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The Endometrial Factor A Reproductive Precision Medicine Approach
 9781498740395, 1498740391, 9781498740401, 1498740405

Table of contents :
Content: Preface. Foreword: An eye to the future. Highlights on the dynamism and multifunctionality of human endometrium in the era of precision medicine. Diagnosis of the Endometrial Factor: New imaging diagnostics. Molecular diagnosis of endometrial receptivity. Molecular diagnosis of endometriosis. Microbiological diagnosis: The human endometrial microbiome-endometritis. Hysteroscopy: An invasive diagnosis. Personalization of the Endometrial Factor: The endometrium in Polycystic Ovary Syndrome. Obesity and the endometrium. Uterine fibroids and the endometrium. New knowledge about adenomyosis. Therapeutic Options of the Endometrial Factor: Hormonal regulation of the endometrium and the effects of hormonal therapies. Abnormal Uterine Bleeding (previously Dysfunctional Uterine Bleeding): How to manage. Inflammation of the endometrium in reproduction. Empirical treatments to improve receptivity: Why not?. Endometrial scratching. Stem Cell Therapies for Atrophic Endometrium and Ashermans Syndrome. Gestational surrogacy. Uterine transplantation. The Ultimate Goal: Embryo/fetal-maternal cross-talk. Embryo transfer: Fresh, deferred, personalized? Reproductive and obstetrical outcomes.

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The Endometrial Factor A Reproductive Precision Medicine Approach

Edited by

Carlos Simón MD, PhD and Linda C. Giudice MD, PhD

CRC Press Taylor & Francis Group 6000 Broken Sound Parkway NW, Suite 300 Boca Raton, FL 33487-2742 © 2017 by Taylor & Francis Group, LLC CRC Press is an imprint of Taylor & Francis Group, an Informa business No claim to original U.S. Government works Printed on acid-free paper International Standard Book Number-13: 978-1-4987-4039-5 (Hardback) This book contains information obtained from authentic and highly regarded sources. While all reasonable efforts have been made to publish reliable data and information, neither the author[s] nor the publisher can accept any legal responsibility or liability for any errors or omissions that may be made. The publishers wish to make clear that any views or opinions expressed in this book by individual editors, authors or contributors are personal to them and do not necessarily reflect the views/opinions of the publishers. The information or guidance contained in this book is intended for use by medical, scientific or health-care professionals and is provided strictly as a supplement to the medical or other professional’s own judgement, their knowledge of the patient’s medical history, relevant manufacturer’s instructions and the appropriate best practice guidelines. Because of the rapid advances in medical science, any information or advice on dosages, procedures or diagnoses should be independently verified. The reader is strongly urged to consult the drug companies’ printed instructions, and their websites, before administering any of the drugs recommended in this book. This book does not indicate whether a particular treatment is appropriate or suitable for a particular individual. Ultimately it is the sole responsibility of the medical professional to make his or her own professional judgements, so as to advise and treat patients appropriately. The authors and publishers have also attempted to trace the copyright holders of all material reproduced in this publication and apologize to copyright holders if permission to publish in this form has not been obtained. If any copyright material has not been acknowledged please write and let us know so we may rectify in any future reprint. Except as permitted under U.S. Copyright Law, no part of this book may be reprinted, reproduced, transmitted, or ­utilized in any form by any ­electronic, mechanical, or other means, now known or hereafter invented, including p ­ hotocopying, microfilming, and recording, or in any ­information storage or retrieval system, without written permission from the publishers. For permission to photocopy or use material electronically from this work, please access www.copyright.com (http://www.copyright.com/) or contact the Copyright Clearance Center, Inc. (CCC), 222 Rosewood Drive, Danvers, MA 01923, 978-750-8400. CCC is a not-for-profit organization that provides licenses and registration for a variety of users. For ­organizations that have been granted a photocopy license by the CCC, a separate system of payment has been arranged. Trademark Notice: Product or corporate names may be trademarks or registered trademarks, and are used only for identification and explanation without intent to infringe. Library of Congress Cataloging-in-Publication Data Simón, Carlos, editor ; Giudice, Linda, editor. The endometrial factor : a reproductive precision medicine approach / edited by Carlos Simón, Linda C. Giudice. Boca Raton, FL : CRC Press/Taylor & Francis Group, [2017] Includes bibliographical references and index. LCCN 2016041251| ISBN 9781498740395 (hardback : alk. paper) | ISBN 9781498740401 (ebook) Subjects: | MESH: Uterine Diseases--diagnosis | Endometrium--physiopathology | Uterine Diseases--complications | Uterine Diseases--therapy Classification: LCC RG316 | NLM WP 440 | DDC 618.1/4--dc23 LC record available at https://lccn.loc.gov/2016041251 Visit the Taylor & Francis Web site at http://www.taylorandfrancis.com and the CRC Press Web site at http://www.crcpress.com

Contents Preface v Foreword: An eye to the future vii Contributors ix 1

Highlights on the dynamism and multifunctionality of human endometrium in the era of precision medicine Linda C. Giudice

1

Section I  DIAGNOSIS OF THE ENDOMETRIAL FACTOR 2 3 4 5 6

New imaging diagnostics Sanja Kupesic Plavsic Molecular diagnosis of endometrial receptivity Jose Miravet-Valenciano, Nuria Balaguer, Felipe Vilella, and Carlos Simón Molecular diagnosis of endometriosis Lusine Aghajanova and Linda C. Giudice Microbiological diagnosis: The human endometrial microbiome—Endometritis Inmaculada Moreno and Carlos Simón Hysteroscopy: An invasive diagnosis Natasha Pritchard, Shavi Fernando, and Luk Rombauts

15 36 50 65 78

Section II  PERSONALIZATION OF THE ENDOMETRIAL FACTOR 7 8 9 10

The endometrium in polycystic ovary syndrome Terhi T. Piltonen Obesity and the endometrium Ioanna A. Comstock, Sun H. Kim, and Ruth B. Lathi Uterine fibroids and the endometrium Deborah E. Ikhena and Serdar E. Bulun New knowledge about adenomyosis Giuseppe Benagiano, Beatrice Ermini, Marwan Habiba, and Ivo Brosens

97 111 120 130

Section III THERAPEUTIC OPTIONS OF THE ENDOMETRIAL FACTOR 11 12 13 14 15 16 17 18

Hormonal regulation of the endometrium and the effects of hormonal therapies Gulcin Sahin Ersoy, Monica Modi, Myles Alderman, and Hugh S. Taylor Abnormal uterine bleeding (the old dysfunctional uterine bleeding): How to manage? Hilary O. D. Critchley and Lucy Whitaker Inflammation of the endometrium in reproduction John Groth, Dana McQueen, and Mary Stephenson Empirical treatments to improve receptivity: Why not? Nick S. Macklon Endometrial scratching Claire Bourgain, Samuel Santos-Ribeiro, and Christophe Blockeel Stem cell therapies for atrophic endometrium and Asherman’s syndrome Benjamin J. Seifer, Hanyia Naqvi, Elham Neisani Samani, Graciela Krikun, and Hugh S. Taylor Gestational surrogacy Molly Quinn and Heather Huddleston Uterine transplantation Mats Brännström

161 177 190 201 208 217 228 234

iii

iv Contents Section IV THE ULTIMATE GOAL 19 20

Embryo/fetal–maternal cross talk Nuria Balaguer, Francisco Dominguez, Carlos Simón, and Felipe Vilella Embryo transfer: Fresh, deferred, personalized? Reproductive and obstetrical outcomes Siladitya Bhattacharya

243 256

Index265

Preface The relevance of the endometrial factor in reproductive medicine is gradually gaining momentum. The Endometrial Factor was carefully conceived with world experts to be a compendium of critically evaluated, stateof-the-art basic and clinical knowledge about the human endometrium that impacts or will impact clinical practice. This new text updates the advances in the diagnosis and treatment of the uterine or endometrial factor in the management of the infertile patient in the ­context of ­precision medicine. Section I is devoted to the description of “all you need to know” about the human ­endometrium, by Linda C. Giudice, coeditor of this book. It is f­ollowed by an update of diagnostic possibilities: noninvasive diagnosis using imaging, by Sanja Kupesic Plavsic; molecular diagnosis of endometrial receptivity, by Jose Miravet-Valenciano; molecular diagnosis of endometriosis, by Lusine Aghajanova; microbiological diagnosis of the human endometrial microbioma—endometritis, by Inmaculada Moreno; and invasive diagnosis through ­hysteroscopy, by Luk Rombauts. Section II focuses on specific reproductive and medical disorders and their clinical impact on the endometrial ­factor in a personalized manner. The endometrium is ­analyzed in polycystic ovary syndrome patients by Terhi T. Piltonen, in obesity by Ruth B. Lathi, in the presence of fibroids by Serdar Bulun and adenomyosis by Ivo Brosens. Section III updates current therapeutic options for the endometrial factor in a critical manner: from effects of classical hormonal treatments by Hugh S. Taylor and the management of dysfunctional uterine bleeding by

Hilary Critchley, to immunological treatment by Mary Stephenson, analysis of empirical unproven treatments by Nick S. Macklon, and endometrial scratching by Claire Bourgain. New stem cell therapies in the atrophic endometrium and Asherman’s syndrome are addressed by Hugh S. Taylor. Finally, we discuss what to do when the uterus is absent or not functional, including surrogacy by Molly Quinn and Heather Huddleston and uterine ­transplantation by Mats Brännström. Section IV addresses the ultimate function of the endometrium—modulating and facilitating embryonic ­ implantation. The interaction between the endometrium and the preimplantation embryo is presented by Felipe Vilella, and the establishment of pregnancy through different embryo transfer strategies by Siladitya Bhattacharya. The book is introduced by the views of a pioneer, Peter A. W. Rogers, in pursuit of a better understanding of ­endometrial function on the hoped-for pathway to ­precision medicine. We express our utmost appreciation to all of our international best-in-class colleagues who have graciously contributed to the realization of this publication; we are grateful for the time and effort they devoted. We hope that readers will find the contents of this book useful as a ­reference and a valuable tool for continued advancement in the understanding and management of the endometrial factor in reproductive medicine. Carlos Simón and Linda C. Giudice 2016

v

Foreword: An eye to the future PETER A. W. ROGERS The advent of in vitro fertilization (IVF) more than 35  years ago revolutionized reproductive medicine. While the goal was to treat infertile ­couples, the techniques that were developed generated a huge amount of new information on reproductive endocrinology, ovarian function, ovulation, fertilization, preimplantation embryology, implantation, and early pregnancy. This knowledge dividend from IVF continues today, as exemplified by chapters in this book covering advances in areas such as pelvic ultrasound, the impact of obesity on the endometrium, and the rapidly emerging field of the microbiome. The importance of this reproductive medicine research is brought sharply into focus by a recent analysis of data from the 2010 Global Burden of Disease Study (1). This work reported a systematic analysis of “years lived with disability” (YLD) for 1160 sequelae of 289 diseases and injuries. Within the list, gynecological diseases contribute 146 YLD per 100,000 years, with major diseases being fibroids (44 YLD), polycystic ovary syndrome (40 YLD), genital prolapse (26 YLD), premenstrual syndrome (18 YLD), and endometriosis (8 YLD). To put this gynecological disability burden in perspective, the same study reports 161 YLD per 100,000 years for HIV/AIDS and tuberculosis combined, 99 YLD for Alzheimer’s and other dementias, and 55 YLD for rheumatoid ­arthritis. Most would agree that the investment by government into improving outcomes for gynecological diseases does not match that for these other higher-profile diseases with similar YLD impacts. This is an issue that requires ongoing lobbying of government by all patients, clinicians, and researchers with an interest in gynecological medicine. This book will help to raise awareness and understanding of these problems. A significant proportion of gynecological disease is directly linked to uterine pathophysiology, including heavy menstrual bleeding, endometriosis, adenomyosis, polyps, endometrial cancer, endometritis, pelvic pain, reduced uterine receptivity leading to failed implantation, and early pregnancy loss. Many of these disorders are addressed by leading experts in this book. Uterine research, and in particular endometrial research, has taken something of a backseat when compared with the advances made in improving oocyte, sperm, and embryo quality over the past 35 years. While we understood many years ago that both a viable embryo and a receptive uterus were required for successful implantation (2), much of the focus since then has been on improving embryo viability rather than uterine receptivity. The lack of investment in uterine and endometrial research is perplexing given the pressing need for improved patient outcomes both in infertility specifically and in gynecology more generally. Recently, a clinical test for uterine receptivity has been developed harnessing earlier basic studies that undertook

molecular profiling of endometrial gene expression. Future potential developments around the endometrial receptivity assay are discussed in detail in Chapter 9. The limited mechanistic and evidence-based understanding that we have of endometrial function is highlighted in two chapters toward the end of the book, where the clinical value of “endometrial scratching” to increase endometrial receptivity is questioned. Many of the current treatments for endometrial factors in subfertile patients are based on empirical approaches due to our incomplete understanding of the molecular regulation of endometrial receptivity. The chapters in this book cover advances in the ­diagnosis and treatment of uterine and endometrial factors in the management of women with gynecological disorders, including infertility. In many sections, there is an increasing focus on a personalized or individualized approach, as part of an overall vision that sees precision medicine as an integral component of healthcare (3). However, there is still much to be done before the goal of precision medicine in gynecology becomes the norm. When looking to the future, it can be instructive to look at the past as a guide to the challenges we face and the progress we might expect to make in solving them. When reviewing what we have learned about endometrial function since the advent of IVF more than 35 years ago, it is not that unfair to suggest that we have learned more about what we do not know than what we do. This comment is made in the context of the limited number of new gynecological treatment paradigms that have reached clinical practice in this time. Over the past decade, there have been important publications that have illustrated the gaps in our knowledge and, as a consequence, dramatically changed our understanding of how much more there still is to discover about the complexity of uterine function. A study that significantly altered my thinking involved genome-wide analysis of estrogen receptor binding sites (4). While the researchers used a breast cancer cell line for this work, the findings were generalizable to all sex steroid–responsive tissues. The major finding was that only 4% of estrogen receptor binding sites mapped as expected to the 1 kb promoterproximal regions of genes. In other words, 96% of in vivo estrogen receptor binding events occurred in regions previously unannotated as cis-­regulatory elements within the genome. To put this s­imply, this work published in 2006 demonstrated that our previous concept of estrogen regulation through estrogen response elements in gene promoters did not account for the vast majority of regulatory events that occur in response to estrogen. At that time, this paper emphasized to me just how little we understood about the molecular mechanisms by which the uterus responds to estrogen. vii

viii Foreword

Another fascinating insight into the complexity of endometrial function came from investigation of the evolution of pregnancy in mammals (5,6). This work initially identified 1532 genes that had been recruited into endometrial expression in the evolution of pregnancy in placental mammals. Many of these genes, including ones that mediate maternal–fetal communication and immunotolerance, contain cis-regulatory elements derived from ancient mammalian transposable elements. The authors suggest that mammalian pregnancy evolved from these DNA sequences being co-opted into hormone-responsive regulatory elements distributed throughout the genome. How and why this first occurred as a coordinated process is difficult to comprehend; however, it has resulted in a large number of endometrial genes being “rewired” to perform functions unique to reproduction and pregnancy. As a consequence of this work, we can begin to understand why gene function and pathway analysis based on data from genes in nonreproductive tissues is often of little value when trying to work out what is happening in the same genes that have been rewired for reproductive function. The third and final example of the depth of our ­ignorance is not confined to just reproductive function. Genome-wide association studies (GWASs) have played an important part in identifying genomic loci that influence phenotype, with most interest being on loci that confer increased susceptibility to complex diseases. However, approximately 88% of GWAS loci turn out to be in noncoding (intergenic or intronic) regions of the DNA (7), which begs the question as to how they exert their influence on phenotype. The fact that parts of what was previously considered “junk DNA” have functional roles has forced a reexamination of our understanding of how gene expression is regulated. If we hope to harness the full power of genetic and genomic information in precision medicine approaches, a far better understanding of the underlying molecular mechanisms is required. So, where does this newfound lack of knowledge about the uterus leave us in our pursuit of a better understanding

of endometrial function on the hoped for pathway to precision medicine? All is not bleak, and some major advances in both fundamental understanding and new treatment paradigms for gynecological disease are in the pipeline. Many of these ideas are contained in the excellent chapters in this book, which should serve to both inform and inspire readers to continue pushing the boundaries of endometrial research. REFERENCES

1. Vos T, Flaxman AD, Naghavi M et al. Years lived with disability (YLDs) for 1160 sequelae of 289 ­diseases and injuries 1990–2010: A systematic analysis for the Global Burden of Disease Study 2010. Lancet 2012;380(9859):2163–96. 2. Rogers PA, Milne BJ, Trounson AO. A model to show human uterine receptivity and embryo viability ­ following ovarian stimulation for in vitro fertilization. J In Vitro Fert Embryo Transf 1986;3(2):93–8. 3. Aronson SJ, Rehm HL. Building the foundation for genomics in precision medicine. Nature 2015;526(7573):336–42. 4. Carroll JS, Meyer CA, Song J et  al. Genome-wide analysis of estrogen receptor binding sites. Nat Genet 2006;38(11):1289–97. 5. Lynch VJ, Leclerc RD, May G, Wagner GP. Transposon-mediated rewiring of gene regulatory networks contributed to the evolution of pregnancy in mammals. Nat Genet 2011;43(11):1154–9. 6. Lynch VJ, Nnamani MC, Kapusta A et  al. Ancient transposable elements transformed the uterine regulatory landscape and transcriptome during the evolution of mammalian pregnancy. Cell Rep 2015;10(4):551–61. 7. Edwards SL, Beesley J, French JD, Dunning AM. Beyond GWASs: Illuminating the dark road from association to function. Am J Hum Genet 2013;93(5):779–97.

