There may also be a role for interventional radiology in such cases. 

the tumour has a predominant intraabdominal component, a two-step procedures will be needed with an anterior approach followed by a posterior one. In cases in which it is unclear if the mass can be removed using exclusively the posterior approach, this should be attempted first. In our experience, a good proportion of tumour with intraabdominal or intrapelvic components can be removed via a posterior approach, particularly if there is a large cystic component.60,61 In terms of the surgery, after introducing a urinary catheter, the child should be positioned in the prone position and a skin incision made with the principle of preserving as much skin as possible.62 The mass should be removed following its capsule, without leaving residual tumour. However, careful dissection is needed to minimise damage, particularly at the level of the sphincter and the rectum.57,63 The latter can also be identified by introducing a Hegar dilator into the anus. If the rectum is damaged, closure with absorbable sutures should be conducted. In case of extensive damage, a stoma formation may become necessary, but it is usually avoidable. The coccyx should be removed together with the mass to avoid recurrence.55,59 Anterior to the coccyx, careful dissection is required to ligate the vascular supply to the tumour.61 Skin closure should be done, trying to give an acceptable cosmetic appearance. In our experience, the best results are obtained using the technique described by Fishman and associates.62 Fetuses with tumours diagnosed in utero without fetal hydrops and severe prematurity have an excellent prognosis with a survival rate in excess of 90%.44,64 Most of the tumours are benign, and when they are completely excised, recurrence is rare. They may contain immature elements but are rarely malignant if removed in the neonatal period. If undetected and resected later in life, there is a higher risk for malignant recurrence, particularly if the AFP is high.56,63 In terms of follow-up, slightly different approaches have been suggested but in general should consist of physical examination (including rectal) and serum markers every 3 months for at least 2 years and then every 6 months for the next 2 years. Ultrasound can be conducted every 3 or 6 months depending on the local protocol.58,65 Recurrence of the tumour is usually local, but metastatic lesions can be present in cases of malignancy. Surgical follow-up should be maintained for at least the first 4 to 5 years to monitor bowel and urinary function. In general, about 30% of children undergoing SCT resection present with problems, ranging from constipation to incontinence, and parents should be made aware of these risks at prenatal consultation.57,65 

Postnatal Management and Outcomes

Conclusion

At birth, if the patient is stable, postnatal imaging should be obtained in preparation for surgery which should be ideally performed in a semi-elective setting during the first week of life.40,55 An ultrasound is usually enough to evaluate the extension of the mass and distinguish the four SCT subtypes. MRI can be used to better define the anatomy but is usually not necessary.56 Care should be taken in managing the teratoma before the operation, and cling film should be used to avoid ulcerations and minimise the risk for bleeding.57 α-Fetoprotein (AFP) and β-human chorionic gonadotropin should be obtained both before surgery and in the early postoperative phase. Their values would be particularly important in the follow-up for early detection of recurrence.58 The SCT subtype it useful in deciding the surgical approach. Specifically, types I and II are mostly resectable using a posterior approach with the patient in a prone position.59 Conversely, if

Fetal tumours are extremely rare, but with more widespread ultrasound screening in pregnancy, the incidence of prenatal diagnosis is increasing. Management of such cases should be in fetal medicine centres working with an MDT of specialists to optimise perinatal outcome. Masses arising anteriorly from the fetal neck are most commonly lymphangiomas or teratomas. Differentiation can be difficult antenatally. MRI can be a useful imaging modality in these cases and is of particular use for soft tissue definition. Fetal neck masses can cause significant airway obstruction, and careful antenatal surveillance is required. In large masses, especially teratomas, an EXIT procedure should be planned at delivery. The treatment for teratomas is usually surgical resection, but lymphovascular abnormalities may require a combination of treatments, including sclerotherapy, aspiration and surgical debulking.

Intrapartum Management

CHAPTER 37  Fetal Tumours

Sacrococcygeal teratomas are the commonest form of congenital tumour. Careful antenatal assessment is required with serial ultrasound. High vascularity, rapid growth or a large solid component to the teratoma are associated with poor antenatal outcomes. In fetuses with hydrops at less than 28weeks’ gestation, in utero therapies may improve outcomes but are associated with high rates of comorbidity. Postnatally, resection of the SCT is the preferred treatment. Follow-up into childhood is required because there is a risk for recurrence, and up to one third of children may have complications, particularly related to bladder and bowel function.

