Fertility, Pregnancy, and Wellness [1 ed.] 0128183098, 9780128183090

Table of contents :
Front Cover
Fertility, Pregnancy, and Wellness
Copyright Page
Contents
List of contributors
About the editors
Preface
1 The importance of healthy lifestyles in helping achieving wellbeing
Relation between lifestyle and health
The study of number of publications: bibliometric analysis
Relationship between lifestyle, habits, and wellbeing
Role of wellbeing and lifestyles in women’s reproduction
Conclusions
References
I. Background
2 Male reproductive system
Anatomy of the male reproductive system
Testis and epididymis
Spermatic cord
Ductus deferens
Seminal vesicle and ejaculatory duct
Prostate
Bulbourethral glands
Penis
Urethra
Scrotum
Perineum
Physiology of the male reproductive system
Endocrine regulation of the reproductive tract
Spermatogenesis and semen
Erectile and ejaculatory functions
Semen analysis: a short primer
References
3 Female reproductive system
Female anatomy and introduction to physiology for the female reproductive system
Internal organs
Ovary
Female reproductive tract
Fallopian tube
Uterus
Vagina
External organs
Perineum
Vulva
Detailed physiological aspects of the hypothalamic–pituitary–ovarian–endometrial axis
Introduction
Hypothalamus and pituitary gland
GnRH neuronal system
The GnRH pulse generator
Kisspeptin neurons
The ovarian cycle
Development of ovarian follicles: gonadotropin-independent growth phase
Development of ovarian follicles: gonadotropin-dependent follicular growth
Follicular recruitment, selection, and dominance
Ovulation: the LH surge and its consequences
The corpus luteum
The endometrial cycle: proliferative and secretory phases and establishment of embryo receptivity (window of implantation)
Conclusion
References
4 Conception and pregnancy
Introduction
Short review of male and female anatomy and physiology for understanding conception and pregnancy
Male
Scrotum and testis
Testis and spermatogenesis
Male reproductive tract and sperm pathway during ejaculation
Female
Overview of the female gamete: steps to ovulation and fertilization
Follicle-stimulating hormone, luteinizing hormone, and Estradiol-17β
Progesterone (P4) and ovulation of a mature ovum
Fertilization
Implantation and pregnancy
Maternal changes in pregnancy
Embryo development
Zygote to hatching blastocyst, sometimes called the preimplantation embryo
Implantation
Formation of bilaminar embryo, placenta, amnion, chorion and yolk sac
Gastrulation or trilaminar embryo formation
Embryonic folding
Organogenesis
Heart development and fetal circulation
Digestive system development and lung development
Muscular skeletal development
Central nervous system development
The placenta as an endocrine organ
Fetal development
Parturition
Term pregnancy
Phases of labor
First stage
Second stage
Third stage
Conclusion
References
5 Genetics and epigenetics of healthy gametes, conception, and pregnancy establishment: embryo, mtDNA, and disease
Gametogenesis and genetic healthy gametes
Gametic mitochondrial DNA and embryo quality
The genetics of fertilization
Embryonic genome activation
Embryo implantation
Mitochondrial diseases
Epigenetic modifications
Genomic imprinting and developmental disorders
Epigenetic reprogramming in the gametes and embryo
Environmental factors and epigenetic changes
Conclusions
Acknowledgments
References
II. Fertility - IIA
6 A contemporary view on global fertility, infertility, and assisted reproductive techniques
Introduction
Definitions of infertility
Prevalence of infertility
Classification of infertility
Male infertility
Causes of male infertility
Lifestyle practices and male infertility
Smoking
Alcohol
Diabetes mellitus and male infertility
Genetic mutations and male infertility
Female infertility
Causes of female infertility
Anatomical abnormalities and female infertility
Ovulatory and menstrual disorders
Polycystic ovary syndrome
Lifestyle practices and female infertility
Smoking
Alcohol
Genetic mutations and female infertility
Overview of fertility evaluation
Clinical history and physical examination
Female infertility evaluation
Ovarian reserve testing and female reproductive ageing
Evaluation of the ovarian reserve
Follicle stimulating hormone analysis
Estradiol quantification
Anti-Müllerian hormone test
Counting ovarian antral follicles by ultrasound
Detection of ovulation
Evaluation of tubal diseases
Evaluation of cervical integrity
Uterine evaluation
Endometriosis
Male infertility evaluation
Medical history
Physical examination
Laboratory examination
Sperm analysis
Endocrine evaluation
Supplementary examination
Reactive oxygen species and DNA integrity
Sperm antibodies
Genetic evaluation of male infertility
Assisted reproductive technology
Low complexity assisted reproductive technology
Intrauterine insemination
Programmed intercourse
High complexity assisted reproductive technology
Conventional in vitro fertilization and intracytoplasmic sperm injection
Oocyte and embryo vitrification
In vitro embryo culture
Assisted hatching
Preimplantation genetic testing
The effects of stress on fertility and assisted reproductive technology outcome
Conclusion
Acknowledgement
References
7 Epidemiologic evidence of the impact of diverse chemicals on fertility
Historical background
Collective intoxications affecting reproduction
Current patterns of change in female employment
Occupational risks and reproduction
Legislative aspects to be protected
Classification and labeling of chemical substances which affect reproduction: mutagenic substances