Contributors Lusine Aghajanova  MD PhD Department of Obstetrics, Gynecology, and Reproductive Sciences University of California, San Francisco San Francisco, California

Ioanna A. Comstock  MD Department of Obstetrics and Gynecology George Washington University Washington, DC

Myles Alderman  BS Yale School of Medicine Yale University New Haven, Connecticut

Hilary O. D. Critchley  MBCHB(Hons) MD FRCOG MRC Centre for Reproductive Health The Queen’s Medical Research Institute University of Edinburgh Edinburgh, United Kingdom

Nuria Balaguer  BS Fundación Instituto Valenciano de Infertilidad (FIVI) Valencia University and Instituto Universitario IVI/INCLIVA Valencia, Spain

Francisco Dominguez  PhD Fundación Instituto Valenciano de Infertilidad (FIVI) Valencia University and Instituto Universitario IVI/INCLIVA Valencia, Spain

Giuseppe Benagiano  MD PhD FACOG FICOG FRCOG Department of Gynecology, Obstetrics, and Urology University of Rome Rome, Italy

Beatrice Ermini  MD Department of Gynecology, Obstetrics, and Urology University of Rome Rome, Italy

Siladitya Bhattacharya  MD FRCOG Institute of Applied Health Sciences School of Medicine, Medical Sciences and Nutrition University of Aberdeen Aberdeen, United Kingdom Christophe Blockeel  MD PhD Centre for Reproductive Medicine Universitair Ziekenhuis Brussel, Vrije Universiteit Brussel Brussels, Belgium Claire Bourgain  MD PhD Department for Pathology University Hospital Leuven Leuven, Belgium and Department for Pathology Imelda Hospital Bonheiden, Belgium Mats Brännström  MD PhD Department of Obstetrics and Gynecology University of Gothenburg Gothenburg, Sweden and Stockholm IVF Stockholm, Sweden

Gulcin Sahin Ersoy  MD Yale School of Medicine Yale University New Haven, Connecticut Shavi Fernando  MBBS(hon) BMedSc(hon) FRANZCOG The Ritchie Centre Department of Obstetrics and Gynecology Monash University and Hudson Institute of Medical Research and Monash Health Department of Obstetrics and Gynaecology Clayton, Australia Linda C. Giudice  MD PhD Department of Obstetrics, Gynecology, and Reproductive Sciences University of California, San Francisco San Francisco, California John Groth  MD PhD Department of Pathology University of Illinois at Chicago Chicago, Illinois

Ivo Brosens  MD PhD FRCOG Leuven Institute for Fertility and Embryology Leuven, Belgium

Marwan Habiba  PhD FRCOG Department of Health Sciences University of Leicester Leicester, United Kingdom

Serdar E. Bulun  MD Department of Obstetrics and Gynecology Prentice Women’s Hospital Northwestern Memorial Hospital Chicago, Illinois

Heather Huddleston  MD Department of Obstetrics, Gynecology, and Reproductive Sciences University of California San Francisco, California ix

x Contributors Deborah E. Ikhena  MD Division of Reproductive Endocrinology and Infertility Department of Obstetrics and Gynecology Prentice Women’s Hospital Northwestern Memorial Hospital Chicago, Illinois Sun H. Kim  MD MS Department of Medicine Stanford University School of Medicine Stanford, California

Natasha Pritchard  MBBS The Mercy Hospital for Women Heidelberg, Australia Terhi T. Piltonen  MD PhD Department of Obstetrics and Gynecology PEDEGO Research Unit Medical Research Center Oulu University Hospital University of Oulu Oulu, Finland

Graciela Krikun  PhD Yale School of Medicine Yale University New Haven, Connecticut

Molly Quinn  MD Department of Obstetrics, Gynecology, and Reproductive Sciences University of California San Francisco, California

Sanja Kupesic Plavsic  MD MSc PhD Department of Obstetrics and Gynecology Paul L. Foster School of Medicine Texas Tech University Health Sciences Center El Paso El Paso, Texas

Peter A.W. Rogers  PhD Department of Obstetrics and Gynaecology University of Melbourne and Royal Women’s Hospital Melbourne, Australia

Ruth B. Lathi  MD Department of Obstetrics and Gynecology Division of Reproductive Endocrinology Stanford University School of Medicine Stanford, California

Luk Rombauts  MD PhD FRANZCOG CREI Department of Obstetrics and Gynaecology Monash University and Group Medical Director, Monash IVF Group and Head of Reproductive Medicine, Monash Health and Vice President, Fertility Society Australia and Board Member, World Endometriosis Society Melbourne, Australia

Nick S. Macklon  MD PhD Department of Obstetrics and Gynaecology Zealand University Hospital University of Copenhagen Copenhagen, Denmark Dana McQueen  MD MAS Department of Obstetrics and Gynecology University of Illinois at Chicago Chicago, Illinois Jose Miravet-Valenciano Igenomix Parc Cientific Valencia University Valencia, Spain Monica Modi  MD Yale School of Medicine Yale University New Haven, Connecticut Inmaculada Moreno  R&D Department Igenomix Valencia, Spain

PhD

Hanyia Naqvi  MS BS Yale School of Medicine Yale University New Haven, Connecticut

Elham Neisani Samani  MD Yale School of Medicine Yale University New Haven, Connecticut Samuel Santos-Ribeiro  MD Centre for Reproductive Medicine Universitair Ziekenhuis Brussel, Vrije Universiteit Brussel Brussels, Belgium and Department of Obstetrics, Gynaecology, and Reproductive Medicine Hospital Universitário de Santa Maria Lisbon, Portugal Benjamin J. Seifer Yale School of Medicine Yale University New Haven, Connecticut

Contributors xi Carlos Simón  MD PhD Igenomix S.L. and Professor of Obstetrics and Gynecology Universidad de Valencia Instituto Universitario IVI/INCLIVA Valencia, Spain

Felipe Vilella  PhD Fundación Instituto Valenciano de Infertilidad (FIVI) Valencia University and Instituto Universitario IVI/INCLIVA and Department of Pediatrics, Obstetrics/Gynecology Valencia School of Medicine Valencia, Spain

and

Lucy Whitaker  MBChB MSc MRCOG MRC Centre for Reproductive Health The Queen’s Medical Research Institute University of Edinburgh Edinburgh, United Kingdom

Department of Obstetrics and Gynecology School of Medicine, Stanford University Stanford, California Mary Stephenson  MD MS Department of Obstetrics and Gynecology University of Illinois at Chicago Chicago, Illinois Hugh S. Taylor  MD Yale School of Medicine Yale University New Haven, Connecticut

Highlights on the dynamism and multifunctionality of human endometrium in the era of precision medicine

1

LINDA C. GIUDICE

INTRODUCTION

Precision medicine is an emerging field for disease prevention, classification, diagnosis, and targeted (individualized) treatment based on individual characteristics, including genetic variations, environmental exposures, lifestyle, and experiences across the life span (1). It derives from big science (“omics”), bioinformatics, and deep phenotyping. Big science includes the genome, epigenome, transcriptome, metabolome, microbiome, exposome, receptome, and other “omes” in which global profiling captures huge numbers of data points (e.g., whole genome sequencing). Bioinformatics is the interdisciplinary field that combines statistics, mathematics, engineering, and computer science to interpret biological data. Deep phenotyping involves extensive patient characteristics (e.g., age, body mass index (BMI), smoking, environmental exposures, zip code, socioeconomic status, pregnancy history, comorbidities, severities, symptoms, family history, and lifestyle) that get incorporated via bioinformatics to interpret findings of the big data. The end result is to diagnose and understand diseases at the individual level so that therapies are based on cross-referencing an individual’s personal history and biology with patterns found across large populations, utilizing the knowledge network to deliver care that is preventive, targeted, effective, and timely. The field is ideal for women’s health, where causes for most disorders are not well understood and there is great variability woman to woman in responses to treatments and also in outcomes. This is particularly true for the female reproductive tract and especially the endometrium, where there are unpredictable responses to the steroid hormone milieu and other factors that result in poor reproductive and other health outcomes. A long-term goal is to abandon empiric and “one-size-fits-all” therapies and adopt precision in fertility treatments, contraceptive and hormonal therapies, treating disorders of the female reproductive tract, and optimizing pregnancy outcomes. This chapter highlights the human endometrium in this context in the dawn of the age of precision medicine. Human endometrium (modern Latin, from endo [“within”] + Greek mētra [“womb/uterus”]) (2), the anatomic prerequisite for continuing the species and a sentinel for protecting the upper female reproductive tract against mucosal infection, is a dynamic tissue responding

cyclically to changing circulating ovarian-derived estradiol (E2) and progesterone (P4) (Figure 1.1). It undergoes cyclic cellular proliferation, differentiation, lymphangiogenesis, immune cell trafficking, and, in the absence of pregnancy, tissue desquamation and hemostasis, followed by regeneration (3). The goals of orchestrated events in endometrium are to permit successful nidation, growth, and survival of the semiallogeneic conceptus for 9 months, followed by involution and subsequent regeneration postpartum (4). Also, it has a major responsibility as a mucosal surface to protect the organism from infections by a variety of microbes (5). Abnormalities in one or more mechanisms underlying endometrial functionality during development, during a women’s reproductive years, during pregnancy, and postmenopause have profound consequences for fertility, pregnancy outcome, and women’s health more broadly. Hardly an “innocent bystander,” the tissue is affected, directly or indirectly, by systemic homeostasis, disease, stress, environment, nutrition, genetics, and hormonal status (age, endogenous, and exogenously administered). Herein, highlights of this remarkable tissue’s origin, physiology, metabolism, evolution, ecology, and plasticity are provided, with an eye to precision medicine, and serving as a backdrop to chapters that follow wherein some topics are presented in more detail. Also, it is hoped that the reader will be inspired to consider the complexities of the human endometrium in clinical practice and women’s well-being and in research in an effort to advance women’s reproductive health beyond empirical therapies, unpredictable endometrial responses, and unpredictable p ­ rognoses for contraception, fertility, pregnancy o ­ utcomes, and endometrial morbidities. EMBRYOLOGY AND VULNERABILITY TO ENDOCRINEDISRUPTING CHEMICALS

The uterus derives from the paramesonephric ducts first described by Muller in 1830 (6). The mesodermally derived paramesonephric (Mullerian) ducts differentiate into the luminal and glandular epithelial cell types of the endometrium, oviduct, cervix, and upper vagina, and the surrounding urogenital ridge mesenchyme differentiates into the endometrial stroma and inner and outer myometrial layers (7). Interactions between the mesenchyme 1

2  Highlights on the dynamism and multifunctionality of human endometrium in the era of precision medicine

P Uterine cycle

E2

Menses

Menses

LH + 6–10 1

14

Menstrual phase

28

WOI

LH surge Proliferative phase

Cellular proliferation growth angiogenesis Postmenstrual repair (E2 independent)

2 mm

11 mm

Secretory phase Glandular secretion

Stromal decidualization

Immune cell proliferation migration

14 mm

Figure 1.1  Circulating hormonal profiles of estradiol (E2) and progesterone (P4) and the endometrial response across the menstrual cycle phases and substages. Endometrial growth and patterns in early and late proliferative and midsecretory phases on transvaginal ultrasound are shown in panels A–C. (From Giudice LC. Reprod Biol Endocrinol 2006;4(Suppl 1):S4. With permission.) and epithelium are essential for female reproductive tract formation and differentiation, and members of the Wnt and Hox gene families are important in these processes. In addition, mesenchymal and epithelial interactions and estrogen receptor (ER) signaling play important roles in adenogenesis and smooth muscle differentiation (7,8). The developing female reproductive tract is highly vulnerable to estrogens and estrogen mimetics (9), and the human experience with diethylstilbesterol (DES) and several experimental studies in animals demonstrate longterm adverse reproductive effects in adults with in utero exposure to this endocrine-disrupting chemical (EDC). In humans, in utero DES exposure causes abnormal vaginal adenosis and uterine structural abnormalities in DES daughters (10). Similar results have been found in mice with in utero exposure to bisphenol A (BPA), another EDC structurally similar to DES and also signaling through the ERα pathway (11). In utero DES exposure results in changes in expression of WNT 7a, HOXA 10, and HOXA11 and altered uterine morphogenesis in female offspring (12,13), as well as causing epigenetic alterations in the uterus (hypermethylation of the HOXA 10 promoter and overexpression of DNA methyl transferases in the mouse (14)). Altered DNA methylation of HOXA 10

and ERα promoters is a recently recognized mechanism of BPA-induced altered reproductive tract developmental programming (15). It has been proposed that some abnormalities in ER-mediated events during female reproductive tract development may have their origins in various environmental (e.g., endocrine disrupters) or genetic factors, resulting in altered critical gene expression in stem or progenitor cells, giving rise to persistent abnormalities in the endometrium of adult women (16). Some examples include hypermethylation of HOXA 10 and hypomethylation of steroidogenic factor (SF) 1 and ERβ promoters, and progesterone receptor (PR) genetic variants in endometriosis (16). Thus, the endometrium and other components of the female reproductive tract are highly vulnerable to EDC exposure in utero, with potentially long-lasting effects on reproductive potential in adulthood that should be considered in preventive health strategies and optimizing reproductive health in the long term. HORMONAL CONTROL

Endometrium comprises several cell types, including glandular epithelium, luminal epithelium, stromal fibroblasts, vascular smooth muscle, and endothelium; an array of immune cells; and stem or progenitor cells (4).