453

The outlook for fetuses with these rare tumours is improving. Key to this is the management of these pregnancies in an experienced fetal medicine centre with easy access to antenatal input from the associated paediatric and adult specialties. Access the complete reference list online at ExpertConsult.com. Self-assessment questions available at ExpertConsult.com.

References 1. Kadlub N, Touma J, Leboulanger N, et al. Head and neck teratoma: from diagnosis to treatment. J Cranio-Maxillofacial Surg. 2014;42:1598–1603. 2. Howarth ES, Draper ES, Budd JL, et al. Population-based study of the outcome following the prenatal diagnosis of cystic hygroma. Prenat Diagn. 2005;25:286–291. 3. Forrester MB, Merz RD. Descriptive epidemiology of teratoma in infants, Hawaii, 1986-2001. Paediatr Perinat Epidemiol. 2006;20:54–58. 4. Oliver G. Lymphatic vasculature development. Nat Rev Immunol. 2004;4:35–45. 5. Molina FS, Avgidou K, Kagan KO, et al. Cystic hygromas, nuchal edema, and nuchal translucency at 11–14 weeks of gestation. Obstet Gynecol. 2006;107:678–683. 6. Croonen EA, Nillesen W, Schrander C, et al. Noonan syndrome: comparing mutation-positive with mutation-negative Dutch patients. Mol Syndromol. 2013;4:227–234. 7. Shaffer LG, Rosenfeld JA, Dabell MP, et al. Detection rates of clinically significant genomic alterations by microarray analysis for specific anomalies detected by ultrasound. Prenat Diagn. 2012;32:986–995. 8. Aubin A, Pondaven S, Bakhos D,L, et  al. Oropharyngeal teratomas in newborns: management and outcome. Eur Ann Otorhinolaryngol Head Neck Dis. 2014;131:271–275. 9. Kathary N, Bulas DI, Newman KD, Schonberg RL. MRI imaging of fetal neck masses with airway compromise: utility in delivery planning. Pediatr Radiol. 2001;31:727–731. 10. Lazar DA, Cassady CI, Olutoye OO, et al. Tracheoesophageal displacement index and predictors of airway obstruction for fetuses with neck masses. J Pediatr Surg. 2012;47:46–50. 11. Santos XM, Papanna R, Johnson A, et  al. The use of combined ultrasound and magnetic resonance imaging in the detection of fetal anomalies. Prenat Diagn. 2010;30:402–407. 12. Lall NU, Meyers ML, Mirsky DM. MRI of fetal and maternal diseases in pregnancy. Springer International Publishing, Switzerland; 2016:119–137. 13. Saleem SN. Fetal MRI: an approach to practice: a review. J Adv Res. 2014;5:507–523. 14. Hubbard A, Crombleholme T, Adzick N. Prenatal MRI evaluation of giant neck masses in preparation for the fetal EXIT procedure. Am J Perinatol. 2008;15:253–257. 15. Wolfe K, Lewis D, Witte D, et  al. Fetal cervical teratoma: what is the role of fetal MRI in predicting pulmonary hypoplasia? Fetal Diagn Ther. 2013;33:252–256. 16. Werner H, Lopes dos Santos JR, Fontes R, et al. Virtual bronchoscopy for evaluating cervical tumors of the fetus. Ultrasound Obstet Gynecol. 2013;41:90–94. 17. Laje P, Peranteau WH, Hedrick HL, et al. Ex utero intrapartum treatment (EXIT) in the management of cervical lymphatic malformation. J Pediatr Surg. 2015;50:311–314. 18. Sheikh F, Akinkuotu A, Olutoye OO, et  al. Prenatally diagnosed neck masses: long-term outcomes and quality of life. J Pediatr Surg. 2015;50:1210–1213. 19. Ogita K, Suita S, Taguchi T, et al. Outcome of fetal cystic hygroma and experience of intrauterine treatment. Fetal Diagn Ther. 16:105–110 20. Chen M, Chen CP, Shih JC, et al. Antenatal treatment of chylothorax and cystic hygroma with OK-432 in nonimmune hydrops fetalis. Fetal Diagn Ther. 2005;20:309–315. 21. Hirose S, Sydorak RM, Tsao K, et  al. Spectrum of intrapartum management strategies for giant fetal cervical teratoma. J Pediatr Surg. 2003;38:446–450. 22. Kontopoulos EV, Gualtieri M, Quintero RA. Successful in utero treatment of an oral teratoma via operative fetoscopy: case report and review of the literature. Am J Obstet Gynecol. 2012;207:e12–e15. 23. Cruz-Martinez R, Moreno-Alvarez O, Garcia M, et al. Fetal endoscopic tracheal intubation: a new fetoscopic procedure to ensure extrauterine tracheal permeability in a case with congenital cervical teratoma. Fetal Diagn Ther. 2014;38:154–158. 24. Kelly MF, Berenholz L, Rizzo KA, et al. Approach for oxygenation of the newborn with airway obstruction due to a cervical mass. Ann Otol Rhinol Laryngol. 1990;99:179–182. 25. Catalano PJ, Urken ML, Alvarez M, et  al. New approach to the management of airway obstruction in “high risk” neonates. Arch Otolaryngol Head Neck Surg. 1992;118:306–309. 26. Deprest J, Gratacos E, Nicolaides KH. Fetoscopic tracheal occlusion(FETO) for severe congenital diaphragmatic hernia: evolution of a technique and preliminary results. Ultrasound Obstet Gynecol. 2004;24:121–126.