Toxicological aspects of interest: toxic-kinetic, maximum allowable concentration and quantitative structure–activity relat...
Prevalence of adverse effects on reproduction
Chemical agents capable of causing effects on fertility
Risk assessment on reproduction
Female
Male
Reproductive toxicity test
Conclusion
References
8 Effect of exercise and lifestyles on male reproductive potential
Introduction
Exercise-related consequences of resistance and endurance training
Resistance training
Resistance training, HPT axis response and improvement of male reproductive potential
Resistance training versus pelvic floor muscles versus erectile dysfunction
Endurance training
Endurance training to improve male reproductive potential
Endurance training versus oxidative stress versus male reproductive potential
Medical conditions and male reproductive potential: related exercise consequences
Obesity
Type 2 diabetes versus male reproductive potential
Training proposed for subfertile subjects with obesity and/or diabetes
Microtraumatism versus reproductive potential
Thermal stress versus reproductive potential
General nutritional recommendations to improve reproductive potential
Importance of correcting unhealthy lifestyles
Environmental factors: endocrine disruptors and male fertility
Conclusion
References
9 Nutrition and exercise intervention for female fertility
Introduction
Fertility
Fertility and the overall diet considerations
Fertility and specific nutrients
Prepregnancy and pregnancy
Prepregnancy, pregnancy and nutrition
Micronutrients considerations
Special nutritional considerations in pregnancy
Body weight status, fertility, and energy intake (macronutrient) needs with pregnancy
Exercise considerations
Exercise, fertility and prepregnancy
In vitro fertilization and exercise
Exercise and nutrient needs
Additional considerations
Type 2 diabetes mellitus
Sleep
Stress
Exposure to endocrine disruptors
Conclusions
References
II. Fertility - IIB
10 Psychological distress and infertility: prevalence, impact, and interventions
Infertility
Definition and related terminology
Prevalence rates
Etiology and risk factors
Reproductive medical treatment
Definitions
Prevalence rates
Outcomes
Consequences
Infertility-related distress
Definition
Prevalence rates
Risk factors
Consequences
Psychosocial support and infertility-related distress
Assessment: recommendations for the clinician
First appointment
Evidence-based psychological interventions for infertility
Additional supports for those experiencing infertility-related distress
Self-help suggestions
Self-help resources
Self-administered interventions
Additional evidence-based interventions to consider
Psychopharmacology
Conclusion
References
11 Mindfulness and yoga approach for fertility: the benefits of mindfulness in human reproduction treatments
Introduction
Mindfulness: origin and meaning
Yoga: origin and meaning
Effect of mindfulness and yoga practice in assisted reproduction treatment
Implications to the field and conclusions
References
12 Epigenetic and biological consequences of unmanaged stress during prenatal, perinatal and early childhood periods
Psychoemotional health and well-being in the perinatal period
The negative consequences of stress and distress
Maternal stress and health behaviors
Effects of maternal prenatal stress on the offspring
Biological and epigenetic basis for alterations
Childhood maltreatment as intergenerational transmitter of fetal programming
HPA axis alterations, inflammation, telomere shortening, and well-being
Conclusion
References
13 Traditional Chinese medicine, Ayurveda, and fertility
Traditional Chinese medicine and Ayurvedic approaches to fertility
The body as a unified whole, not a series of parts
Personalized treatment and balance
Focus on root causes of imbalance
Require active participation of the patient
Focus on prevention of illness and optimizing well-being
Optimizing fertility with traditional Chinese medicine
Till the soil before planting the seed
Fundamental elements of traditional Chinese medicine
Yin and Yang
The four substances: Qi (energy), Xue (blood), Jinye (fluids), Jing (essence)
The internal organs
Common patterns of imbalance
Imbalances of Qi
Qi stagnation
Qi deficiency
Phlegm and Dampness
Imbalances of blood
Blood deficiency
Blood stasis
Imbalances of Yin and Yang
Jing deficiency
Causes of illness
External causes of illness
Internal causes of illness
Miscellaneous (noninternal, nonexternal) causes of illness
Tools of traditional Chinese medicine diagnosis
Visual examination
Inquiry
Tongue and pulse diagnosis
Palpation
Basal body temperature chart analysis
Traditional Chinese medicine pattern diagnosis
One disease, different patterns
One pattern, different diseases
Traditional Chinese medicine treatment methods
Acupuncture
Acupuncture and mechanism of action
Acupuncture and in vitro fertilization research
Complexities of acupuncture and fertility research
Acupuncture and polycystic ovary syndrome research
Acupuncture to promote natural fertility research
Acupuncture and stress of infertility research
Acupuncture and sperm research
Whole systems traditional Chinese medicine and fertility research
Chinese herbal medicine
Chinese herbal medicine research
Nutrition
Lifestyle guidance
Sleep
Smoking and vaping
Movement
Taoist sexual practices
Retention of semen
Pelvic floor strengthening
Regulating emotions
Specific guidelines for women
Specific guidelines for men
Pattern-specific nutrition and lifestyle guidelines
Summary
Ayurveda: general concept