Histology 3

The glandular and stromal compartments undergo profound changes in response to circulating E2 and P4 across the cycle (Figure 1.1). A brief description of these changes is included here to underscore the events and to highlight how abnormalities can profoundly impact normal hormone responsiveness and thus function and dysfunction of the tissue. Also, abnormal uterine bleeding and “breakthrough” bleeding accompanying a variety of contraceptive steroids or (peri-)menopausal hormone therapy reflect endometrial dysfunctionality in ways that are sometimes unpredictable. While we have not yet achieved the goals of precision medicine to define endometrial abnormalities in the context of genetics, epigenetics, and environmental factors, this is a challenge for more personalized care of common women’s health issues related to the endometrium, as well as fertility potential and pregnancy ­outcomes. It is with this lens that the normal histology gains a new perspective. HISTOLOGY

In the early proliferative phase (days 4–7) (Figure 1.2) (17), the surface epithelium has short, straight glands, the stroma is compact, and there is minimal mitotic (a)

activity in both compartments. By the midproliferative phase (days 8–10), the surface epithelium is columnar and there are longer curving glands, variable stromal edema, and numerous mitoses. In the late proliferative phase (days 11–14), there is extensive mitotic activity resulting in undulation of the surface epithelium, increasingly coiled glands and blood vessels, and dense stroma (18). These changes occur largely in response to increasing circulating levels of E2. In concert with the latter are increases in ERs (mainly ERα) in nearly all cells in the tissue, peaking in the late proliferative phase, with concomitant increases in PR (mainly progesterone receptor A [PRA]) (19). With the onset of and shortly after the luteinizing hormone (LH) surge, circulating E2 begins to decrease, and P4 increases, the latter inhibiting cellular proliferation and inducing “secretory activity” of the glands and differentiation (decidualization) of the stromal fibroblasts (20). Interval endometrium (day 15) looks histologically much like that of the late proliferative phase, but with some scattered subnuclear vacuoles in the glands. By days 16–17, ER and PR begin to decrease and Ki-67 mitotic activity is reduced (19), reflecting the initiation of the proliferative to secretory transition. By day 17, the glands exhibit

(b)

Early proliferative (days 4–7)

(d)

(c)

Midproliferative endometrium Ki-67 staining (e)

Late proliferative (days 11–14)

(f )

Day 17 endometrium, with reduced Ki-67 staining and tortuous glands (g)

(h)

Days 20–21: maximum secretion

(i)

Days 23–24

(j)

Day 25

Day 28

Figure 1.2  Histology of the endometrium across the menstrual cycle. (a) Early proliferative (days 4–7). (b) Midproliferative endometrium Ki-67 staining. (c) Late proliferative (days 11–14). (d–f) Day 17 endometrium, with reduced Ki-67 staining and tortuous glands. (g) Maximal secretion (days 20–21). (h) Days 23–24. (i) Day 25. (j) Day 28. (Panels b and e from Ferenczy A, Mutter G. The endometrial cycle. Carlisle, UK: Global Library of Women’s Medicine; 2008. With permission. Panels c and f from http://www.pathologyoutlines. com/topic/uterusdating.html. With permission. All others from the University of California–San Francisco National Institutes of Health Human Endometrial Tissue and DNA Bank, courtesy of Juan C. Irwin.)

4  Highlights on the dynamism and multifunctionality of human endometrium in the era of precision medicine

regular tortuosity with subnuclear glycogen vacuoles that transition to luminal vacuoles, and by day 19, intraluminal secretion commences, maximally occurring on days 20–21 (20) (Figure 1.2). Remarkably, glycogen metabolism is almost nonexistent in the proliferative phase and increases beginning in the early secretory phase and is maximal by days 20–21 (21). The early secretory phase glycogen metabolism is compromised (decreased glycogen synthetase and phosphorylase) in women with infertility (21), reflecting perhaps P4 resistance during the proliferative-secretory transition, which is also a time of P4 resistance in endometrium of women with endometriosis (22). The metabolome of human endometrium across the cycle has yet to be defined, although a recent preliminary report has described the metabolome and lipidome of exosomelike vessels (ELVs) derived from uterine fluid and plasma from women with and without endometrial cancer (23). This study demonstrated a high percentage of phospholipids, peptides, and nucleotides within ELVs that differed in women with and without disease. This approach offers promise to defining the metabolome of human endometrium in other endometrial disorders and also across the cycle in normal women to determine potential prognostic and diagnostic profiles for endometrial health, fertility, and pregnancy outcomes in the future. On day 22, there is maximal stromal edema, and days 23 and 24 are characterized by prominent spiral arterioles and perivascular decidualization (20). By days 23–24, the glands begin to show regressive changes, and by day 25, subepithelial decidualization is prominent, with stromal confluence and an influx of lymphocytes by day 26. By day 27, there are prominent stromal granulocytes and focal necrosis and hemorrhage. On day 28, glandular or stromal breakdown and tissue necrosis and hemorrhage occur, with stromal and glandular “exhaustion,” intravascular fibrin thrombi, and hemorrhage. In the absence of implantation, orderly shedding of the tissue and hemostasis are essential to prevent morbidities of hypermenorrhea, menorrhagia, and anemia. Multiple factors can contribute to disrupted endometrial signaling pathways, cell–cell communications, and biological performance, which warrant identification through big data, bioinformatics, and deep phenotyping. IMMUNE CELLS

The endometrium is a tissue in which the immune and endocrine systems directly interact (5), although interactions with the environment are relatively unexplored. Components of the endometrial immune system are directly regulated by E2 and P4 and have been adapted to achieving the endometrium’s two major goals: successful pregnancy and defense against sexually transmitted pathogens. Lymphocytes comprise a large proportion of endometrial leukocytes, with natural killer (NK) cells and T cells representing the two major subsets, about 25% and 35%–50%, respectively (24). T cells decrease slightly in the secretory versus proliferative phase, and neutrophils, dendritic cells, and B cells (the latter two in low abundance) do

not display cycle dependence (24). However, uterine NK (uNK) cells increase across the secretory phase, especially midcycle, and are important in receptivity, angiogenesis, and pregnancy success, and macrophages influx dramatically during the late secretory phase, in preparation for menses (24). In the proliferative phase, E2 stimulates stromal fibroblast hepatocyte growth factor (HGF), CCL2, and interleukin (IL)-8, which are chemotactic to T cells, and secretory leukocyte protease inhibitor (SLPI), elafin, and beta defensins (HBD2) in epithelial cells for antiviral and antibacterial activity. The effects of E2 and P4 on epithelial cells, fibroblasts, and immune cells and the specific pathogen-killing and inactivation properties are shown in Figure 1.3. The innate immune system is critical as the first line of defense to infection and consists of mechanical, chemical, and cellular components (25,26). The mechanical barrier includes a mucus lining secreted by the epithelium and overlying it, preventing direct contact with microbes, as well as tight junctions between cells that ensure mucosal integrity. The chemical barrier comprises natural antimicrobial peptides (e.g., defensins, elafin, SLPI, lactoferrin, and lysozyme), pattern recognition receptors (e.g., toll-like receptors), and cytokines or chemokines (25,26). The adaptive immune system involves TH1 cell–mediated and TH2 humoral components (25). As the innate and adaptive mucosal immune systems in the endometrium are hormonally driven, the question arises about the effects of, e.g., estrogen mimetics or EDCs, extremes of E2 and P4 in stimulation cycles for fertility, as well as contraceptive steroids that may alter immune defenses against sexually transmitted infections (27). Regarding the latter, Wira and colleagues (5) have postulated that there is a window of vulnerability that corresponds to the window of implantation (WOI) during which immune defenses are altered for successful embryo implantation at the concomitant expense of increased risk of sexually transmitted infections. Furthermore, while human endometrium and placenta traditionally have been considered sterile, challenges to this concept have been raised recently (Chapter 11 (28)) and the role of the endometrial immune defense in this setting is important. The endometrial microbiome is an emerging concept that warrants consideration in fertility assessment and prognostics for fertility potential and pregnancy outcomes, in addition to potential roles in endometrial disorders more broadly. Opportunities for specific immune modulation to minimize pathogens in unique hormonal settings is wanting for better understanding of patient-to-patient variability in immune responses due to genetics, lifestyle, or environmental exposures. DECIDUALIZATION

Progesterone induces endometrial stromal fibroblasts to decidualize (i.e., differentiate) to cells with characteristic morphology, transcriptome, and secretory profiles (29,30). Decidualization is initiated independent of pregnancy in humans and is a master regulator with functions in

Decidualization 5 Immune function Pathogen killing and inactivation

Lumen

CCL20, HBD2, SLPI, elafin, IgA and IgG (plgR and FcRn)

Columnar epithelial cells

Macrophage

Macrophage

T-cell NK-cell

Lymphoid aggregate

Macrophage

T-cell

T-cell NK-cell

Lymphoid aggregate

Macrophage CX, CL1, CCL2, IL-8, HBD2 CCL20, SLPI, elafin, TGFβ

Epithelial cells

Immune cell recruitment, innate immune protection and tolerogenic nature

Fibroblasts

Immune cell recruitment Regulation of epithelial cells



CXCL12



HGF

Immune cells

Pathogen killing

↓ ↓

CD8+ CTL activity NK-cell activity (IFNγ) CD163+ macrophages



CD4+ T cell susceptibility

Protection against viruses Protection against bacteria Infection by HIV



Figure 1.3  Immune functions, cells, and effector molecules involved in pathogen killing and inactivation in human endometrium (see text). (From Wira CR, Rodriguez-Garcia M, Patel MV. Nat Reviews Immunol 2015;15:217–30. With permission.)

cycling endometrium, as well as in pregnancy. In cycling endometrium, it governs coordinated tissue differentiation for normal menstrual shedding and tissue regeneration in the absence of pregnancy. In conception cycles, it is essential for early pregnancy establishment (attachment and invasion), normal placentation, vascularization, immune tolerance, pregnancy maintenance, and outcome (30). As noted above, decidualization begins in the midsecretory phase in stromal fibroblasts surrounding the terminal spiral arteries and located subepithelially, and in the late secretory phase, there is stromal confluence. These decidualized stromal fibroblasts create unique niches for angiogenesis and leukocyte recruitment in endometrium preparing for embryo implantation, immune tolerance, and increased blood supply to support a conceptus (Figure 1.4), and some of the players involved are shown in Table 1.1 (31,32). Recent studies have shown the evolutionary importance of regulatory transposable DNA elements that govern the P4 and phosphokinase A (cyclic AMP) signaling pathways driving the decidualization process and expression of key decidua-specific genes, including FOX01, C/EBPb, HOXA10, HOXA11, PRL, and IGFBP1 (33,34). These elements are only present in placental mammals and are believed to have enabled the ability of

the endometrium to accommodate the deep trophoblast invasion that occurs in humans (35). During the decidualization process (35), there is a pro-inflammatory profile, which is followed by an anti-inflammatory response and downregulation of several chemokines and inflammatory mediators (36), some of which prevent effector T cells from entering the decidua (37). In parallel, continued P4 signaling upregulates 11β-hydroxysteroid dehydrogenase type I (11βHSD1), which converts cortisone to cortisol (38), and it has been postulated that there is a cortisol gradient that provides an implanting conceptus protection from maternal immune rejection (39). Human endometrial stromal fibroblasts express multiple steroid hormone receptors, including ER, PR, the androgen receptor (AR), as well as glucocorticoid (GR) and mineralocorticoid (MR) receptors (38). Activation of the P4-11βHSD1-MR axis induces expression of enzymes involved in retinoic acid biosynthe­sis and storage and lipid metabolism, which have been proposed to be important to support embryo development before placental perfusion occurs in the late first trimester (40). The effects on these ­processes of, e.g., maternal and fetal genetic variants, epigenomes, metabolism, and the microbiome, have yet to be explored and may play a major role in fertility potential and p ­ regnancy outcomes.

6  Highlights on the dynamism and multifunctionality of human endometrium in the era of precision medicine (a) Epithelium basement membrane

Blastocyst adhesion

Syncytiotrophoblast

Invasion

Cytotrophoblast

Villus formation

Interstitial trophoblasts

DSC DIC Endovascular trophoblasts Endometrial gland

DEC

Spiral arteries

Macrophage Spiral artery

T cell NK cell

DSC: decidual stromal cell DIC: decidual immune cell DEC: decidual epithelial cell

(b)

Villous cytotrophoblast cells (vCT)

Cytotrophoblast

CXCL14 Extravillous cytotrophoblast cells (EVT)

Syncytiotrophoblast cells CCR3 CCR6 decoy receptors

CCR1 CCL17 CXCR7

CXCR4 Cell column CXCR1 CXCR3 decoy receptors

CX3CR1

CCL2 CCL4 CCL7 CCL14 CCL16 CCL17 CXCL10 CXCL14 CX3CL1 CCR3 CCR10 DSC

CCR10 CCR4 iEVT

DEC

CX3CL1 CCL14 CCL7 CCL16 CCL4

CCR2 CCR5 CXCR6 10%–15% CCR2 CCR4 CCR5 CXCR6 50%–70% CCR2 CCR5 CXCR3

Macrophage 10%–15% DIC

T cell NK cell

Figure 1.4  Events at the maternal–fetal interface in human pregnancy. (a)  Dynamic process of forming the maternal–fetal i­nterface in early human pregnancy. (b) Chemokines and chemokine receptors at the human maternal–fetal interface in mid–late first trimester. (From Du M-R, Wang S-C, Li D-J. Cell Mol Immunol 2014;11:438–48. With permission.)

WINDOW OF IMPLANTATION, NIDATION, AND UTERINE PLASTICITY

In humans, the receptive phase for embryo nidation occurs on average from cycle days 20 to 23 (41). It is characterized histologically and metabolically as described above. While this WOI is a temporally and spatially defined time in the endometrial cycle in which blastocyst implantation can begin, events prior to it are critical in optimizing endometrial receptivity to embryonic implantation in this time frame. In addition, events post the implantation window in a conception cycle reflect extensive paracrine cross talk between the decidual fibroblasts and immune cells and

the invading placental trophoblasts ((42), Chapter 12 in this book, and Figure  1.4). Miscommunication between the endometrium and the implanting embryo can lead to miscarriage and other poor pregnancy outcomes. How to improve receptivity and the maternal–placental dialogue to optimize pregnancy outcomes has long been a topic of great interest, although this goal is yet to be achieved. Recently, however, the endometrial receptivity array (ERA), based on endometrial transcriptomics and highdimensional data analysis (43,44), has demonstrated the personalized optimal timing for embryo transfer after in vitro fertilization (IVF) and those whose receptivity is not

Window of implantation, nidation, and uterine plasticity  7

Table 1.1  Functions of decidualized endometrial stromal fibroblasts • Secrete chemokines that • Directionally stimulate immune cell adhesion, migration (e.g., uNKs) • Promote T-cell activation/differentiation • Are potent mediators of angiogenesis (e.g., IL-8) • Secrete cytokines and other modulators of the immune response • IL-17 (recruit Th17 cells → proliferation and invasion of TB) • IDO and PGE2 inhibit NK killer activity and dendritic cell differentiation • IL-15, IL-11, and IL-33 increase uNK proliferation and differentiation • Express and secrete miRNAs that regulate gene expression • Secrete unique biomarkers and modulators (IGFBP-1, PRL) • Secrete a unique extracellular matrix Note: TB, trophoblast; IDO, indoleamine 2,3-dioxygenase; PGE2, prostaglandin E2.

optimal (Chapter 9), with embryo transfer (ET) in artificial cycles with a receptive ERA profile. In natural or stimulated cycles with abnormalities in the WOI, therapies to mitigate these comprise an area of great promise for future investigation and in the context of precision medicine. The WOI is one of the most highly investigated stages of human endometrium. It is well known that it demonstrates plasticity in that it can be delayed or advanced,

depending on the hormonal milieu (45). A relatively new and provocative hypothesis about uterine plasticity and reproductive fitness has recently been put forward by Lucas and colleagues (46). They cite the high incidence of chromosomally abnormal and developmentally compromised preimplantation embryos in humans (47) and the teleological need for the maternal response to protect against prolonged investment in supporting invasive but developmentally abnormal embryos. They propose five stages (Figure 1.5). In “preparation,” spontaneous decidualization, described above, is first characterized by a proinflammatory profile, followed by an anti-inflammatory profile, and a proposed cortisol gradient purportedly providing further protection of the invading trophoblast against maternal immune rejection. The second phase of “counterattack” includes encapsulation of the embryo, once it has breached the epithelium, by decidualized stromal fibroblasts, which then leads to “quality control”—i.e., identification of a suitable or unsuitable embryo, and if the latter is detected, then “rejection,” along with shedding and menstruation, as in a nonconception cycle. The final stage involves adult endometrial stem cells in their perivascular niche for “regeneration” of the tissue for a more suitable conceptus the next time around. It is proposed that pregnancy failure “should be the norm” in healthy couples, and that reproductive success is a testimony to the plasticity of the uterus and endometrium (46). While an attractive hypothesis, signals of embryo quality recognition by the decidual stromal cells have yet to be determined and offer

Immune system Complement system Innate Metabolism Biosynthesis Cholestrol Amino acids Fatty acids PGs/prostanoids Acetyl CoA Steroids Transporters

DNA synthesis Cell division Angiogenesis Ion channels Cell adhesion

Inflammation NK cells

Detox Antioxidants Metallothionines Cell-cell comm. Secretory products Response to chemicals environment wounding stress

Matrix Matrix degrading enzymes and their inhibitors CeII-matrix

LH surge

Regeneration Proliferative phase

Secretory phase

Cellular proliferation Growth Angiogenesis

Glandular secretion

Stromal decidualization

Menstrual phase

Immune ceII proliferation migration

Figure 1.5  Summary of events occurring across the menstrual cycle, deduced from microarray analyses. (From Giudice LC. Reprod Biol Endocrinol 2006;4(Suppl 1):S4. With permission.)