27. Laje P, Johnson MP, Howell LJ, et  al. Ex utero intrapartum treatment in the management of giant cervical teratomas. J Pediatr Surg. 2012;47:1208–1216. 28. Lin EE, Moldenhauer JS, Tran KM, et al. Anesthetic management of 65 cases of ex utero intrapartum therapy. Anesth Analg. 2016;123:411–417. 29. Boat A, Mahmoud M, Michelfelder EC, et al. Supplementing desflurane with intravenous anesthesia reduces fetal cardiac dysfunction during open fetal surgery. Paediatr Anaesth. 2010;20:748–756. 30. Ryan G, Somme S, Crombleholme TM. Airway compromise in the fetus and neonate: prenatal assessment and perinatal management. Semin Fetal Neonatal Med. 2016;21:230–239. 31. Barthod G, Teissier N, Bellarbi N, et  al. Fetal airway management on placental support: limitations and ethical considerations in seven cases. J Obstet Gynaecol. 2013;33:787–794. 32. Cheng J, Bastidas N. Considerations for management of head and neck lymphatic malformations in children. J Craniofac Surg. 2016;27:908– 912. 33. Morgan P, Keller R, Patel K. Evidence-based management of vascular malformations. Facial Plast Surg. 2016;32:162–176. 34. Bagrodia N, Defnet AM, Kandel JJ. Management of lymphatic malformations in children. Curr Opin Pediatr. 2015;27:356–363. 35. Horbach SE, Lokhorst MM, Saeed P, et al. Sclerotherapy for low-flow vascular malformations of the head and neck: a systematic review of sclerosing agents. J Plast Reconstr Aesthetic Surg. 2016;69:295–304. 36. Hoff SR, Rastatter JC, Richter GT. Head and neck vascular lesions. Otolaryngol Clin North Am. 2015;48:29–45. 37. Elluru RG, Azizkhan RG. Cervicofacial vascular anomalies. II. Vascular malformations. Semin Pediatr Surg. 2006;15:133–139. 38. Adams MT, Saltzman B, Perkins JA. Head and neck lymphatic malformation treatment: a systematic review. Otolaryngol Head Neck Surg. 2012;147:627–639. 39. Lazar DA, Olutoye OO, Moise Jr KJ, et al. Ex-utero intrapartum treatment procedure for giant neck masses—fetal and maternal outcomes. J Pediatr Surg. 2011;46:817–822. 40. Peiró JL, Sbragia L, Scorletti F, et  al. Management of fetal teratomas. Pediatr Surg Int. 2016;32:635–647. 41. Altman RP, Randolph JG, Lilly JR. Sacrococcygeal teratoma: American Academy of Pediatrics Surgical Section Survey—1973. J Pediatr Surg. 1974;9:389–398. 42. Derikx JP, De Backer A, van de Schoot L, et al. Long-term functional sequelae of sacrococcygeal teratoma: a national study in the Netherlands. J Pediatr Surg. 2007;42:1122–1126. 43. Partridge EA, Canning D, Long C, et al. Urologic and anorectal complications of sacrococcygeal teratomas: prenatal and postnatal predictors. J Pediatr Surg. 2014;49:139–143. 44. Akinkuotu AC, Coleman A, Shue E, et al. Predictors of poor prognosis in prenatally diagnosed sacrococcygeal teratoma: a multiinstitutional review. J Pediatr Surg. 2015;50:771–774. 45. Rodriguez MA, Cass DL, Lazar DA, et al. Tumor volume to fetal weight ratio as an early prognostic classification for fetal sacrococcygeal teratoma. J Pediatr Surg. 2011;46:1182–1185. 46. Shue E, Bolouri M, Jelin EB, et al. Tumor metrics and morphology predict poor prognosis in prenatally diagnosed sacrococcygeal teratoma: a 25-year experience at a single institution. J Pediatr Surg. 2013;48:1225– 1231. 47. Coleman A, Kline-Fath B, Keswani S, Lim FY. Prenatal solid tumor volume index: novel prenatal predictor of adverse outcome in sacrococcygeal teratoma. J Surg Res. 2013;184:330–336. 48. Westerburg B, Feldstein VA, Sandberg PL, et al. Sonographic prognostic factors in fetuses with sacrococcygeal teratoma. J Pediatr Surg. 2000;35:322–326. 49. Benachi A, Durin L, Vasseur Maurer S, et  al. Prenatally diagnosed ­sacrococcygeal teratoma: a prognostic classification. J Pediatr Surg. 2006; 41:1517–1521. 50. Coleman A, Shaaban A, Keswani S, Lim FY. Sacrococcygeal teratoma growth rate predicts adverse outcomes. J Pediatr Surg. 2014;49:985–989. 51. Ayed A, Tonks AM, Lander A, Kilby MD. A review of pregnancies complicated by congenital sacrococcygeal teratoma in the West Midlands region over an 18-year period: population-based, cohort study. Prenat Diagn. 2015;35:1037–1047. 52. Van Mieghem T, Al-Ibrahim A, Deprest J, et al. Minimally invasive therapy for fetal sacrococcygeal teratoma: case series and systematic review of the literature. Ultrasound Obstet Gynecol. 2014;43:611–619. 53. Sananes N, Javadian P, Schwach Werneck Britto I, et al. Technical aspects and effectiveness of percutaneous fetal therapies for large sacrococcygeal teratomas: cohort study and literature review. Ultrasound Obstet Gynecol. 2016;47:712–719.