Fundamental principles of Ayurveda
Prakriti
Ayurveda in health maintenance
Ayurvedic vision of diet
Food to pacify vata, pitta, and kapha
Diet to pacify vata
Diet to pacify pitta
Diet to pacify kapha
Ayurveda in the treatment of disease
Panchakarma
Ayurveda and reproduction
Fertility
Fertility disorders
Ayurveda and herbal compounds
Aphrodisiac herbal compounds
Ayurvedic compounds for the treatment of infertility
Ayurvedic herbal compounds for male infertility
Mucuna pruriens
Withania somnifera
Pedalium murex
Cassia auriculata
Tribulus terrestris
Curculigo orchioides
Anacyclus pyrethrum and Ionidium suffruticosum
Syzygium aromaticum
Centella asiatica
Asparagus racemosus, Chlorophytum borivilianum, Dactylorhiza hatagirea
Polyherbal complexes
Ayurvedic herbal compounds for female infertility
Asparagus racemosus and Aloe barbadensis in polycystic ovary syndrome
Phyllanthus emblica
Summary
Acknowledgments
References
14 Biofield and manipulative therapies for emotional wellbeing and fertility
Introduction
Biofield and manipulative therapies and birthing
Biofield therapies for fertility
Energy therapies
Reiki
Gemotherapy/crystal therapy
Therapeutic touch/healing touch/spiritual touch
Biomagnetic therapy
Qi Gong
Flower remedies
Metamorphic therapy
Manipulative therapies for fertility and pregnancy
Physical therapy
Osteopathy
Chiropractic
Chiropractic in fertility care
Chiropractic in pregnancy care
Conclusions
Acknowledgment
References
15 Diverse complementary therapies for fertility-related emotional and physical wellbeing
Introduction
Fertility and infertility—causes and consequences of stress and emotional challenge
Hypnosis and emotional wellbeing in relation to fertility
Aromatherapy and emotional/mood issues in fertility
Aromatherapy and specific mental health and emotional challenges for fertility
Touch, massage, and emotional wellbeing
Sound and music and fertility
Ayurveda and emotional wellbeing in relation to fertility
What is Ayurveda?
Ayurveda and infertility
Conclusion
References
Further reading
16 Phytotherapeutic support for infertility: evaluating the evidence
Gametogenesis and early embryogenesis
Spermatogenesis and male reproduction
Oogenesis, folliculogenesis, and early embryogenesis
Factors in infertility
Male infertility, subfertility, and dietary factors
Female infertility, subfertility, and dietary factors
Reproductive disorders, infertility, and preconception considerations
Nutraceuticals overview and regulation
Historical applications and infertility
Contemporary nutraceuticals in fertility management
Ashwagandha (Withania somnifera)
Astragalus (Astragalus membranaceus)
Black cohosh (Cimicifuga racemose L.)
Blackcurrant (Ribes nigrum L.)
Chasteberry (Vitex agnus-castus)
Dong quai (Angelica sinensis)
Ginseng (Panax ginseng)
Fennel seeds (Foeniculum vulgare Mill.)
Maca (Lepidium meyenii)
Oats (Avena sativa)
Tea (Camellia sinensis)
Tongkat ali (Eurycoma longifolia)
Tribulus terrestris L
Yerba maté (Ilex paraguariensis St. Hilare)
Summary of key points
Conclusion
References
III. Pregnancy - IIIA
17 Nutrition for a healthy pregnancy and environment
Introduction
What do we understand by a “healthy” diet in the adult?
The most relevant modifications during pregnancy with an impact on the nutritional needs of the expectant mother
Modifications in the uterus and ovaries
Modifications in the breast
Cardiovascular modifications and hematologic changes
Modifications in the digestive tract
Modifications in the urinary system
Endocrine modifications
Changes in weight
Nutritional needs in expectant women
Energy requirements
Carbohydrate requirements
Lipid requirements
Protein requirements
Micronutrient (vitamins and minerals) requirements
Consumption of other substances
Dietary recommendations for the expectant mother
Take home message
References
18 Exercise and pregnancy
Introduction
Physical and physiological changes during pregnancy
Prepregnancy exercise
Exercise recommendations during pregnancy
When to stop exercising during pregnancy
Acute responses to exercise during pregnancy
Absolute and relative contraindications to exercise during pregnancy
Weight gain and exercise during pregnancy
Exercise in clinical conditions and common discomforts
Exercise and morning sickness/hyperemesis gravidarium
Exercise and preeclampsia
Exercise to prevent and/or treat gestational diabetes
Bed rest
Benefits of exercise for mother and baby
Elite athletes and pregnancy
Exercise in the postpartum period
Female pelvis and pelvic floor
Postural evolution during pregnancy and delivery and its relationship with the pelvic floor
Characteristics of the main pelvic floor dysfunctions
Postural evolution and pelvic floor consequences
Individual factors related to pelvic floor disorders
Pre- and postpartum pelvic floor exercises
Conclusion
References
III. Pregnancy - IIIB - Holistic, mind-body and complementary approaches for a healthy pregnancy
19 The importance of a healthy psychological approach to life and how trauma can influence health, pregnancy, and children
What is good psychological health? What is good mental health?
What are the factors that influence one’s mental health, and how does maternal mental health impact overall health and psyc...
To which extent does mental health impact the fetus and infant into childhood?
What is trauma and how does it impact mental health?
Trauma across the generations and the role of epigenetics
Benefits of addressing maternal mental health
Beneficial strategies to improve maternal mental health
Labor, delivery, and lactation