8  Highlights on the dynamism and multifunctionality of human endometrium in the era of precision medicine

great promise in enhancing reproductive performance, fertility, and positive pregnancy outcomes. MENSTRUATION AND REGENERATION

Menstruation only occurs in women and other primates (mainly old world monkeys, the elephant shrew, and bats) (48)—species where endometrial decidualization occurs spontaneously (49). The process of menstruation, refreshing and renewing the endometrium for conception, and the length of human pregnancy have been the subject of much research, and the question has arisen as to why menstruation occurs rather than tissue resorption, as in other mammals. Strassman (50) has proposed that cyclical regression and renewal are energetically less costly than maintaining the endometrium in the metabolically active state required for embryo implantation, and remarkably, in the regressed state, oxygen consumption (per milligram of protein per hour) in human endometrium declines nearly sevenfold (50). In addition, a woman’s metabolic rate is about 7% lower during the proliferative phase than during the secretory phase, reflecting an estimated energy savings of 53 MJ over four cycles, or nearly 6 days’ worth of food. Thus, it has been suggested that the menstrual cycle, with its proliferative, secretory, and menstrual phases, economizes the energy costs of reproduction (50). Hallmarks of menstruation are the influx of leukocytes, tissue degradation, and tissue regeneration (49). In the absence of a conceptus to maintain the corpus luteum and its production of E2 and P4, changes occur in the tissue as the levels of these steroid hormones decline. P4 withdrawal triggers release of matrix-degrading enzymes from the decidualized stromal fibroblasts (51), accompanied by loss of blood vessel and interstitial stromal extracellular matrix integrity. Leukocytes (macrophages mostly) and their proinflammatory cytokines and chemokines are key players for leukocyte recruitment and tissue shedding, as well as hemostasis involving cyclooxygenases and prostaglandins (49). Shedding and repair occur simultaneously at adjacent sites, with about two-thirds of the fuctionalis of shed and repair occurring in the absence of blood clotting or scarring. One of the biggest challenges in g­ ynecology is abnormal uterine bleeding, for which there is yet to be a “silver bullet” for management. However, there is much promise for personalized approaches in the dawn of p ­ recision medicine for specific and targeted therapies for individual women. Stem or progenitor cells of epithelial, mesenchymal, and endothelial origin all likely contribute to rapid endometrial cyclic growth and regeneration (52). Reepithelization of the endometrium starts immediately after the onset of menses (53). The remaining stumps of endometrial glands after endometrial breakdown proliferate rapidly, forming marginal collars. By cycle day 6, this proliferative process results in a continuous layer of fusiform cuboidal epithelial cells that cover the entire endometrial surface of the uterine cavity. Which tissue and extra-tissue stem cells contribute to total reconstitution of the endometrium is still the subject of debate (52). That said, the dynamics of

epithelial growth is rapid, and ciliogenesis occurs in some surface endometrial cells (53). Much research has focused recently on endometrial stem cells as cell therapeutics for multiple disorders (52,54,55). The endometrial mesenchymal stem cells (eMSCs), in particular, have properties of other mesenchymal stems or progenitors with regard to evading the immune system, and thus heterologous transplantation without immunosuppressants is an attractive therapeutic strategy (55). Some disorders being proposed include direct use of eMSCs for pelvic organ prolapse (56) and Asherman’s syndrome (57), and differentiation of eMSCs down various lineages to treat type I diabetes (58) and Parkinson’s disease (59) in animal models. Cell therapeutics involving endometrial stem cells is less invasive than bone marrow-derived mesenchymal stem cells, e.g., and offers great promise for a variety of applications, although the safety of these approaches needs validation before adapting them to humans. GLOBAL CHANGES

Functional genomics has enabled global assessment of gene expression in endometrial tissue in a changing hormonal milieu, leading to further insight into biological processes and signaling pathways in health and disease (3,60–63) (Figure 1.6). In the proliferative phase, genes involved in DNA synthesis and cell cycle regulation, ion channels, cell adhesion, and angiogenic factors are expressed. After the LH surge, different profiles are uniquely observed in the early, mid-, and late secretory phases. The early secretory phase is notable for upregulation of multiple genes and gene families involved in cellular metabolism, steroid hormone metabolism, and secreted glycoproteins. The midsecretory phase is characterized by multiple biological processes, including upregulation of genes encoding secreted glycoproteins, immune response genes (innate immunity), and genes involved in detoxification mechanisms key to the receptivity of an implanting embryo. In the late secretory phase, in the setting of declining E2 and P4, as the tissue prepares for desquamation, there is a marked upregulation of an inflammatory response, along with matrixdegrading enzymes and genes involved in hemostasis. This approach has validated histological evaluation of the endometrium and has added a dimension of pathways and biological processes involved (62). ENDOMETRIAL ECOLOGY

Ecology is the branch of biology that deals with the relations of organisms to one another and to their physical surroundings (2). Traditionally, the endometrium is described as a “steroid hormone-responsive” tissue with a focus on its responses to ovarian-derived E2 and P4, However, the tissue exists in a milieu of multiple other effectors, including genetics, systemic homeostasis (e.g., endocrine system and immune system), disease, and stress—nutritional, psychosocial, and environmental— all of which can affect ovarian function and perhaps

References 9 Preparation Counter-attack Quality control Regeneration Rejection

Cycle day 17

19

21

23

25

27

1

Secretory phase

3

5

Menstruation

7

9

11

13

15

Proliferative phase

Figure 1.6  Various stages of embryo implantation, including quality control (see text). (Reproduced from Lucas ES, Salker MS, Brosens JJ. Reprod Biomed Online, 2013;27:506–14. http://dx.doi.org/10.1016/j.rbmo.2013.06.012. With permission.)

regulate, directly or indirectly, endometrial function. The question arises about how much endometrial function is independent of the ovaries (64) programmed into its stem cells or modified by environmental cues? Hypothalamic amenorrhea, e.g., can affect endometrium indirectly, and hyperinsulinemia can affect it directly, as there are insulin and insulin-like growth factor (IGF)-1 receptors in the tissue with known effects on cellular function ((3), Chapter 5 of this book). In addition, with similar circulating levels of E2 accompanying controlled ovarian stimulation, endometrial thickness and pattern vary widely among women, without a direct dose–response relationship (65), and in postmenopausal women, endometrial thickness varies with BMI (66). Of note, there are inter- and intrapopulation variations in ovarian steroid hormone levels, related to energy expenditure, energy balance, nutritional status, and developmental conditions that can affect endometrial functionality ((64), review). Clinical conditions affecting endometrial function and pregnancy outcomes include endometrial polyps, uterine septum, infection, scarring, anomalies, infertility therapies, metabolic syndrome, and in utero EDC exposures. In addition, immune stress and psychosocial stress play roles in fertility, fecundity, and pregnancy outcomes, with inflammation as a common underlying process (64). It is remarkable that inflammation (systemic and endometrial based) is not infrequent in women with endometrial disorders (67)— e.g., endometritis, endometriosis, uterine fibroids, polycystic ovarian syndrome, obesity, hydrosalpinges, and adenomyosis—all of which have dedicated chapters in this book. The potent role of inflammation in endometrial disease, fertility, and pregnancy outcomes (68) warrants future assessment of patient-specific risk factors for inflammation in women with poor endometrial-based reproductive performance and endometrial disorders.

CONCLUDING REMARKS AND FUTURE PERSPECTIVES

A normally functioning endometrium is essential for reproductive success and the well-being of women. In its multiple roles, including quality control for implantation and pregnancy, a huge energy commitment for a long gestation, orderly development, and synchronized processes resulting in tissue homeostasis for more than 400 cycles in a woman’s lifetime, and as a sentinel for upper reproductive tract infections, it is important to consider this tissue as a master regulator of reproductive performance and women’s health. It has been suggested that ecology, ovarian function, and age are likely to be major determinants of endometrial variation, function, and reproductive success (64). So, what is the role of genetics, epigenetics, metabolomics, the microbiomes of the gut and reproductive tract, and other omics in endometrial responsiveness to ovarian hormones and independent of ovarian hormones? I posit these will be elucidated with deep phenotyping clinically and big data from large numbers of women across the life span and across the globe. Indeed, precision medicine holds great promise to optimize women’s reproductive health and women’s health more broadly in the foreseeable future for current and future generations. REFERENCES

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10  Highlights on the dynamism and multifunctionality of human endometrium in the era of precision medicine

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19. Konishi I, Fujii S, Nonogaki H et al. Immunohistochemical analysis of estrogen receptors, progesterone receptors, Ki-67 antigen, and human papillomavirus DNA in normal and neoplastic epithelium of the uterine cervix. Cancer 1991;68:1340–50. 20. Rock J, Bartlett MK. Biopsy studies of human endometrium: Criteria of dating and information about amenorrhea, menorrhagia, and time of ovulation. J Am Med Assoc 1937;108:2022–8. 21. Mildwidsky A, Palti Z, Gutman A. Glycogen metabolism of the human endometrium. J Clin Endocrinol Metab 1980;51:765–70. 22. Burney RO, Talbi S, Hamilton AE et al. Gene expression analysis of endometrium reveals progesterone resistance and candidate susceptibility genes in women with endometriosis. Endocrinology 2007;148:3814–26. 23. Altadill T, Campoy I, Lanau L et al. Enabling metabolomics based biomarker discovery studies using molecular phenotyping of exosome-like vesicles. PLoS ONE 2016;11(3):e0151339. doi:10.1371/journal.pone.0151339 24. Wira CR, Fahey JV, Schaefer TM et  al. Innate and adaptive immunity in the human female reproductive tract: Influence of the menstrual cycle and menopause on the mucosal immune system in the uterus. In: Aplin JD, Fazleabas AT, Glasser SR, Giudice LC, eds. The Endometrium: Molecular, Cellular, and Clinical Perspectives. Second ed. London: Taylor & Francis, 2008:493–6. 25. Hickey DK, Patel MV, Fahey JV, Wira CR. Innate and adaptive immunity at mucosal surfaces of the female reproductive tract: Stratification and integration of immune protection against the transmission of sexually transmitted infections. J Reprod Immunol 2011;88:185–94. 26. Amjadi F, Salehi E, Mehdizadeh M, Aflatoonian R. Role of the innate immunity in female reproductive tract. Adv Biomed Res 2014;3:1–12. 27. Goldfien GA, Barragan F, Chen J et  al. Progestincontaining contraceptives alter expression of host defense-related genes of the endometrium and cervix. Reprod Sci 2015;22:814–28. 28. Aagaard K, Ma J, Antony KM et  al. The placenta harbors a unique microbiome. Sci Transl Med 2014;6:237ra65–5. 29. Irwin J, Giudice L. The decidua. In: Knobil E, Neill J, eds. Encyclopedia of Reproduction. San Diego: Academic Press; 1998:823–35. 30. Gellersen B, Brosens J. Cyclic decidualization of the human endometrium in reproductive health and failure. Endocr Rev 2014;35:851–905. 31. Du M-R, Wang S-C, Li D-J. The integrative roles of chemokines at the maternal-fetal interface in early pregnancy. Cell Mol Immunol 2014;11:438–48. 32. Wu H-X, Jin L-P, Xu B et al. Decidual stromal cells recruit Th17 cells into decidua to promote proliferation and invasion of human trophoblast cells by secreting IL-17. Cell Mol Immunol 2014;11:253–62.

References 11

33. Lynch VJ, Leclerc RD, May G, Wagner GP. Transposon-mediated rewiring of gene regulatory networks contributed to the evolution of pregnancy in mammals. Nat Genet 2011;43:1154–9. 34. Lynch VJ, May G, Wagner GP. Regulatory evolution through divergence of a phosphoswitch in the transcription factor CEBPB. Nature 2011;480:383–6. 35. Kin K, Nnamani MC, Lynch VJ et al. Cell-type phylogenetics and the origin of endometrial stromal cells. Cell Rep 2015;10:1398–409. 36. Salker M, Teklenburg G, Molokhia M et al. Natural selection of human embryos: Impaired decidualization of the endometrium disables embryo-maternal interactions and causes recurrent pregnancy loss. PLoS One 2010;5:e10287. 37. Nancy P, Tagliani E, Tay CS et  al. Chemokine gene silencing in decidual stromal cells limits T cell access to the maternal-fetal interface. Science. 2012;336:1317–21. 38. Kuroda K, Venkatakrishnan R, Salker MS et  al. Induction of 11beta-HSD 1 and activation of distinct mineralocorticoid receptor- and glucocorticoid receptor-dependent gene networks in decidualizing human endometrial stromal cells. Mol Endocrinol 2013;27:192–207. 39. Gilmour JS, Coutinho AE, Cailhail JF et  al. Local amplification of glucocorticoids by 11 beta-­ hydroxsteroid dehydrogenase type I promotes macrophage phagocytosis of apoptotic leukocytes. J Immunol 2006;176:7604–11. 40. Burton GJ, Scioscia M, Rademacher TW. Endometrial secretions: Creating a stimulatory microenvironment within the human early placenta and implications for the aetiopathogenesis of preeclampsia. J Reprod Immunol 2011;89:118–25. 41. Wilcox AJ, Baird DD, Weinberg CR. Time of implantation of the conceptus and loss of pregnancy. N Engl J Med 1999;340:1796–9. 42. Genbecev OD, Prakobphol A, Foulk RA et  al. Trophoblast L-selectin-mediated adhesion at the maternal-fetal interface. Science 2003;299:405–8. 43. Díaz-Gimeno P, Horcajadas JA, Martínez-Conejero JA, Esteban FJ, Alamá P, Pellicer A, Simón C. A genomic diagnostic tool for human endometrial receptivity based on the transcriptomic signature. Fertil Steril.2011;95(1):50–60, 60.e1–15. 44. Ruiz-Alonso M, Blesa D, Diaz-Gimeno P et al. The endometrial receptivity array for diagnosis and personalized embryo transfer as a treatment for patients with repeated implantation failure. Fertil Steril 2013;100:818–24. 45. Voorhis BJ, Dokras A. Delayed blastocyst transfer: Is the window shutting? Fertil Steril 2008;89:31–2. 46. Lucas ES, Salker MS, Brosens JJ. Uterine plasticity and reproductive fitness. Reprod Biomed Online 2013;27:506–14. http://dx.doi.org/10.1016/j. rbmo.2013.06.012.