453.e1

453.e2 R E F E REN C ES

54. Roybal JL, Moldenhauer JS, Khalek N, et al. Early delivery as an alternative management strategy for selected high-risk fetal sacrococcygeal teratomas. J Pediatr Surg. 2011;46:1325–1332. 55. Flake AW. Fetal sacrococcygeal teratoma. Semin Pediatr Surg. 1993; 2:113–120. 56. Schmidt B, Haberlik A, Uray E, et al. Sacrococcygeal teratoma: clinical course and prognosis with a special view to long-term functional results. Pediatr Surg Int. 1999;15:573–576. 57. Malone PS, Spitz L, Kiely EM, et al. The functional sequelae of sacrococcygeal teratoma. J Pediatr Surg. 1990;25:679–680. 58. Yao W, Li K, Zheng S, et al. Analysis of recurrence risks for sacrococcygeal teratoma in children. J Pediatr Surg. 2014;49:1839–1842. 59. Cohen L, Lauer SJ, Ablin A, et al. Complete surgical excision is effective treatment for children with immature teratomas with or without malignant elements: a Pediatric Oncology Group/Children’s Cancer Group Intergroup Study. J Clin Oncol. 1999;17:2137–2143. 60. Hedrick HL, Flake AW, Crombleholme TM, et al. Sacrococcygeal teratoma: prenatal assessment, fetal intervention, and outcome. J Pediatr Surg. 2004;39:430–438. -8.