Conclusion
References
20 The importance of yoga and mindfulness during pregnancy
Introduction
What is yoga?
Background
Pranayama and breath in prenatal yoga asana
Effects on the mother and fetus
Prenatal yoga benefits on the mother at the physical level
Yoga modifications to accommodate for changes in the different trimesters and benefits
Prenatal yoga benefits on the mother at the psychoemotional level
Effects on the fetus
Prenatal yoga practice
General guidelines before engaging in a prenatal yoga practice
Recommended asanas per trimester
First trimester
Second trimester
Third trimester
Sequencing of prenatal poses for the trimesters
Yoga types and asanas not recommended in pregnancy
Yoga in clinical conditions and common discomforts
Back pain
Gestational diabetes
Yoga asana and pranayama in preparation for labor
Yoga asana and pranayama in the postpartum phase
What is mindfulness?
Background
Principles and objectives in mindfulness
Types of mindfulness practices and intervention models
Levels of action and benefits of interventions through mindfulness
Effect at the neurological and hormonal levels
Meditation as the basis of mindfulness
Mindfulness and pregnancy
Pregnancy-specific mindfulness programs
Mindfulness and maternal-fetal bonding
Mindfulness and prenatal stress and depression
Mindfulness and maternal health
Mindful eating in pregnancy
Gestational diabetes mellitus
Cardiovascular health
Preterm complications
Limitations and strengths of mindfulness-based intervention studies
Conclusion
Acknowledgment
References
21 Traditional Chinese medicine and Ayurvedic care during pregnancy
Introduction
Part one: Traditional Chinese medicine
Preparing the body for pregnancy
Yin and Yang
The four substances
General guidelines during pregnancy
Fetal education
Emotions
Diet
Movement
Common patterns of imbalance during pregnancy
Issues relating to TCM research
Safety considerations
Acupuncture
Forbidden points
Chinese Herbal Medicine (CHM)
Traditional Chinese medicine treatment for common issues in pregnancy
Threatened and recurrent miscarriage
Nausea and vomiting
Low back pain, neck pain, headache, pelvic pain
Breech presentation
Normalizing birth and preparing for labor
Anxiety and depression
Hypertension and preeclampsia
Acupuncture for various symptoms of pregnancy
Acupuncture and acupressure in labor and delivery
Postpartum
Conclusion to traditional Chinese medicine
Part two: Ayurveda
Ayurveda in pregnancy
Align with the sunrise to start your day
Practice upon waking
Morning cleanse
Exercise and meditation
Breakfast, lunch, and dinner
Mealtime practice
Afternoon meditation
Social time
Preparing for bed
Sleep
Diet and Pregnancy
Risks and drawbacks of Ayurvedic medicine
Ayurveda in the treatment of conditions and disease that interfere with reproduction and gestation
Anxiety and stress
Hypertension
Preeclampsia
Diabetes
Obesity
Postpartum
Other uses of Ayurvedic medicine: contraception
Male contraceptives
Female contraceptives
Conclusion
Acknowledgments
References
22 Prenatal bonding: the importance of connecting with body and baby
Introduction
Human stress responses
Effects of stress, attachment, and love on physical, psychological, and social wellbeing
Women of the childbearing age and preconception
Fetal development
Postpartum depression and infant wellbeing
Promoting connections between parents and unborn children
Connections before birth
Examining myths of mindfulness practices
Addressing mindfulness as a spiritual practice
Addressing mindfulness as a required state of relaxation
Addressing mindfulness’s contemporary popularity
Take-home message
Key points
Conclusion
References
23 Aromatherapy, music, and acupuncture for optimizing emotional wellbeing in pregnancy
Introduction
Complementary medicine and pregnancy
Aromatherapy for emotional wellbeing during pregnancy
Enhancing emotional and psychological wellbeing during pregnancy using aromatherapy
Combining aromatherapy with other therapies
Using aromatherapy when medical complications affect the pregnancy experience
Using sound and music for emotional wellbeing during pregnancy
Acupuncture, moxibustion, and acupressure in traditional Asian treatments
Conclusion
References
24 Nutraceutical and phytotherapeutic support in pregnancy
Supporting maternal wellbeing with nutraceuticals
Chamomile
German chamomile (Matricaria chamomilla)
Roman chamomile (Anthemis nobilis)
Echinacea
Garlic (Allium sativum L.)
Ginger (Zingiber officinale Roscoe)
Lavender (Lavandula angustifolia Mill.)
Lemon balm (Melissa officinalis L.)
Mallow (Malva sylvestris)
Marshmallow (Althaea officinalis)
Mint
Psyllium (Plantago ovata)
Witch hazel (Hamamelis virginiana)
Other herbs and phytotherapeutic plants
Limitations, precautions, contraindications, and special considerations
Summary of key points
Conclusion
References
25 Ethics in fertility and pregnancy management
Ethics of fertility treatments
Introduction on assisted reproductive techniques
Principles of medical ethics
Counseling and shared decision making
Examples of fertility treatments for ethical considerations
Fertility procedures
Disclosure of success rates
Ovulation induction cycles
In vitro fertilization: number of embryos to transfer
Futility continuing treatment
In vitro fertilization: preimplantation genetic testing
Embryo sex selection
Donor gametes
Posthumous reproduction
Uterus transplantation
Mitochondrial DNA transfer
Germline gene editing
Misconceptions
Summary
Ethics, informed consent, and childbearing
References
26 Conclusion
Index
Back Cover