47. Macklon NS, Geraedts JP, Fauser BC. Conception to ongoing pregnancy: The “black box”: Of early  ­pregnancy loss. Hum Reprod Update 2002;8:333–43. 48. Martin RD. The evolution of human reproduction: A primatological perspective. Am J Phys Anthropol Suppl 2007;45:59–84. 49. Salamonsen LA, Giudice LC. “The curse”: A 21st century perspective of models of its molecular basis. Endocrinology 2010;151:4092–5. 50. Strassman B. The evolution of endometrial cycles and menstruation. Quant Rev Biol 1996;71:181–220. 51. Irwin JC, Kirk D, Gwatkin RB et al. Human ­endometrial matrix metalloproteinase-2, a putative menstrual proteinase. J Clin Invest 1006;97:438–47. 52. Gargett CE, Masuda H. Adult stem cells in the endometrium. Mol Hum Reprod 2010;16:818–34. 53. Ludwig H, Metzger H. The re-epithelization of endometrium after menstrual desquamation. Arch Gynakol 1976;221:51–60. 54. Verdi J, Tan A, Shoae-Hassani A, Seifalian AM. Endometrial stem cells in regenerative medicine. J Biol Eng 2014;8:20–9. 55. Mutlu L, Hufnagel D, Taylor H. The endometrium as a source of mesenchymal stem cells for regenerative medicine. Biol Reprod 2015;92:138. 56. Ulrich D, Edwards SL, Su K et al. Human endometrial mesenchymal stem cells modulate the tissue response and mechanical behavior of polyamide mesh implants for pelvic organ prolapse repair. Tissue Eng Part A 2014;20:785–98. 57. Alawadhi F, Du H, Cakmak H, Taylor HS. Bone marrow-derived stem cell (BMDSC) transplantation improves fertility in a murine model of Asherman’s syndrome. PloS One 2014;9(5):e96662. 58. Santamaria X, Massasa EE, Feng Y et al. Derivation of insulin producing cells from human endometrial stromal stem cells and use in the treatment of murine diabetes. Mol Ther 2011;19:2065–71. 59. Wolff EF, Gao X-B, Yao KV et al. Endometrial stem cell transplantation restores dopamine production in a Parkinson’s disease model. J Cell Mol Med 2011;15:747–55. 60. Talbi S, Hamilton AE, Vo KC et al. Molecular phenotyping of human endometrium distinguishes menstrual cycle phases and underlying biological processes in normo-ovulatory women. Endocrinology 2006;147:1097–121. 61. Ponnampalam AP, Weston GC, Trajstman AC et al. Molecular classification of human endometrial cycle stages by transcriptional profiling. Mol Hum Rep rod.2004;10:879–93. 62. Giudice LC, Talbi S, Hamilton A, Lessey BA. Transcriptomics. In: Aplin JD, Fazleabas AT, Glasser SR, Giudice LC, The Endometrium: Molecular, Cellular, and Clinical Perspectives. Second ed. London: Taylor & Francis, 2008:3–18.

12  Highlights on the dynamism and multifunctionality of human endometrium in the era of precision medicine

63. Altmae S, Estaban FF, Stavreus-Evers A et  al. Guidelines for the design, analysis and interpretation of “omics” data: Focus on human endometrium. Hum Reprod Update 2014;20:12–28. 64. Clancy KBH. Reproductive ecology and the endometrium: Physiology, variation, and new directions. Yearb Phys Anthropol 2009;52:137–54. 65. Amir W, Micha B, Ariel H et  al. Predicting factors for endometrial thickness during treatment with assisted reproductive technology. Fertil Steril 2007;87:799–804.

66. Andolf E, Aspenberg P. Age, weight, and body mass index and endometrial thickness in postmenopausal women. Acta Obstet Gynaecol Scan 1996;75:867–8. 67. Maybina JA, Critchley HOD, Jabbour HN. Inflam­ matory pathways in endometrial disorders. Mol Cell Endocrinol 2011;335:42–51. 68. Vannuccini S, Clifton VL, Fraser IS et al. Infertility and reproductive disorders: Impact of hormonal and inflammatory mechanisms on pregnancy ­outcomes. Hum Reprod Update 2016;22(1):104–15.

I

Section   

Diagnosis of the Endometrial Factor

2

New imaging diagnostics SANJA KUPESIC PLAVSIC

INTRODUCTION

Pelvic ultrasound (US) enables high-resolution imaging of the female pelvis in patients of all ages. Due to its high resolution, low cost, widespread use, and lack of exposure to ionizing radiation, in the majority of patients US may be the only diagnostic examination required for evaluation of the uterus and endometrium. For this reason, this chapter focuses on sonographic images of normal endometrium and most common endometrial pathologic findings. Sonographic images of the endometrium are reviewed in the context of the patient’s age; phase of the menstrual cycle; premenarchal, menopausal, or pregnancy status; and hormonal therapy. Although US is the modality of choice for visualization of the endometrium, often it is complemented with x-ray hysterosalpingography (HSG), magnetic resonance imaging (MRI), and computed tomography (CT). Familiarity with ultrasonographic findings of the endometrium from prepubertal to postmenopausal years aids in better differentiation between endometrial physiologic changes and pathologic conditions. This chapter aims to present the actual possibilities of US imaging of the endometrium, looking for possible future applications of this method in the assessment of patients with infertility, abnormal uterine bleeding, endometrial thickening, intracavitary lesions, and Müllerian duct anomalies (MDAs). ULTRASOUND IMAGING OF SEQUENTIAL ENDOMETRIAL CHANGES

The endometrium is a specialized mucosa that varies in echogenicity, thickness, and composition through the menstrual cycle (1). It shows a wide spectrum of appearances reflecting the influence of reproductive hormones. In prepubertal and postmenopausal years, the endometrium remains thin. Sonographic measurements of the endometrium should be made in the longitudinal axis of the uterus, including the layers of the endometrium on the anterior and posterior walls. In normal patients, the endometrial thickness ranges from approximately 1 mm immediately after menstruation to about 14 mm immediately before menstruation. The endometrial thickness is measured as the sum of the two endometrial layers on a longitudinal transvaginal US scan at the site of maximum thickness, which is usually in the area of the uterine fundus. The endometrium is composed of a superficial layer (zona functionalis) and a deep basal layer. During the menstrual phase, the endometrium assumes variable thickness and a heterogeneous appearance due to the presence of menstrual blood and sheets of sloughed portions of endometria (Figure 2.1a). In the postmenstrual phase, the endometrium is typically thin and hyperechogenic. Due

to the estrogen effect, the endometrial thickness increases during the proliferative phase. Increased echogenicity of the basal layer and decreased echogenicity of the functional layer lead to the most characteristic sign of the late proliferative endometrium—triple-line or multilayered appearance. The central echogenic line represents a touching zone of the anterior and posterior layers (Figure 2.1b). The hypoechoic functional layer reflects the development of glands, vessels, and stroma, while the outer hyperechogenic lines represent the basal layer (2). When measuring the endometrium, the outermost, hypoechoic layer (­subendometrial halo) should be excluded, because it is myometrial in origin (3). Progesterone secretion during the luteal phase of the menstrual cycle differentiates the endometrial glands for secreting glycoproteins. Following ovulation, the functional layer becomes thickened and edematous, and the spiral arteries become tortuous. On US examination, secretory endometrium appears homogeneous and hyperechogenic (Figure 2.1c). For this reason, assessment of the endometrium for focal abnormalities should not be performed in this phase of the menstrual cycle (2). Characteristic histomorphologic and sonographic appearances of the endometrium are presented in Table 2.1. Disruption of the central hyperechoic line and heterogeneity of the endometrium may indicate focal abnormalities such as an endometrial polyp, intracavitary or submucosal leiomyoma, or intrauterine adhesions (4). COLOR AND SPECTRAL DOPPLER IMAGING OF ENDOMETRIUM

Endometrial blood flow originates from the radial arteries, which after passing through the myometrial–endometrial junction divide to the basal arteries that supply the basal portion of the endometrium, and the spiral arteries that continue up toward the endometrium. Color Doppler demonstrates the average flow velocities over time within the endometrium and provides directional information, determining whether the flow is toward or away from the transducer (5). Spectral or pulsed Doppler displays the peak velocity of flow in endometrial vessels. Spectral Doppler will present as either a positive or negative shift above or below the baseline, indicating the direction of flow within the evaluated vessel. Direction of flow is more important when evaluating larger blood vessels and can be quite challenging for smaller spiral arteries supplying the endometrium. Figure 2.2a–c illustrates color and pulsed Doppler assessment of the spiral arteries in d ­ifferent phases of the menstrual cycle. The quality of endometrial perfusion is highly dependent upon the uterine, arcuate, and radial artery blood 15

16  New imaging diagnostics (a)

(b)

(c)

Figure 2.1  (a) Transvaginal US of the uterus during menstrual phase, demonstrating the technique for measuring endometrial thickness in the presence of intracavitary fluid. Note that each endometrial layer is individually measured and added to reflect the double endometrial thickness. Note that intracavitary fluid is not included in the measurement. (b) Transvaginal US of periovulatory endometrium. Increased echogenicity of the basal layer and decreased echogenicity of the functional layer lead to a tripleline (multilayered) appearance. The central echogenic line represents a touching zone of the anterior and posterior layers (arrow). (c) Transvaginal US of secretory endometrium. The endometrium is homogeneously echogenic, and the central interface could not be appreciated. Typically, the endometrium reaches a maximum thickness during the midsecretory phase.

flow. Spiral artery blood flow velocity waveforms are characterized by lower velocity and lower impedance to blood flow than are observed in the uterine arteries (6). It is hypothesized that the features of endometrial blood flow may be used to predict the implantation success rate and reveal unexplained infertility problems (7).

Table 2.1  Endometrial changes during the menstrual cycle Menstrual phase

Days of cycle

Thickness (double layer/mm)

Menstrual Postmenstrual

1–5 6–9

Variable 1–4

Proliferative

10–13

5–8

Secretory

14–28

9–14

Endometrial echo pattern Heterogeneous Thin echogenic line Multilayered/ triple line Homogeneously echogenic

3-D ULTRASOUND OF ENDOMETRIUM

Three-dimensional (3-D) US has been introduced into a routine practice, enabling storage of complete sets of volume data. Once stored, these datasets can be accessed at any time without deterioration in quality, and the entire dataset can be manipulated to render the multiplanar, surface, or transparent views (8). There are many advantages to using 3-D US in evaluation of the endometrium. The sonographer can navigate through the stored volume in all three planes with tomographic precision, which provides better insight into the spatial relationship of the endometrium with the surrounding myometrium (Figure 2.3). Visualization of a coronal plane enables instantaneous visualization of the uterine cavity, myometrium, fundus, and cervix, which facilitates diagnosis and differentiation of different types of uterine anomalies and abnormalities (9–11). By providing multiple tomographic sections of the uterine cavity, endometrial polyps, intrauterine adhesions, and intracavitary and submucosal

3-D ultrasound of endometrium  17 (a)

(b)

(c)

Figure 2.2  (a) Transvaginal color Doppler scan of periovulatory endometrium. Blood flow velocity waveforms extracted from subendometrial arteries demonstrate moderate vascular resistance (RI = 0.68). (b) Transvaginal color Doppler scan demonstrating secretory endometrium with coiling appearance of the spiral arteries. (c) Transvaginal color Doppler scan of a secretory endometrium. Higher velocity and lower vascular resistance (RI = 0.43) are detected in the spiral arteries during the secretory phase of the menstrual cycle.

Figure 2.3  3-D multiplanar US image of normal uterine cavity. fibroids become easily visible. 3-D volumetric sonography enables repeatable and reproducible measurements of endometrial volumes. Quantification of the endometrial volume in combination with blood flow studies contributes to the assessment of the endometrial receptivity,

and may have a potential to predict pregnancy rates in assisted reproductive techniques (12). Combined evaluations of morphology and neovascularization by 3-D power Doppler may improve the detection of uterine malignancy (13).

18  New imaging diagnostics

Digital volume storage allows retrospective analysis of the volumes and independent review by an expert sonographer. De-identified datasets may also be efficiently used for training purposes. 2-D AND 3-D SALINE INFUSION SONOGRAPHY (HYSTEROSONOGRAPHY)

Assessment of the uterine cavity can be improved with the use of hysterosonography, a technique that involves distension of the uterine cavity with the injection of sterile saline (saline infusion sonography [SIS]) (14). Figure 2.4a and b shows normal uterine morphology assessed by twodimensional (2-D) and 3-D SIS or hysterosonography. Smooth and echogenic endometrium is visualized along the periphery of the uterine cavity distended with sonolucent fluid. 3-D hysterosonography allows simultaneous

assessment of the uterine cavity in longitudinal, transverse, and coronal planes (Figure 2.4b). PREMENARCHAL ENDOMETRIUM

During the neonatal period, childhood and adolescent transabdominal US is the primary imaging technique for the assessment of female reproductive organs. In the neonatal period, the ratio of the uterine body to the cervix is 1:2, and thickened endometrium may appear due to stimulation of maternal hormones (4). During childhood, the endometrium appears as a thin echogenic line within the central portion of the uterus, and the ratio of the uterine body to the cervix is about 1:1. At puberty, the uterus is transformed from a tubular to a pear-shaped organ. The endometrium becomes more prominent, and the ratio of the uterine body to the cervix is between 1.5:1 and 2:1.

(a)

(b)

Figure 2.4  (a) 2-D hysterosonography (SIS) of the normal endometrium. Smooth and echogenic endometrium is visualized along the periphery of the uterine cavity distended with sonolucent fluid. Catheter is visualized in the inferior portion of the uterus. (b) 3-D hysterosonographic image of the normal uterine cavity. Multiplanar imaging allows simultaneous assessment of the uterine cavity in longitudinal, transverse, and coronal planes. Lower right image demonstrates surface rendering of the triangular uterine cavity.

Postmenopausal endometrium  19

Figure 2.5 

Transabdominal US image of the uterus in a normal peripubertal, 12-year-old girl. The uterus is pear shaped, with the uterine body wider than the cervix. Note the angulation of the uterine body with respect to the cervix (consistent with anteflexion).