61. Kremer ME, Wellens LM, Derikx JP, et al. Hemorrhage is the most common cause of neonatal mortality in patients with sacrococcygeal teratoma. J Pediatr Surg. 2016;51:1826–1829. 62. Fishman SJ, Jennings RW, Johnson SM, Kim HB. Contouring buttock reconstruction after sacrococcygeal teratoma resection. J Pediatr Surg. 2004;39:439–441. 63. Rescorla FJ, Sawin RS, Coran AG, et al. Long-term outcome for infants and children with sacrococcygeal teratoma: a report from the Children’s Cancer Group. J Pediatr Surg. 1998;33:171–176. 64. Brace V, Grant SR, Brackley KJ, et al. Prenatal diagnosis and outcome in sacrococcygeal teratomas: a review of cases between 1992 and 1998. Prenat Diagn. 2000;20:51–55. 65. Shalaby MS, Walker G, O’Toole S, et  al. The long-term outcome of patients diagnosed with sacrococcygeal teratoma in childhood. A study of a national cohort. Arch Dis Child. 2014;99:1009–1013.

38

Open Fetal Surgery LUC JOYEUX, FRANK VAN CALENBERGH, ROLAND DEVLIEGER, LUC DE CATTE AND JAN DEPREST

KEY POINTS

Selection Criteria for Open Fetal Surgery

• Most fetuses with a prenatal diagnosis of a congenital abnormality can be managed expectantly. For some conditions, in utero referral is mandatory for planned delivery and management after birth. • Fetal surgery is only required for conditions that cannot await therapy after birth and when there is enough evidence that prenatal surgery partly reverses the natural course. • Open fetal surgery (OFS) is one modality to perform operations on fetuses. OFS is invasive, with maternal morbidity and an impact on the uterus in the index and future pregnancies, and it increases the risk for preterm delivery. • Open spina bifida is the only nonlethal condition operated in utero, yet it became the most frequent indication based on level I evidence. OFS improves the outcome of children with spina bifida, but it is not a cure.

Open fetal surgery is currently being offered in highly specialised multidisciplinary fetal centres for highly selected fetuses with a condition that is, without any intervention, either lethal or in which the subsequent organ function loss leads to an extremely poor quality of life after birth. The International Fetal Medicine and Surgery Society (IFMSS) defined five criteria required to justify fetal surgery (Table 38.1).2 

Introduction Definition Open fetal surgery (OFS) is the type of fetal surgery that is performed via hysterotomy. It is also referred as open maternal-fetal surgery or fetal surgery by open access. 

Rationale of Open Fetal Surgery for Congenital Abnormalities Pathophysiology and Natural History Since the introduction of ultrasound (US) and later other imaging modalities and the increased interest for congenital anomalies (CAs) and their outcomes in relation to prenatal findings, it has become possible to define the natural history of several potentially correctable CAs.1 Also, researchers developed appropriate animal models for the disease of interest to study both the pathophysiology as well as the surgical interventions contemplated. These studies, particularly in primates, have been used for developing the anaesthetic, tocolytic and surgical protocols for hysterotomy and OFS.  454

Indications A Nonlethal Condition: Spina Bifida Aperta The epidemiology, pathophysiology and natural history have been addressed in Chapter 28. Within routine screening US programs neural tube defects should be diagnosed prenatally. An invasive fetal procedure for spina bifida aperta (SBA) seems to be justified because of the significant lifelong neurologic disabilities, the prenatal progression of findings and the experimental validation of the two-hit pathophysiology.3 Experimental research and early clinical experience suggest that ongoing damage to the exposed malformed spinal cord and developing brain is alleviated by prenatal repair.1,4 The Management of Myelomeningocele Study (MOMS) was a multicentre randomized controlled trial that unequivocally demonstrated that prenatal surgery for SBA improves outcome compared with standard postnatal repair.5 Table 38.2 displays the initial and current indications and contraindications for in utero SBA repair. Fetal surgery was shown to lessen or reverse hindbrain herniation, reduce the postnatal ventriculoperitoneal shunt rate at 1 year of age and improve neurofunctional outcome at the age of 30 months. 