Citation preview

Fertility, Pregnancy, and Wellness

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Fertility, Pregnancy, and Wellness Edited by

Diana Vaamonde Morphological Sciences Department, School of Medicine and Nursing, Universidad de Cordoba, Cordoba, Spain; INPEF—International Network on Physical Exercise and Fertility, Cordoba, Spain

Anthony C. Hackney University of North Carolina, Chapel Hill, NC, USA

Juan Manuel Garcia-Manso University of Las Palmas de Gran Canaria, Canary Islands, Spain

Elsevier Radarweg 29, PO Box 211, 1000 AE Amsterdam, Netherlands The Boulevard, Langford Lane, Kidlington, Oxford OX5 1GB, United Kingdom 50 Hampshire Street, 5th Floor, Cambridge, MA 02139, United States Copyright © 2022 Elsevier Inc. All rights reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Details on how to seek permission, further information about the Publisher’s permissions policies and our arrangements with organizations such as the Copyright Clearance Center and the Copyright Licensing Agency, can be found at our website: www.elsevier.com/permissions. This book and the individual contributions contained in it are protected under copyright by the Publisher (other than as may be noted herein). Notices Knowledge and best practice in this field are constantly changing. As new research and experience broaden our understanding, changes in research methods, professional practices, or medical treatment may become necessary. Practitioners and researchers must always rely on their own experience and knowledge in evaluating and using any information, methods, compounds, or experiments described herein. In using such information or methods they should be mindful of their own safety and the safety of others, including parties for whom they have a professional responsibility. To the fullest extent of the law, neither the Publisher nor the authors, contributors, or editors, assume any liability for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions, or ideas contained in the material herein. British Library Cataloguing-in-Publication Data A catalogue record for this book is available from the British Library Library of Congress Cataloging-in-Publication Data A catalog record for this book is available from the Library of Congress ISBN: 978-0-12-818309-0 For Information on all Elsevier publications visit our website at https://www.elsevier.com/books-and-journals

Publisher: Stacy Masucci Acquisitions Editor: Patricia Osborne Editorial Project Manager: Pat Gonzalez Production Project Manager: Kiruthika Govindaraju Cover Designer: Matthew Limbert Typeset by MPS Limited, Chennai, India

Contents

List of contributors About the editors Preface

xiii xv xvii

1. The importance of healthy lifestyles in helping achieving wellbeing 1 Manuel Vaquero-Abellan, Francisco Genil Marquez and Pilar Aparicio Martı´nez Relation between lifestyle and health The study of number of publications: bibliometric analysis Relationship between lifestyle, habits, and wellbeing Role of wellbeing and lifestyles in women’s reproduction Conclusions References

1 2 4 14 16 16

Part I Background

21

2. Male reproductive system

23

Juan Andre´s Ramı´rez-Gonza´lez and Andrea Sansone Anatomy of the male reproductive system Testis and epididymis Spermatic cord Ductus deferens Seminal vesicle and ejaculatory duct Prostate Bulbourethral glands Penis Urethra Scrotum Perineum Physiology of the male reproductive system Endocrine regulation of the reproductive tract

23 23 24 25 25 27 28 29 30 31 31 32 32

Spermatogenesis and semen Erectile and ejaculatory functions Semen analysis: a short primer References

3. Female reproductive system

33 34 35 35

37

Joao Sabino Cunha Filho, R. James Swanson, Bo Liu and Sergio Oehninger Female anatomy and introduction to physiology for the female reproductive system Internal organs External organs Detailed physiological aspects of the hypothalamic
pituitary
ovarian
endometrial axis Introduction The ovarian cycle Development of ovarian follicles: gonadotropin-independent growth phase Development of ovarian follicles: gonadotropin-dependent follicular growth Follicular recruitment, selection, and dominance Ovulation: the LH surge and its consequences The corpus luteum The endometrial cycle: proliferative and secretory phases and establishment of embryo receptivity (window of implantation) Conclusion References

4. Conception and pregnancy

37 38 39 40 40 44 44 45 45 46 47 48 49 49

53

R. James Swanson and Bo Liu Introduction Short review of male and female anatomy and physiology for understanding conception and pregnancy Fertilization