Ratio assessment changes confirm that, as puberty progresses, the uterine corpus grows more than the cervix. Figure 2.5 shows the endometrial appearance in a normal peripubertal girl. Assessment of the endometrial thickness by 2-D US and endometrial volume by 3-D US may aid in the assessment of patients with clinical signs of precocious puberty, along with gonadotropin-releasing hormone (GnRH) testing, growth velocity, and bone age studies. POSTMENOPAUSAL ENDOMETRIUM

Transvaginal US is routinely performed for the assessment of endometrial thickness in postmenopausal women. The postmenopausal endometrium typically appears as a thin echogenic line (Figure 2.6a). A small amount of endometrial fluid may be seen in postmenopausal patients due to mild cervical stenosis. This fluid should be excluded from the endometrial thickness measurement (Figure 2.6b). (a)

Several studies have demonstrated that a thin endometrium (10 mm; empty gestational sac empty gestational sac Lack of uterine or gestational sac growth and Lack of embryonic or fetal heart action lack of embryonic or fetal heart action Residual products of conception Heterogeneous intrauterine collection Bright color signals with low vascular resistance beneath endometrium or within myometrium Arteriovenous malformation Endometrial heterogenicity with small Mosaic vascular pattern with high-velocity and anechoic areas in the myometrium low-resistance turbulent flow Prominent peripheral flow with low vascular Molar pregnancy Endometrial mass with multiple sonolucent impedance cysts with posterior acoustic enfacement (snowstorm appearance) Choriocarcinoma Heterogeneous uterine mass Neovascular signals (high velocity and low vascular resistance) Sources: Kasar P, Andonotopo W, Kupesic Plavsic S. Donald School J Ultrasound Obstet Gynecol 2015;9(2):175–8; Kupesic Plavsic S, Kurjak A, Baston K. In: Kupesic Plavsic S, ed. Color Doppler, 3D & 4D Ultrasound in Gynecology, Infertility and Obstetrics. New Delhi: Jaypee Publishers; 2011:243–51; Tie W, Tajnert K, Kupesic Plavsic S. Donald School J Ultrasound Obstet Gynecol 2013;7(1):105–12; Tullius TG Jr et  al. J Clin  Ultrasound 2015;43(5):327–34; Kurjak A, Kupesic S J Placenta 1998;19:619–23.

power Doppler parameters (vascularization index [VI], flow index [FI], and vascularization flow index [VFI]) in differentiation between endometrial polyps and endometrial cancer (42,43). Endometrial polyp Endometrial polyps are benign lesions composed of endometrial glands, fibrous stroma, and vascular channels (35). They may be clinically asymptomatic, or may cause vaginal spotting or postcoital or intermenstrual bleeding. On transvaginal US, polyps appear as hyperechoic ovoid lesions causing focal endometrial thickening. When reproductive age patients are evaluated during the second phase of the menstrual cycle, endometrial polyps can be easily missed because their echogenicity is similar to that of the surrounding secretory endometrium. Therefore, endometrial polyps are best imaged during the early proliferative

phase of the menstrual cycle when there is a contrasting, more hypoechoic endometrium, or after injection of the negative contrast medium (saline) into the uterine cavity (Figure 2.12a) (44). They may contain small cystic spaces and are vascularized by regularly separated feeding vessels within the stalk (Figure 2.12b and c). 3-D SIS can better visualize the uterine cavity and the endometrial thickness than transvaginal sonography or 2-D SIS. Using multiplanar view, polypoid structures are clearly demonstrated, allowing for the optimal plane to present their pedicle. The surface rendering mode allows visualization of the polypoid structure in continuity with the endometrial lining (45,46). Although the measurement of endometrial thickness assessed by transvaginal US is similar in patients with endometrial hyperplasia (diffuse endometrial thickening) and polyps (focal endometrial thickening), endometrial

Transvaginal US Postmenopausal patients: at random Premenopausal patients: early postmenstrual phase

Postmenopausal patients: ≥5 mm Premenopausal endometrium: ≥7 mm

Postmenopausal patients: 2-fold, respectively (75). Several P4-regulated genes (Table 4.1), normally upregulated during the window of implantation, were downregulated in MSE of women with endometriosis (75). In a subsequent study, we performed global analysis of the eutopic endometrial transcriptome throughout the

menstrual cycle from women with and without moderate to severe endometriosis (34). Interestingly, comparative analysis of different cycle phases with and without endometriosis revealed that the largest number of statistically significantly differentially expressed genes between the two groups was in ESE (34). On principal component analysis (PCA), ESE samples from women with endometriosis clustered closer to PE samples rather than MSE samples. Several P4-regulated genes (Table 4.1) were downregulated in ESE and MSE in women with disease, while PR was upregulated in ESE, suggesting resistance to the actions of endogenous P4 (34). Remarkably, evaluation of LSE in women with endometriosis compared with disease-free subjects is not informative (76). Absenger et al. (77) also reported on differential gene expression in the eutopic endometrium from women with endometriosis compared with controls in the proliferative and secretory cycle phases that suggested Cyr61 (cysteine-rich angiogenic inducer 61, CCN1) as a cycle-independent biomarker for endometriosis. It was also upregulated in endometriotic lesions, and the results were confirmed in a nude mouse xenograft model of endometriosis (77). The proposed role for Cyr61 in the pathogenesis of endometriosis is likely

Table 4.1  Select gene expression changes in microarray studies on human eutopic endometrial tissue from women with versus without endometriosis Cycle phase

Dysregulated in endometriosis

Endometriosis stage

Reference

MIG6 GPX3 MAOA Metallothioneins DKK1

ESE, MSE ESE, MSE/LSE ESE, MSE ESE, MSE/LSE ESE, MSE, LSE

− –/+ − − −

Moderate/severe Moderate/severe Moderate/severe Moderate/severe Mild/moderate/severe

FOXO1A STC1 MUC1 PR IL-15 Glycodelin (PP14) GlcNAc6ST G0S2 PNP B61 (ephrin A1) PRH1 SEMA3C OLFM1 KHDRBS3 PDEGF ITGA2 PTPRR PCDH17 IL6ST CYR61

ESE, MSE ESE, MSE/LSE ESE, MSE ESE MSE ESE, MSE MSE ESE, MSE MSE MSE MSE MSE/ESE ESE, MSE MSE MSE MSE/LSE ESE, MSE/LSE LSE LSE PE, SE/LSE

− −/+ − + − − − − − − − +/– + + − –/+ –/+ + + +

Moderate/severe Moderate/severe Moderate/severe Moderate/severe Mild/moderate Mild/moderate Mild/moderate Mild/moderate Mild/moderate Mild/moderate Mild/moderate Mild/moderate Mild/moderate Mild/moderate Mild/moderate Mild/moderate Mild/moderate/severe Mild/moderate/severe Mild/moderate/severe Unknown

Burney et al. (34) Burney et al. (34); Fassbender et al. (127,128) Burney et al. (34) Burney et al. (34); Fassbender et al. (127,128) Kao et al. (75); Burney et al. (34); Fassbender et al. (127,128) Burney et al. (34) Burney et al. (34); Fassbender et al. (127,128) Burney et al. (34) Burney et al. (34) Kao et al. (75) Kao et al. (75); Burney et al. (34) Kao et al. (75) Kao et al. (75); Burney et al. (34) Kao et al. (75) Kao et al. (75) Kao et al. (75) Kao et al. (75); Burney et al. (34) Kao et al. (75); Burney et al. (34) Kao et al. (75) Kao et al. (75) Kao et al. (75); Fassbender et al. (127,128) Burney et al. (34); Sherwin et al. (76) Sherwin et al. (76) Sherwin et al. (76) Absenger et al. (77); Fassbender et al. (127,128)

Gene

Note: SE, secretory phase; +, upregulation; −, downregulation.

Epigenetics 55

associated with facilitating adhesions and angiogenesis (77). As a consequence, we believe that genomic profiling of endometrial tissue could potentially revolutionize the noninvasive diagnostics of endometriosis. Thus, together with estrogen dependence, progesterone resistance is one of the hallmarks of pathophysiology of endometriosis. While this phenomenon is not specific to endometriosis (reported in endometrium from women with PCOS and endometrial hyperplasia (78–80)), diagnostic platforms for molecular diagnosis of endometriosis are likely to be based on the array of genes and factors affected by progesterone resistance (see below). EPIGENETICS

Dysregulation of miRNA expression in endometrium and lesions Endometrium is believed to be under epigenetic regulation of small (22n) noncoding RNAs, miRNAs (81,82), via posttranscriptional regulation of gene expression by either degrading or translationally repressing mRNAs (83,84). Almost 1000 miRNAs have been identified and validated in humans. It has been estimated that each miRNA potentially regulates up to hundreds of mRNA targets (85). Within the past decade, research on miRNA roles in reproductive tissues in health and disease has intensified and has been summarized in a recent review (86). Not surprisingly, miRNAs in human endometrial tissue demonstrate cyclic variation and are regulated by ovarian steroids in endometrial cells in culture (87,88). Moreover, miRNAs have been proposed as novel biomarkers of human endometrial receptivity (89). Paired analysis of eutopic and ectopic endometrium has demonstrated differential expression of miRNAs in these tissues (88,90). Downregulation of miR-196b in ectopic endometriotic tissue was consistently demonstrated, and its overexpression inhibits cell proliferation and induces apoptosis in endometriotic cells (90,91). Global miRNA array analysis of eutopic endometrium from women with and without endometriosis revealed significant downregulation of members of the miR-9 and miR-34 families in ESE from women with endometriosis, associated with the biological processes of cell death, cell cycle, and cellular assembly and organization (92). Laudanski et al. (93) found miR-483-5p and miR-629* to be downregulated in eutopic endometrium from women with endometriomas (93) (Table 4.2). Joshi et al. (94) demonstrated significant downregulation of miR-451 in eutopic endometrium from women with endometriosis compared with no-disease controls and baboons before and 3 months after induction of endometriosis. Overexpression of miR-145 correlated with decreased cell proliferation (94). Peritoneal fluid from subjects with endometriosis has been shown to modify miRNA profiles of eutopic endometrial stromal cells in vitro (95). To date, only a few studies have applied next-­generation sequencing or deep sequencing (RNA sequencing, in which all miRNAs, including unknowns, are sequenced)

to identify miRNA expression profiles in human endometrium from women with endometriosis. Analysis of miRNA profiles between ovarian endometriomas and eutopic endometrium from women without endometriosis, coupled with transcriptome profiling and in silico microRNA targeting predictions, discovered dysregulation of multiple biologically important pathways, with validation of function of miR-29c, which targets several extracellular matrix genes in endometriomas (96). Highthroughput miRNA sequencing of paired peritoneal endometriotic lesions and matched healthy surrounding peritoneal tissues, together with the eutopic endometria from the same patients, demonstrated upregulation of miR-34c, miR-449a, miR-200a, miR-200b, and miR-141 in diseased peritoneal samples (97). Based on the above data, endometrial miRNAs play a crucial role in the development and progression of endometriosis, with proliferation, apoptosis, and invasion being but a few of the regulated cell functions. Current research is focused on developing diagnostic and therapeutic miRNA targets for this disease. Circulating miRNAs It has been suggested that miRNAs are shed from tissues and released into circulation (98,99), making them appealing as noninvasive diagnostic markers of endometriosis. MicroRNA analysis of plasma samples from women with and without disease showed significantly higher plasma levels of miR-16, miR-191, and miR-195 in women with endometriosis, and other study showed significant reduction in levels of miR-17-5p, miR-20a, and miR-22 levels in subjects with severe disease (100,101) (Table 4.2). These data suggest that plasma miRNA shed from abnormal eutopic endometrium or disease lesions hold promise as a noninvasive molecular diagnostic of endometriosis. However, the low correlation with tissue miRNA expression to date (Table 4.2) suggests that further studies are needed with large numbers of well-annotated subjects. DNA methylome: Potential future diagnostics There is increasing evidence of epigenetic regulation of steroid hormone action in various tissues (102). Methylation of the carbon-5 position of cytosines in CpG dinucleotides is the main epigenetic modification of DNA. DNA methylation is essential for a properly functioning genome, including chromosome stability maintenance and transcriptional repression, and is important in embryonic and germ cell development and reprogramming (103–105). Epigenetic processes, such as DNA methylation, are complex, and changes in their signals may impact gene activity, which in turn can reflect nongenetic or genetic influences and affect disease pathogenesis and progression. We conducted the first study on global DNA methylation of eutopic endometrium throughout the menstrual cycle (PE, ESE, and MSE) from normal controls without endometriosis demonstrated cycle phase-dependent DNA methylation that correlated with gene expression changes at specific loci, underscoring it as an important

56  Molecular diagnosis of endometriosis

Table 4.2  Validated expression of eutopic endometrial and circulating serum microRNAs, dysregulated in endometriosis compared with nonendometriosis subjects based on microarray studies Endometrial miRNA

Dysregulation

Tissue/cell type

miR-9* miR-34b* miR-34c-5p miR-20a miR-15a miR-181a miR-142-5p miR-99a miR-145 miR-126 miR-200b miR-141 miR-424 miR-200a miR-200b miR-200c miR-182 miR-202 miR-483-5p miR-629* miR-196b

− − − − + + + + − − − + + + − − − − + − − −

miR-183-5p miR-200b miR-29c

− − +

Eutopic endometrium, control vs. disease Eutopic endometrium, control vs. disease Eutopic endometrium, control vs. disease Eutopic endometrium, control vs. disease Eutopic endometrium, control vs. disease Eutopic endometrium, control vs. disease Eutopic endometrium, control vs. disease Eutopic endometrium, control vs. disease Ectopic vs. eutopic endometrium Ectopic vs. eutopic endometrium Ectopic vs. eutopic endometrium Ectopic vs. eutopic endometrium Ectopic vs. eutopic endometrium Ectopic vs. eutopic endometrium Ectopic vs. eutopic endometrium Ectopic vs. eutopic endometrium Ectopic vs. eutopic endometrium Ectopic vs. eutopic endometrium Ectopic vs. eutopic endometrium Ectopic vs. eutopic endometrium Ectopic vs. eutopic endometrium Ectopic vs. eutopic endometrial stromal cells Ectopic vs. eutopic endometrium Ectopic vs. eutopic endometrium Ectopic vs. eutopic endometrium

miR-9

Cycle phase ESE ESE ESE ESE SE SE SE SE PE, SE PE, SE PE, SE PE, SE PE, SE PE, SE PE PE PE PE PE PE PE

Mostly PE Mostly PE

References Burney et al. (92) Burney et al. (92) Burney et al. (92) Burney et al. (92) Pan et al. (88) Pan et al. (88) Pan et al. (88) Pan et al. (88) Ohlsson et al. (90) Ohlsson et al. (90) Ohlsson et al. (90) Ohlsson et al. (90) Ohlsson et al. (90) Ohlsson et al. (90) Filigheddu et al. (170) Filigheddu et al. (170) Filigheddu et al. (170) Filigheddu et al. (170) Filigheddu et al. (170) Laudanski et al. (93) Laudanski et al. (93) Abe et al. (91) Shi et al. (171) Hawkins et al. (96) Hawkins et al. (96)

Circulating miRNA miR-16 miR-191 miR-195 miR-17-5-p miR-20a miR-22 miR-145* miR-141* miR-542-3p miR-9* miR-199a miR-122

+ + + − − − − − − − + +

Suryawanshi et al. (101) Suryawanshi et al. (101) Suryawanshi et al. (101) Jia et al. (100) Jia et al. (100) Jia et al. (100) Wang et al. (172) Wang et al. (172) Wang et al. (172) Wang et al. (172) Wang et al. (172) Wang et al. (172)

Note: Bold highlighted miRNAs demonstrate the correlation between tissue and serum findings.

mechanism of steroid hormone action on this tissue (106). Alteration of endometrial cells influencing their ability to implant may be acquired (environmental and lifestyle) or hereditary (genetic). In endometriosis, epigenetic and concomitant gene expression abnormalities have been reported, including aberrant DNA methylation of genes relevant to the endometrial steroid hormone response (e.g., PGRB, HOXA10, ESR2, SF1, aromatase, and E-cadherin) (71,107–109).