Lethal Conditions Congenital Thoracic Malformations Complicated With Fetal Hydrops. Congenital thoracic malformations (CTMs) are a

heterogeneous group of rare disorders that may involve the airways or lung parenchyma. As an accurate pathological prenatal diagnosis is not possible; a detailed prenatal description of the appearance of the lesion is sufficient and should follow the new CTM classification and nomenclature (see Chapter 30).6 This section focuses on the two most frequent CTMs that are also amenable to OFS: congenital cystic adenomatoid malformation (CCAM) and its related malformation,

CHAPTER 38  Open Fetal Surgery

TABLE Five Criteria for Maternal-Fetal Surgery From 38.1 the International Fetal Medicine and Surgery

Society

1. Accurate diagnosis and staging possible, with exclusion of associated anomalies 2. Natural history of the disease is documented, and individualised prognosis is established 3. Currently no effective postnatal therapy (i.e., improving the condition or curing it) 4. In utero surgery proven feasible in animal models, reversing the deleterious effects of the condition 5. Interventions performed in specialised multidisciplinary fetal treatment centres within strict protocols and approval of the local ethics committee with informed consent of the mother or parents Adapted Harrison MR, Filly RA, Golbus MS, et  al. Fetal treatment 1982. N Engl J Med 307(26):1651–1652, 1982.

  

bronchopulmonary sequestration (BPS). It is important to realise that the exact nature of the condition (or their combination) may only be possible after resection. In fact, lung parenchymal malformations, although superficially heterogeneous in appearance, have significant overlap and seem to share a common embryologic origin.7 According to the European Surveillance of Congenital Anomalies (EUROCAT) registry, the prevalence of CTM between 2008 and 2012 was 4.13 per 10,000 live births. Among the CTMs, the estimated prevalence of CCAM was 1.05 per 10,000 live births, that is, about one quarter of all CTMs.8 The exact prevalence of BPS is unknown and probably lower than for CCAM.9 CTMs may spontaneously regress before birth; the ones that deteriorate in utero and may even lead to fetal death are of relevance to this chapter (see Table 38.2). Congenital Cystic Adenomatoid Malformation. Congenital cystic adenomatoid malformation is a benign cystic intrapulmonary nonfunctioning lung mass that is usually localised in one lobe of the lung and mainly unilateral. CCAM contains cysts ranging from smaller than 1 mm to larger than 10 cm in diameter. Most CCAMs derive their blood supply from the pulmonary circulation. CCAM is histologically characterised by an overgrowth of terminal respiratory bronchioles that form cysts and lack normal alveoli. Many pathologists consider it a hamartoma (i.e., a developmental abnormality with excess of one or several tissue components). Although nonfunctional for normal gas exchange, CCAM parenchyma has connections with the tracheobronchial tree as evidenced by air trapping that can develop during postnatal resuscitative efforts.10 There are different prenatal classifications; however, postnatally, typically four Stocker types (I–IV) are described. Congenital cystic adenomatoid malformation growth usually reaches a plateau by 28 weeks of gestation and may even nearly disappear by birth.10 Others may cause fetal hydrops and in utero death. The impact on normal lung development and postnatal function has not been properly studied. Prenatally, the size of the lesion, the cyst size, the growth and perfusion and the secondary signs have all been used to describe the severity of the impact on the fetus. In general, fetal hydrops is the single accepted criterion for fetal therapy. A CCAM volume ratio (CVR) (CCAM volume by sonographic measurement using the formula for an ellipse, length × height × width × 0.52 divided by head circumference to correct for differences in fetal size) greater than1.6 has been shown to predict hydrops in 80% of fetuses with CCAM.10 