53

53 60 v

vi

Contents

Implantation and pregnancy Parturition Conclusion References

5. Genetics and epigenetics of healthy gametes, conception, and pregnancy establishment: embryo, mtDNA, and disease

60 69 71 71

73

Ciro Dresch Martinhago and Cristiana Libardi Miranda Furtado Gametogenesis and genetic healthy gametes Gametic mitochondrial DNA and embryo quality The genetics of fertilization Embryonic genome activation Embryo implantation Mitochondrial diseases Epigenetic modifications Genomic imprinting and developmental disorders Epigenetic reprogramming in the gametes and embryo Environmental factors and epigenetic changes Conclusions Acknowledgments References

Part II Fertility - IIA 6. A contemporary view on global fertility, infertility, and assisted reproductive techniques

73 75 75 76 77 78 80 81 83 84 85 85 85

91

93

Natalibeth Barrera, Temidayo S Omolaoye and Stefan S Du Plessis Introduction Definitions of infertility Prevalence of infertility Classification of infertility Male infertility Causes of male infertility Lifestyle practices and male infertility Diabetes mellitus and male infertility Genetic mutations and male infertility Female infertility Causes of female infertility Anatomical abnormalities and female infertility Ovulatory and menstrual disorders Polycystic ovary syndrome Lifestyle practices and female infertility Genetic mutations and female infertility

93 94 94 95 96 96 97 97 97 98 98 98 98 98 98 99

Overview of fertility evaluation Clinical history and physical examination Female infertility evaluation Male infertility evaluation Assisted reproductive technology Low complexity assisted reproductive technology High complexity assisted reproductive technology The effects of stress on fertility and assisted reproductive technology outcome Conclusion Acknowledgement References

7. Epidemiologic evidence of the impact of diverse chemicals on fertility

99 99 100 104 108 108 109 112 113 113 113

121

Manuel Vaquero-Abellan, Fernando Gil Herna´ndez and Pilar Aparicio Martı´nez Historical background Collective intoxications affecting reproduction Current patterns of change in female employment Occupational risks and reproduction Legislative aspects to be protected Classification and labeling of chemical substances which affect reproduction: mutagenic substances Toxicological aspects of interest: toxic-kinetic, maximum allowable concentration and quantitative structure
activity relationship (chemical structure relation activity) Prevalence of adverse effects on reproduction Chemical agents capable of causing effects on fertility Risk assessment on reproduction Female Male Reproductive toxicity test Conclusion References

8. Effect of exercise and lifestyles on male reproductive potential

121 121 122 122 123 123

124 125 125 126 126 127 129 129 129

131

Diana Vaamonde, Juan Manuel Garcia-Manso and Anthony C. Hackney Introduction Exercise-related consequences of resistance and endurance training Resistance training Endurance training

131 131 131 133

Contents

Medical conditions and male reproductive potential: related exercise consequences Obesity Type 2 diabetes versus male reproductive potential Training proposed for subfertile subjects with obesity and/or diabetes Microtraumatism versus reproductive potential Thermal stress versus reproductive potential General nutritional recommendations to improve reproductive potential Importance of correcting unhealthy lifestyles Environmental factors: endocrine disruptors and male fertility Conclusion References

9. Nutrition and exercise intervention for female fertility

134 134 135 135 136 136 137 139 140 141 141

149

Anthony C. Hackney, Diana Vaamonde and Juan Manuel Garcia-Manso Introduction Fertility Fertility and the overall diet considerations Fertility and specific nutrients Prepregnancy and pregnancy Prepregnancy, pregnancy and nutrition Micronutrients considerations Special nutritional considerations in pregnancy Body weight status, fertility, and energy intake (macronutrient) needs with pregnancy Exercise considerations Exercise, fertility and prepregnancy In vitro fertilization and exercise Exercise and nutrient needs Additional considerations Type 2 diabetes mellitus Sleep Stress Exposure to endocrine disruptors Conclusions References

Part II Fertility - IIB

149 149 149 150 151 151 151 151 152 153 153 155 155 156 156 156 156 157 157 157

161

10. Psychological distress and infertility: prevalence, impact, and interventions 163 Jessica Clifton and Alice D. Domar Infertility Definition and related terminology

163 163

Prevalence rates Etiology and risk factors Reproductive medical treatment Definitions Prevalence rates Outcomes Consequences Infertility-related distress Definition Prevalence rates Risk factors Consequences Psychosocial support and infertility-related distress Assessment: recommendations for the clinician First appointment Evidence-based psychological interventions for infertility Additional supports for those experiencing infertility-related distress Self-help suggestions Self-administered interventions Additional evidence-based interventions to consider Psychopharmacology Conclusion References

vii

164 165 166 166 166 167 167 168 168 169 169 169 170 171 171 172 173 173 173 174 174 175 175

11. Mindfulness and yoga approach for fertility: the benefits of mindfulness in human reproduction treatments 183 Rachel M.M. Tardin, Pilar Aparicio Martı´nez, Marı´lia Porto Bonow and Alessandro Schuffner Introduction Mindfulness: origin and meaning Yoga: origin and meaning Effect of mindfulness and yoga practice in assisted reproduction treatment Implications to the field and conclusions References

183 184 185 186 188 189

12. Epigenetic and biological consequences of unmanaged stress during prenatal, perinatal and early childhood periods 193 Diana Vaamonde, Carolina Algar-Santacruz and Dana M. Dillard Psychoemotional health and well-being in the perinatal period The negative consequences of stress and distress