Other findings include an epigenetic switch for GATA expression (110), and abnormal eutopic DNA methylation profiles, in a limited number of studies, platforms, and sample sizes (110–112). Genome-wide profiling of methylated promoters in endometriosis lesions and eutopic endometrium revealed hyper- and hypomethylation unique to these groups, and similarities in DNA methylation profiles between eutopic endometrium and endometriosis lesions support eutopic endometrium as the origin of ectopic

Neuroangiogenesis and neuronal markers of endometriosis  57

lesions (110,113,114). Naqvi et al. (111) used genome-wide methylation screening of eutopic endometrial samples from women with and without endometriosis and found 59 genes significantly hypermethylated and 61 genes hypomethylated in women with endometriosis, identifying some of the known dysregulated genes, such as HOXA10 and methyltransferase, among others (111). Promoter hypermethylation of PRB, but not PRA, in the epithelial cells of endometriotic implants can explain progesterone resistance and the mechanism of differential expression of PR isoforms in endometriosis—decreased PRB expression and relative increase of PRA expression, as discussed above. This also correlates with the reported hypermethylation of HOXA10 (107,115), and dramatic hypomethylation of NR5A1 (steroidogenic factor-1 [SF-1]) and ESR2 (ERβ) in the eutopic endometrium of women with endometriosis, explaining the striking differences in the expression of these genes between the two groups (108,116,117). Markedly higher levels of ERβ and lower levels of ERα in endometriotic tissues and endometriotic stromal cells result from ERβ promoter hypomethylation, allowing the latter to take over the ERα promoter activity, which results in the suppression of ERα levels in endometriotic tissues and cells (116,118) and development of the endometriotic phenotype (36). These have relevance to the inflammatory phenotype (36) (Figure 4.2) and may provide candidate biomarkers for diagnosing the disease, although confounders of other inflammatory disorders need to be addressed. In all, DNA methylation changes in human ectopic and eutopic endometrium from women with and without endometriosis are intriguing, and so far data have been promising as to association between gene expression changes and methylation patterns. It is anticipated that additional global DNA methylome profiling and gene expression analyses will advance understanding of endometriosis pathogenesis and potentially offer diagnostic and therapeutic applications. ENDOMETRIAL PROTEOME AND SECRETOME

Gene expression and biological functions are linked via complex protein synthesis and gene interaction pathways. Thus, proteomic techniques have been applied to screen and identify endometriosis-specific proteins in biological fluids (serum, urine, peritoneal fluid, and menstrual blood) and eutopic and ectopic endometrium in search for potential biomarkers of the disease, with the majority of studies focusing on serum and eutopic endometrium (reviewed in (119)). Initial studies showed differential expression of proteins involved in cytoskeleton formation, regulation of cell cycle, signal transduction, or immunological function in serum and eutopic endometrium from women with versus without endometriosis, with some correlation between the two (120). Differential expression of protein peaks in secretory phase endometrium from women with endometriosis versus controls has been reported (121–123), with 91.7% sensitivity and 90% specificity in the diagnosis of endometriosis (124).

Interestingly, analysis of the differentially expressed proteins in the two-dimensional gel electrophoresis studies with eutopic endometrium revealed few proteins reliably seen in at least three of the studies so far, and include vimentin, peroxiredoxins, HSP70, HSP90, annexins, actins, and 14-3-3 family proteins, suggesting these as potential candidates in the pathology of endometriosis (120,123,125,126). Proteomic analysis of plasma samples obtained during the menstrual phase enabled the diagnosis of endometriosis undetectable by ultrasonography with high sensitivity and specificity (75% and 86% for minimal–mild and 98% and 81% for moderate–severe endometriosis, respectively) (127). Fassbender et al. performed paired assessment of the endometrial transcriptome and proteome and described a panel of differentially expressed peptide peaks in the ESE proteome of women with endometriosis versus controls without endometriosis but with other pelvic pathologies, as diagnostic of endometriosis of all stages with 91% sensitivity and 80% specificity (128). To date, only one study has validated their serum results of differentially expressed proteins in subjects with endometriosis in a separate cohort of patients, revealing haptoglobin (HP), immunoglobulin kappa chain C (IGKC) region, and alpha-1B-glycoprotein (A1BG) as candidate serum markers for the diagnosis of Stage II, III, and IV endometriosis, and IGKC and HP for the diagnosis of Stage I disease (129). Proteomics assessment of serum or endometrial tissue holds great promise in the development of noninvasive diagnostic tools for endometriosis. NEUROANGIOGENESIS AND NEURONAL MARKERS OF ENDOMETRIOSIS

A key mechanism for endometrial cell survival in ectopic locations is establishing a blood supply (130). This process (neoangiogenesis) occurs concomitantly with neuronal sprouting, and the two together comprise neuroangiogenesis (131). It is supported by high levels of vascular endothelial growth factor (VEGF) and other angiogenic factors in peritoneal fluid and endometrium from women with endometriosis compared with healthy controls (132–135). Endothelial endometrial cells from women with endometriosis also express higher proliferation ability and distinct gene expression patterns compared with nonendometriosis controls (136). A transcript for pleiotrophin, an angiogenesis-associated peptide, was significantly upregulated in eutopic endometrium from women with severe endometriosis (137); however, its usefulness as a potential marker has not been evaluated. Proangiogenic E2-regulated Cyr61 has been proposed as a potential cycle-independent marker of endometriosis (see above and (77)). Neuronal fibers in the eutopic endometrium of women with endometriosis and ectopic disease are candidates for the pathophysiology of dysmenorrhea and pelvic pain associated with endometriosis (138). Using neuronal protein markers, nerve fibers have been confirmed in endometrium from women with endometriosis, but not in

58  Molecular diagnosis of endometriosis

those with endometritis, leiomyomata, or endometrial polyps (139–142). Nerve fibers have also been detected in animal models of endometriotic lesion innervation (143– 145). Genes involved in endometriosis-related pain, such as tyrosine kinase receptor B (TRkB) in epithelial cells and serotonin transporter (5HTT) and mu opioid receptor (MOR), were upregulated in stromal fibroblasts (146), and axon signaling pathways were dysregulated in eutopic endometrium from women with endometriosis in microarray studies (147). Therapy with progestogens or combined oral contraceptives significantly decreased nerve fiber density and nerve growth factor (NGF) expression in deep infiltrating endometriosis lesions (148), suggesting it as an important mechanism of hormonal therapy control of pain symptoms. While there are conflicting results with regard to nerve fibers in endometrial biopsy samples as a noninvasive diagnostic for endometriosis (149,150), this is a promising area of investigation that warrants inclusion of hormonal status in such analyses. MINIMALLY INVASIVE DIAGNOSTICS AND DISEASE CLASSIFIER

A systematic review of potential endometrial biomarkers of endometriosis identified 182 articles with more than 200 potential biomarkers, including hormones and their receptors (n = 29), cytokines (n = 25), and factors identified through proteomics (n = 8) and histology (n = 10), with the reported sensitivity and specificity in only 32 articles ranging from 0% to 100% (151). Recently, we reported on classifiers to diagnose and stage endometriosis, with high accuracy (152). Whole genome microarray data of 148 endometrial specimens from women with confirmed endometriosis or other benign surgically confirmed gynecologic pathology (leiomyoma, endometrial polyp, and hydrosalpinx) and women with no uterine pathology (confirmed surgically) throughout the menstrual cycle were analyzed (152). Importantly, the performance of the classifier was evaluated on an independent sample set. Best-performing classifiers identified endometriosis with 90%–100% accuracy and were cycle phase specific or independent (152). Remarkably, a relatively small number of genes (less than 100) were sufficient to separate endometriosis from other uterine pathologies and to classify disease by severity. In particular, PE and ESE phasespecific disease classifiers achieved 100% accuracy using less than 100 genes for each disease classification decision. Pathway analyses revealed immune activation, altered steroid and thyroid hormone signaling and metabolism, and growth factor signaling in the endometrium of women with endometriosis, confirming earlier findings (see above and (152)). While these results are promising, their validation in a large, prospective multicenter independent cohort is necessary. INFERTILITY

Endometriosis is believed to contribute to infertility by anatomic distortion or scarring, a pro-inflammatory environment that is toxic to sperm and embryos, and a

decreased ovarian reserve, and at the level of the endometrium (3,153–156). Endometrial receptivity in endometriosis The endometrial receptivity array (ERA), which is a validated and currently the only diagnostic assay for clinical evaluation of human endometrial receptivity during the window of implantation (157,158), has recently been applied to the patients with endometriosis (159). The authors compared endometrial receptivity signatures in infertile women with endometriosis and infertile women without endometriosis and found that hierarchical clustering and PCA of 238 ERA gene expression data sets showed no clustering of samples based on the presence or absence of endometriosis or endometriosis stage (159). The conclusion was that the possible impact of endometriosis on endometrial receptivity, or at least on the molecular signature of it, from a clinical point of view may be nonsignificant. Whole genome transcriptomics of MSE from women with severe endometriosis versus control women with no uterine pathology showed that of 238 known ERA genes, only 8 (IL2RB, C11orf8c, CDC20c, CDAc, NKG7, CTSW, CFDc, and RPRMc) were upregulated and 1 (MMP26) downregulated beyond the ERA threefold threshold (160,161), supporting the findings by Garcia-Velasco et al. (159). However, the above findings do not rule out the possibility that endometriosis may impair endometrial receptivity (161), as the ERA does not pinpoint molecular abnormalities since it does not cover all the established “receptivity-associated” markers such as HOXA10, HOXA11, and KLF9, which have been shown to be altered in midsecretory eutopic endometrium from women with endometriosis (162–165). On the other hand, leukemia inhibitory factor (LIF), which is an established receptivity marker that is downregulated in MSE from women with unexplained infertility, has not been unequivocally shown to be dysregulated in endometriosis (165,166); hence, the absence of its dysregulation in the ERA panel in subjects with endometriosis is not a surprising finding. A potential reason for decreased implantation potential in women with endometriosis is the dysregulation of endometrial stromal cell decidualization, a well-established phenomenon discussed above, which is not detected by the ERA test. In addition, it is often assumed that there is correlation between the levels of mRNA and protein. However, the opposite has been reported, including in human endometrium (128,167–169). The reasons are not entirely understood and include variable and complex posttranscriptional and posttranslational mechanisms, such as temperature-dependent RNA structural changes, regulatory proteins, and translation efficiency (167). In human eutopic endometrium, a recent comparative study did not show differences at the transcriptome level between women with and without endometriosis, although proteomic analysis of the luteal phase endometrium in the same samples allowed the diagnosis of endometriosis with high sensitivity and specificity (128). Thus, while the poor correlation between transcript and corresponding protein levels can

References 59

contribute to our inability to identify the precise mechanisms of impaired endometrial receptivity, proteomic and transcriptomic studies await further validation, but hold great promise for future endometriosis diagnostics. CONCLUSIONS

Endometriosis is a common benign chronic and debilitating disease that requires surgery for definitive diagnosis and has no cure. Extensive basic, translational, and clinical research has progressed markedly over the past decade toward understanding the pathophysiology of the disease. However, large-scale studies with large numbers of participants and deep phenotyping are needed for moving the needle on the molecular diagnostics of endometriosis and targeted therapies for pain and infertility associated with this disorder. REFERENCES

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Microbiological diagnosis The human endometrial microbiome—Endometritis

5

INMACULADA MORENO and CARLOS SIMÓN

INTRODUCTION

The microorganisms inhabiting the human body—and their genetic information—make up the human microbiome. Up to 3% of our body weight is thought to account for the microorganisms living inside us; thus, a healthy adult weighing 80 kg hosts approximately 2 kg of microorganisms. Eukaryotes, archaea, bacteria, and viruses all live in association with humans. Bacteria are considered the cornerstone of the human microbiome, as the human body houses 10 times more bacterial cells than human cells, each containing thousands of genes independent of the human genome. When balanced correctly, these commensal bacteria play fundamental roles in human health by aiding in the absorption of nutrients, assisting the body with the production of essential compounds and inflammatory molecules, and supporting immune function, along with many other still unknown roles. In contrast, an imbalance of microbial communities in specific body sites is associated with pathological conditions, such as obesity, Crohn’s disease, and other gut alterations (1–3). HUMAN MICROBIOME

The identification of microorganisms inhabiting the human body has been traditionally performed by in vitro culture of specimens under selective conditions. However, this culturing method presents a major limitation; because only bacteria that are able to grow in the selective conditions can be detected and quantified, nonculturable bacteria remain unaccounted. However, nonculturable species are now detectable thanks to advances in genome sequencing and the publication of sequences from known bacteria (4), which have enabled a DNA sequence-based taxonomical classification of species. Several molecular techniques have been developed to assess bacterial communities present in a single sample. Particularly useful in this classification has been the gene encoding the 16S ribosomal RNA in bacteria and archaea. The gene contains nine variable regions (V1 to V9) placed among highly conserved sequences (5). These variable regions are species specific, and comparing their sequences with known bacterial genomes enables accurate and rapid taxonomical classification. The first method developed for bacterial strain identification involved fingerprinting by polymerase chain reaction (PCR) using specific primers for the 16S rRNA gene. This resulted in the amplification of DNA from all the bacteria present if the primers were

designed to include conserved regions, or specific bacteria could be identified if the primers were designed to cover variable regions of the gene. The major drawback of this technique is that a limited number of known bacterial species can be identified at one time in a sample. To circumvent this problem, genetic sequencing of a specific variable region of the targeted gene in a complex sample was used to provide information about all the taxonomic groups present, even if they were unexpected (6). The most advanced method used to identify bacterial communities from human samples has employed metagenomics sequencing of the bacterial whole genome (7). This method involves random sequencing, and the results obtained may correspond to any DNA region of the microorganisms present within the sample. The sequences are then assembled and mapped to known microbial genomes (8). This method is the most comprehensive, although it does present some limitations if presented with unknown genomes that lack a reference for DNA assembly. This strategy is gaining traction, as it can result in new microbes identified and functional and metabolic information about the bacteria apart from the mere presence of the microorganism (9). The Human Microbiome Project (HMP) (www. hmpdacc.org) was launched in 2008 by the U.S. National Institutes of Health to investigate and characterize the human microbiome and its influence in health and disease. The HMP has provided novel and valuable data through the comprehensive analysis of global microbial communities, shedding light on the roles of individual microbes. A  combination of sequencing of the 16S rRNA bacterial gene, metagenomic whole-genome shotgun (WGS) sequencing, and bioinformatics has been used to simultaneously identify the vast composition of microorganisms in a particular sample and elucidate the functional complexity of the microbial community within each sample. Using this technology, the HMP Consortium identified the core microbiomes of various distinct body sites—the respiratory tract, oral cavity, skin, gastrointestinal tract, and urogenital tract—in 4788 samples collected from 242 asymptomatic donors (129 men and 113 women) (10). The study revealed that independent communities consisting of conserved bacterial genera inhabit different body sites. In healthy individuals, resident communities are varied, with the highest variations found in the skin and the more stable communities found in the oral cavity and the urogenital tract (Figure 5.1). Interestingly, functional 65

66  Microbiological diagnosis All body sites base map of healthy individuals PC2 (7%)

Airways Gastrointestinal tract Oral Skin Urogenital tract

PC1 (16%) PC3 (4%)

Figure 5.1 

Unique microbial communities inhabit different sites within the human body. Principal coordinate (PC) analysis representing the separate microbiome groups found in the airways, gastrointestinal tract, oral cavity, skin, and urogenital tract. (Reprinted from SnapShot: The human microbiome, 158(3), González A, Vázquez-Baeza Y, Knight R, Cell, 690–1, Copyright 2014, with permission from Elsevier.)

and metagenomic analysis of microbial populations showed that major functions and metabolic pathways are conserved in healthy individuals despite the variation in microorganisms identified in each subject (11). MICROBIOME OF THE REPRODUCTIVE TRACT