455

Bronchopulmonary Sequestration. Bronchopulmonary sequestration is defined as a nonfunctioning lung mass that receives a systemic blood supply rather than from a branch of the pulmonary artery. It belongs to the spectrum of congenital foregut malformations arising as an aberrant outpouching from the developing foregut. BPS is believed to be aberrantly located pulmonary mesenchyma that develops apart from the normal lung.7 Bronchopulmonary sequestration is classified into intralobar and extralobar forms. The extralobar (25%) form consists of pulmonary tissue located outside the lung and enveloped in its own pleura without communication with the normal tracheobronchial tree. Around 90% are supradiaphragmatic and 10% infradiaphragmatic, usually left suprarenal. The intralobar form is found within the normal lung tissue with or without communication.9  Other Rarer Lethal Conditions. We will not discuss these even rarer indications, some of which have been operated in utero10: • Hybrid lesions (CCAM and BPS) • Bronchogenic and enteric cysts • Mediastinal cystic teratoma • Congenital lobar emphysema • Haemangioma • Bronchial atresia • Pulmonary leiomyofibroma • Intrathoracic gastric duplication cyst  Sacrococcygeal Teratoma With Hydrops Fetalis. Sacrococcygeal teratoma (SCT) is the most common tumour of newborns with a prevalence of about 0.37 to 0.93 per 10,000 live births.11,12 SCT is uniformly attached to the coccyx and has been classified into four types (I–IV) by the relative amounts of intrapelvic and external tumour. SCTs presenting postnatally have excellent longterm outcomes; conversely, prenatally diagnosed SCTs have a significant perinatal mortality ranging from 25% to 37%. Death occurs mainly in fetuses with fast-growing, solid and highly vascularised teratomas that lead to high-output cardiac failure or haemorrhage.13 The pathophysiological mechanism behind this is explained by the ‘vascular steal’ from the placenta and the fetus and by the mass effect10: 1. SCT acts as a large arteriovenous malformation that deviates high volumes of blood from the fetus and the placenta. Also, bleeding inside the tumour can cause anaemia. The consequence is high-output cardiac failure, which can then lead to placentomegaly, hydrops fetalis, intrauterine fetal demise, preterm birth and neonatal death. This may also cause Ballantyne syndrome (maternal mirror syndrome), which is a dangerous maternal complication. 2. SCT compresses the abdominal and thoracic organs, leading to polyhydramnios (oesophageal and gastric compression) inducing uterine irritability, premature rupture of the membranes (PPROM) and preterm delivery. The tumour may also have an effect on nearby organs, such as obstructive uropathy. Dystocia in undiagnosed large masses is frequently associated with traumatic tumour rupture and haemorrhage during delivery, which is usually fatal. Assessment of tumour size, growth rate and fetal cardiac function by fetal imaging allows the identification of fetuses at particular risk for decompensation. Ultrafast fetal magnetic resonance imaging (MRI) is superior to US in delineating the intrapelvic extent of the tumour, yet this does not contribute to the indication for fetal surgery.14 The currently accepted indications for OFS for this condition are detailed in Table 38.2. 

456

SE C T I O N 7     Diagnosis and Management of Fetal Malformations

TABLE 38.2 Indications for Open Fetal Surgery

Indications for OFS

Type of Malformation

Spina bifida aperta

Rationale for In Utero Therapy

Inclusion Criteria

Exclusion Criteria

MMC or myeloschisis with CM

• Untethering and covering of exposed malformed spinal cord to prevent or reverse functional damage to the cord and nerves54,55 • Cessation of CSF leakage to prevent or reverse hydrocephaly and CM56-60

• Maternal age ≥18 yr • Gestational age 19 + 0-25 + 6weeks • Isolated lesion • Normal karyotype • Level lesion from T1–S1 • Confirmed CM on prenatal US and MRI

• Multiple-gestation pregnancy • Uterine anomaly or corporeal uterine surgery • Additional fetal anomalies unrelated to SBA (chromosomal or not) • Fetal kyphosis ≥30 degrees • Previous spontaneous singleton delivery at