193 193

viii

Contents

Maternal stress and health behaviors Effects of maternal prenatal stress on the offspring Biological and epigenetic basis for alterations Childhood maltreatment as intergenerational transmitter of fetal programming HPA axis alterations, inflammation, telomere shortening, and well-being Conclusion References

13. Traditional Chinese medicine, Ayurveda, and fertility

194 197 198 201 203 205 205

209

Lara Rosenthal, Paula Hernandez and Diana Vaamonde Traditional Chinese medicine and Ayurvedic approaches to fertility The body as a unified whole, not a series of parts Personalized treatment and balance Focus on root causes of imbalance Require active participation of the patient Focus on prevention of illness and optimizing well-being Optimizing fertility with traditional Chinese medicine Till the soil before planting the seed Fundamental elements of traditional Chinese medicine Yin and Yang The four substances: Qi (energy), Xue (blood), Jinye (fluids), Jing (essence) The internal organs Common patterns of imbalance Imbalances of Qi Imbalances of blood Imbalances of Yin and Yang Jing deficiency Causes of illness External causes of illness Internal causes of illness Miscellaneous (noninternal, nonexternal) causes of illness Tools of traditional Chinese medicine diagnosis Visual examination Inquiry Tongue and pulse diagnosis Palpation Basal body temperature chart analysis Traditional Chinese medicine pattern diagnosis One disease, different patterns One pattern, different diseases

209 209 210 210 210 210 210 211 211 211 211 212 212 213 213 214 214 215 215 215 216 216 216 216 216 216 216 217 217 218

Traditional Chinese medicine treatment methods Acupuncture Acupuncture and mechanism of action Acupuncture and in vitro fertilization research Complexities of acupuncture and fertility research Acupuncture and polycystic ovary syndrome research Acupuncture to promote natural fertility research Acupuncture and stress of infertility research Acupuncture and sperm research Whole systems traditional Chinese medicine and fertility research Chinese herbal medicine Chinese herbal medicine research Nutrition Lifestyle guidance Sleep Smoking and vaping Movement Taoist sexual practices Retention of semen Pelvic floor strengthening Regulating emotions Specific guidelines for women Specific guidelines for men Pattern-specific nutrition and lifestyle guidelines Summary Ayurveda: general concept Ayurveda in health maintenance Ayurvedic vision of diet Food to pacify vata, pitta, and kapha Ayurveda in the treatment of disease Panchakarma Ayurveda and reproduction Fertility Fertility disorders Ayurveda and herbal compounds Aphrodisiac herbal compounds Ayurvedic compounds for the treatment of infertility Polyherbal complexes Summary Acknowledgments References

14. Biofield and manipulative therapies for emotional wellbeing and fertility

218 219 219 219 220 220 221 221 221 222 222 224 224 225 225 225 225 225 226 226 226 227 227 227 227 227 229 229 231 233 233 235 235 236 236 236 237 240 242 242 242

249

Alys Einion Introduction

249

Contents

Biofield and manipulative therapies and birthing Biofield therapies for fertility Energy therapies Reiki Gemotherapy/crystal therapy Therapeutic touch/healing touch/spiritual touch Biomagnetic therapy Qi Gong Flower remedies Metamorphic therapy Manipulative therapies for fertility and pregnancy Physical therapy Osteopathy Chiropractic Conclusions Acknowledgment References

15. Diverse complementary therapies for fertility-related emotional and physical wellbeing

251 251 252 252 252 252 253 254 254 255 255 255 256 257 259 260 260

265

Alys Einion Introduction Fertility and infertility—causes and consequences of stress and emotional challenge Hypnosis and emotional wellbeing in relation to fertility Aromatherapy and emotional/mood issues in fertility Aromatherapy and specific mental health and emotional challenges for fertility Touch, massage, and emotional wellbeing Sound and music and fertility Ayurveda and emotional wellbeing in relation to fertility What is Ayurveda? Ayurveda and infertility Conclusion References Further reading

16. Phytotherapeutic support for infertility: evaluating the evidence

283 283 284 284 284 285 285 285 285 285 286 286 286 286 287 287 287 287 287 287 288 288 288

265

266

Part III Pregnancy - IIIA

293

267 269 270 271 272 274 274 275 276 276 280

281

Dana M. Dillard Gametogenesis and early embryogenesis Spermatogenesis and male reproduction Oogenesis, folliculogenesis, and early embryogenesis Factors in infertility

Male infertility, subfertility, and dietary factors Female infertility, subfertility, and dietary factors Reproductive disorders, infertility, and preconception considerations Nutraceuticals overview and regulation Historical applications and infertility Contemporary nutraceuticals in fertility management Ashwagandha (Withania somnifera) Astragalus (Astragalus membranaceus) Black cohosh (Cimicifuga racemose L.) Blackcurrant (Ribes nigrum L.) Chasteberry (Vitex agnus-castus) Dong quai (Angelica sinensis) Ginseng (Panax ginseng) Fennel seeds (Foeniculum vulgare Mill.) Maca (Lepidium meyenii) Oats (Avena sativa) Tea (Camellia sinensis) Tongkat ali (Eurycoma longifolia) Tribulus terrestris L Yerba mate´ (Ilex paraguariensis St. Hilare) Summary of key points Conclusion References

ix

281 281 282 283

17. Nutrition for a healthy pregnancy and environment

295

Guillermo Molina-Recio Introduction What do we understand by a “healthy” diet in the adult? The most relevant modifications during pregnancy with an impact on the nutritional needs of the expectant mother Modifications in the uterus and ovaries Modifications in the breast Cardiovascular modifications and hematologic changes Modifications in the digestive tract Modifications in the urinary system Endocrine modifications Changes in weight Nutritional needs in expectant women Energy requirements Carbohydrate requirements Lipid requirements