The bacteria colonizing the genital tract represent 9% of the total human microbiome (12). Vaginal tissue in healthy women is most populated by Lactobacillus species, followed by bacteria from different families such as Prevotella, Gardnerella, and Atopobium (13) (Figure 5.2). The molecular identification of vaginal nonculturable bacterial species was first published in 2002 and described the amplification of the bacterial 16S rRNA gene in asymptomatic reproductive-age (14) and postmenopausal women (15). Since then, many reports have Urogenital tract Lactobacillus Prevotella Gardnerella Atopobium Sneathia Bifidobacterium Megasphera Anaerococcus Other

Figure 5.2  Urogenital tract microbiome. Taxonomic information at the family level from the urogenital tract microbiome of 250 individuals participating in the HMP. (Reprinted from SnapShot: The human microbiome, 158(3), González A, Vázquez-Baeza Y, Knight R, Cell, 690–1, Copyright 2014, with permission from Elsevier.)

described the vaginal microbiome in women of reproductive age, nonpregnant women from different ethnicities (16,17), pregnant women (18,19), patients undergoing in vitro fertilization (IVF) (20), and patients with recurrent vaginal infections (21). All these reports conclude that the vaginal microbiome of healthy women is dominated by Lactobacillus iners, Lactobacillus crispatus, Lactobacillus gasserii, and Lactobacillus jensenii, and that changes in the bacterial community composition may favor an imbalance in the physiological conditions of the genitourinary tract, ­leading to dysbiosis or a pathological state. Vaginal bacterial communities have been described by Ravel and collaborators using five community state types (CSTs), depending on the major bacteria dominating the microbiota. CSTs I, II, III, and V are dominated by L. crispatus, L. gasserii, L. iners, and L. jensenii, respectively, while CST IV is associated with high levels of strictly anaerobic bacteria such as Aerococcus, Atopobium, Dialister, Gardnerella, Megasphaera, Prevotella, and Sneathia, among others (16). The benefit of a Lactobacillusdominated vaginal microbiome is lactic acid production, which lowers the vaginal pH (22). Under low pH and ­oxygen pressure, Lactobacillus can grow and inhibit other pathogenic bacteria that might cause infections, such as Gardnerella vaginalis, Chlamydia trachomatis, or Neisseria gonorrhoeae (23–26), and protect against viral infections (27,28). Instability among vaginal microbiota may be influenced by sexual practice, hygiene, vaginal douching, and other personal habits, but is not always related (17). It has been observed that some women present very stable vaginal communities over time, while others are more prone to microbial changes. Changes in the vaginal microbiome are affected by hormonal changes over the course of the menstrual cycle, as intervals of high variability occur ­during menses, while more stable microbiomes coincide with the higher levels of estradiol and progesterone during the late follicular and midluteal phases, respectively (17). Recent studies show that up to 20%–30% of asymptomatic women present with high percentages of anaerobic bacteria, to the detriment of Lactobacillus (29). The vaginal microbiome is most commonly altered in reproductive-age women with bacterial vaginosis (BV), caused by the overgrowth of anaerobic bacteria from the Atopobium, Gardnerella, Mobilincus, Mycoplasma, Clostridium, and Prevotella genera (15,30). BV is diagnosed using the Nugent score, based on the morphotypes and proportion of Gramstained bacteria present in vaginal smears. Nugent scoring assigns low scores (0–3) to samples considered to be normal, medium scores (4–6) to intermediate flora, and high scores (7–10) to samples diagnosed as BV (31). This scoring system has a sensitivity of 89% and specificity of 83% (32). About 29.2% of women in the United States are thought to suffer from BV at some point during their lifetime, but its etiology remains unknown (33). Current treatment for BV consists of oral or local administration of metronidazole and clindamycin, but relapse is observed in 30% of women due to the presence of resistant strains, typically

Microbiome of the reproductive tract  67

from the Clostridiales, Megasphaera, Enterobacteriacae, and Staphylococci genera (34). As a result, probiotics are encouraged as supplementary treatment (30,35). Alterations in normal vaginal microflora, particularly those involved in the profile of BV, are thought to be ­associated with an increased risk of spontaneous pregnancy loss and spontaneous preterm birth (36–39), although no solid mechanistic proof has been presented. Additionally, alterations in vaginal microbiota have been shown to increase the risk of sexually transmitted infection transmission, including human immunodeficiency virus type 1 (HIV-1) (40), and the development of pelvic inflammatory disease (41). Vaginal microbiome: Impact on infertility and obstetrical outcomes The influence of vaginal microbiota on women’s reproductive health has garnered lots of interest in recent years. A direct relationship between vaginal pathogens, namely, N. gonorrhoeae, C. trachomatis, and Mycoplasma spp., and infertility has been proposed (12). Abnormal microbiota have been estimated in approximately 39% of infertile patients, with 19% suffering from BV (42). Although controversial, the presence of pathogenic bacteria in the vagina has been correlated with adverse pregnancy o ­ utcomes in patients undergoing IVF (43,44). Other studies, including a recent meta-analysis, suggest that BV does not influence implantation in patients undergoing IVF, but is associated with other obstetrical complications (42,45). These complications have been estimated to be a twofold increased risk of first trimester miscarriage, fivefold increased risk of late miscarriage, threefold increased risk of premature rupture of membranes, and twofold increased risk of preterm birth (prior to 32 weeks of gestation) for pregnant patients with BV (36–46). These risks are important to consider not only for IVF patients, but also for the fertile population w ­ ishing to conceive n ­ aturally, as the ­prevalence of BV in pregnant women has been estimated to vary from 6% to 63% (46), and thus, the same complications may affect every pregnant woman with BV even if they have conceived naturally. Reproductive tract microbiota at the maternal–fetal interface during pregnancy are becoming increasingly relevant to neonatal health, since altered microbial communities have been widely associated with obstetric complications and preterm birth. Comparison of vaginal microbiomes between pregnant and nonpregnant women indicates that pregnancy is associated with a unique vaginal microbiome, enriched in Lactobacillales (L. iners, L. crispatus, L. jensenii, and L. johnsonii), Clostridiales, Bacteroidales, and Actinomycetales (47), with lower bacterial diversity and higher stability. The vaginal microbiome of pregnant women with normal pregnancies who delivered at term was found to be dominated by Lactobacillus spp. of CST I, II, III, or V, and some variation within these communities has been observed over the course of pregnancy (18). The higher stability of the vaginal microbiome during pregnancy can be attributed to different factors including a high concentration of estrogen and

progesterone, increased glycogen levels in the vaginal epithelium (this helps acidify the environment and promotes the growth of Lactobacillus spp.), the absence of menses, or changes in the consistency and composition of cervical secretions. Interestingly, microbial stability increases as pregnancy progresses, with L. crispatus and L. iners ­dominating by the end of pregnancy (48). Bacterial communities of CST IV are rarely observed in pregnant women who deliver at term (18), and the presence of BV-causing bacteria is associated with increased preterm delivery (20,49). A recent study analyzing the temporal and spatial variation of vaginal microbiota ­ during p ­ ­regnancy by culture-independent methods revealed that high Gardnerella or Ureaplasma levels and concomitant low Lactobacillus spp. levels during pregnancy are clear risk factors for preterm birth. This pathological effect is much more striking in women with higher proportions of bacterial pathogens or longer exposure to them (19). However, the relationship between BV and preterm birth is controversial, as studies using different molecular techniques for the identification of bacterial pathogens in the vagina of pregnant women have failed to consistently demonstrate this correlation (50). During pregnancy, bacterial communities in the vagina undergo modifications relevant to the health of the ­newborn, as the microbiota of babies born following vaginal labor resemble the mother’s vaginal microbiota, while babies born after cesarean section present with skinrelated microbiota (51). The transfer of bacterial communities from mother to neonates likely occurs both vertically and horizontally. Vertical transmission was demonstrated a decade ago when the probiotic Lactobacillus strain administered to mothers during pregnancy was found in the stool samples of infants born by vaginal labor (52). Horizontal transmission of the maternal microbiome at delivery and postpartum may also influence the microbiome of the newborn, as the bacterial communities found in the stool samples of healthy infants born at term closely resemble the ones found in the maternal samples (53). Placental microbiota A special mention should be made to the placental microbiome. Contrary to the prevailing idea of a sterile onset of life in the intrauterine environment, it has been reported that 0.0002% of placental weight belongs to bacteria. This is considered a low abundance of microbiota, when compared with the communities inhabiting other human tissues, but its biological function may be of outstanding relevance, as this is the first environment surrounding the growing conceptus. The identification of bacteria in the placenta does not always correlate with uterine infections during pregnancy or other obstetrical complications, suggesting that the healthy placenta has a microbiome all its own. The first report isolating microorganisms in placentas of healthy women who delivered at term was published in 1988 (54) by microbiological culture and subsequently confirmed by molecular techniques (55–57). Aagaard and colleagues (58) have recently characterized

68  Microbiological diagnosis

bacterial communities residing in human placentas of 320 women by WGS sequencing, showing that the vast majority of samples were dominated by commensal bacteria belonging to Proteobacteria (including Escherichia coli and some nonpathogenic species of Neisseria), Actinobacteria (including Bifidobacterium species), Firmicutes (including Lactobacillus species), Bacteroidetes (including Flavobacterium species), Tenericutes (including some Mycoplasma species), and Fusobacteria phyla. Interestingly, the placental microbiome is more similar to the oral cavity microbiome, assessed by sampling the tongue, tonsils, saliva, and gingival plaques, than to the skin, vaginal, or gastrointestinal tracts, suggesting that colonization of the placenta is not the result of ascending bacteria from the lower reproductive tract or contamination during delivery, but rather could be spread through a hematogenous route, starting at the time of vascularization and placentation and finally seeding the placenta (58). Evidence of endometrial microbiota While the vaginal microbiome has been widely studied, there is insufficient evidence to support the presence of an indigenous endometrial microbiome. Implantation occurs in the interface between the endometrium and the embryo, making the microbial state of the endometrium of particular interest to the development of assisted reproductive techniques since it could impact embryo implantation and pregnancy outcomes. The endometrial cavity has been classically considered to be a sterile organ, but reports challenging this dogma support the existence of endometrial microbiota composed of different microorganisms (specifically Lactobacillus spp., Mycoplasma hominis, G. vaginalis, and Enterobacter spp.), which have been isolated by microbiological culture from endometrial samples obtained via the cervicovaginal canal using a double-lumen catheter (59). Also, recent studies have demonstrated that women, especially those with BV, can present polymicrobial G. vaginalis biofilms adhered to the endometrium and also the fallopian tubes, showing that polymicrobial bacterial could be attached to the endometrium and not only as free-floating bacteria (60). Various groups have attempted to assess endometrial microbiota in IVF patients through classical microbiological culture of the distal tip of the embryo transfer catheter, followed by isolation of the grown species. Accumulating evidence links the isolation of Lactobacillus spp. with patients with better chances of IVF success (61–62). In contrast, microbiological culture from patients undergoing IVF has also revealed endometrial bacterial pathogens that negatively affect implantation and pregnancy rates; in fact, Enterobacteriaceae, Streptococcus spp., Staphylococcus spp. (63), E. coli (64), and Gram-negative bacteria (62) have all been associated with decreased implantation rates and poor pregnancy outcomes (62–66) (Table 5.1). A relevant study conducted by Egbase et  al. in 1999 (67) clearly showed the negative impact of endometrial pathogens in implantation and pregnancy outcome. In this study, women undergoing IVF were assessed for

endometrial microbial flora at the time of oocyte retrieval and treated with prophylactic antibiotic therapy. Embryo transfer was performed 48 hours later, and the tip of the transfer catheter was again subjected to microbiological culture. Women with normal flora, or those that responded to antibiotic therapy, had better reproductive outcomes than those who presented with endometrial pathogens at the time of embryo transfer (Figure 5.3), suggesting that pathogenic bacteria present in the cervix adversely impacted pregnancy outcome (67). These studies are limited by the number of bacterial species that can be isolated and identified after culture of the catheter tip and the risk of contamination of the catheter tip in the vagina, ectocervix, or endocervix or during manipulation. Consequently, no consensus has been reached to date regarding the origin and identification of endocervical bacterial pathogens, nor has there been progress in understanding the mechanisms that could interfere with embryonic implantation. Another piece of information supporting the presence of an endometrial microbiome came from the microbiological culture of endometrial samples obtained directly from hysterectomy specimens, which avoid contamination by the cervix (68). Recently, molecular identification of bacterial species in the endometrium of asymptomatic patients undergoing hysterectomy for benign indications confirmed that the uterine cavity is not sterile (69). In addition, a murine model of ascending bacterial infection supports the concept that the endometrium might not be as sterile as previously thought (70) (Figure 5.4). A recent study has reported the endometrial microbiome at the time of embryo transfer by sequencing the bacterial 16S rRNA gene in DNA purified from the transfer catheter tip of 33 patients undergoing IVF. The most represented bacteria found in the overall samples belong to the Lactobacillus and Flavobacterium genera. However, when the endometrial microbiomes of patients with ongoing versus nonongoing pregnancies were compared, the presence or abundance of Lactobacillus was not significantly different between these two groups, while ongoing pregnancy seemed associated with higher abundance of Acinetobacter and Pseudomonas species (71). Our group recently analyzed bacterial communities present in paired endometrial fluid and vaginal aspirate samples from fertile asymptomatic women by pyrosequencing bacterial 16S rRNA (72). The results indicated that the bacterial species colonizing the vagina and ­uterine cavity differ in some women in both structure and ­composition, corroborating the existence of endometrial microbiota that are not identical to vaginal microbiota in every woman (Figure 5.5). One of the main successes of the study was the analysis of endometrial fluid while avoiding contamination with vaginal bacteria. Individual women’s unique endometrial microbiome profiles can be classified as Lactobacillus dominated (LD) or non-Lactobacillus dominated (NLD) based on the relative abundance of bacterial operational taxonomic units (OTUs) found in the endometrial fluid samples. This provides interesting opportunities to study endometrial

Microbiome of the reproductive tract  69

Table 5.1  Assisted reproduction technique outcomes in the patients positive and negative for culture of the catheter tip at the time of embryo transfer No. patients analyzed

Patients positive for culture (%)

Egbase et al. (1996)

110

54 (49.1%)

Fanchin et al. (1998)

279

143 (51.2%)

Egbase et al. (1999)

430

297 (69%)

Moore et al. (2000)

127

109 (85.8%)

Salim et al. (2002)

204

129 (63.2%)

Selman et al. (2007)

152

133 (87.5%)

Study

IR (%) positive vs. negative

Pathogens isolated (% patients) Escherichia coli (25.9%) Klebsiella pneumoniae (9.2%) Staphylococcus sp. (7.4%) Streptococcus sp. (61.1%) Anaerobic (5%) Enterobacteriaceae (5%) Enterococcus sp. (3%) Escherichia coli (64%) Haemophylus sp. (2%) Staphylococcus sp. (3%) Streptococcus sp. (8%) Miscellaneous (10%) Enterococcus sp. (59.4%) Staphylococcus sp. (31.2%) Escherichia coli (1.6%) Mixed growth (7.8%) Anaerobic Gram positive (45%) Enterococcus (27%) Staphylococcus epidermidis (75%) Streptococcus viridans (6%) Anaerobic (29.5%) Escherichia coli (9.3%) Gram-positive bacteria (35.6%) Other Gram negative (5.4%) Enterobacteriaceae (65.1%) Staphylococcus sp.(45%) Streptococcus sp. (28.2%) Other pathogens (18.3%)

PR (%) positive vs. negative

MR (%) positive vs. negative

References

NA

29.6 vs. 57.1 (p