295 295

299 300 300 300 301 301 301 301 302 302 306 308

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Contents

Protein requirements Micronutrient (vitamins and minerals) requirements Consumption of other substances Dietary recommendations for the expectant mother Take home message References

18. Exercise and pregnancy

310 312 314 315 316 316

319

Ritva S. Mikkonen, Daiana P. Rodrigues-de-Souza and Johanna K. Ihalainen Introduction Physical and physiological changes during pregnancy Prepregnancy exercise Exercise recommendations during pregnancy When to stop exercising during pregnancy Acute responses to exercise during pregnancy Absolute and relative contraindications to exercise during pregnancy Weight gain and exercise during pregnancy Exercise in clinical conditions and common discomforts Exercise and morning sickness/hyperemesis gravidarium Exercise and preeclampsia Exercise to prevent and/or treat gestational diabetes Bed rest Benefits of exercise for mother and baby Elite athletes and pregnancy Exercise in the postpartum period Female pelvis and pelvic floor Postural evolution during pregnancy and delivery and its relationship with the pelvic floor Characteristics of the main pelvic floor dysfunctions Postural evolution and pelvic floor consequences Individual factors related to pelvic floor disorders Pre- and postpartum pelvic floor exercises Conclusion References

Part III Pregnancy - IIIB - Holistic, mind-body and complementary approaches for a healthy pregnancy

343

19. The importance of a healthy psychological approach to life and how trauma can influence health, pregnancy, and children

345

Kelly Buckingham and Lynn Gordon 319 320 322 323 324 324 325 326 326 326 326 326 327 327 328 329 329

331 331 331 332 333 334 335

What is good psychological health? What is good mental health? What are the factors that influence one’s mental health, and how does maternal mental health impact overall health and psychology of mother and child? To which extent does mental health impact the fetus and infant into childhood? What is trauma and how does it impact mental health? Trauma across the generations and the role of epigenetics Benefits of addressing maternal mental health Beneficial strategies to improve maternal mental health Labor, delivery, and lactation Conclusion References

20. The importance of yoga and mindfulness during pregnancy

345

347 349 351 353 354 354 360 360 361

367

Diana Vaamonde, Carolina Algar-Santacruz, Jennifer Pettit, Blanca Chacon and Dana M. Dillard Introduction What is yoga? Background Pranayama and breath in prenatal yoga asana Effects on the mother and fetus Prenatal yoga benefits on the mother at the physical level Prenatal yoga benefits on the mother at the psychoemotional level Effects on the fetus Prenatal yoga practice

367 367 367 368 371 371 372 373 373

Contents

General guidelines before engaging in a prenatal yoga practice Recommended asanas per trimester Sequencing of prenatal poses for the trimesters Yoga types and asanas not recommended in pregnancy Yoga in clinical conditions and common discomforts Back pain Gestational diabetes Yoga asana and pranayama in preparation for labor Yoga asana and pranayama in the postpartum phase What is mindfulness? Background Principles and objectives in mindfulness Types of mindfulness practices and intervention models Levels of action and benefits of interventions through mindfulness Effect at the neurological and hormonal levels Meditation as the basis of mindfulness Mindfulness and pregnancy Pregnancy-specific mindfulness programs Mindfulness and maternal-fetal bonding Mindfulness and prenatal stress and depression Mindfulness and maternal health Mindful eating in pregnancy Gestational diabetes mellitus Cardiovascular health Preterm complications Limitations and strengths of mindfulnessbased intervention studies Conclusion Acknowledgment References

373 374 389 390 390 390 391 391 396 397 398 398 399 400 401 401 402 402 403 403 405 405 407 407 407 408 409 410 410

xi

Traditional Chinese medicine treatment for common issues in pregnancy 421 Acupuncture and acupressure in labor and delivery 425 Postpartum 425 Conclusion to traditional Chinese medicine 426 Part two: Ayurveda 426 Ayurveda in pregnancy 426 Align with the sunrise to start your day 427 Practice upon waking 427 Morning cleanse 427 Exercise and meditation 428 Breakfast, lunch, and dinner 428 Mealtime practice 428 Afternoon meditation 429 Social time 429 Preparing for bed 429 Sleep 429 Diet and Pregnancy 429 Risks and drawbacks of Ayurvedic medicine 430 Ayurveda in the treatment of conditions and disease that interfere with reproduction and gestation 431 Anxiety and stress 431 Hypertension 432 Preeclampsia 432 Diabetes 432 Obesity 433 Postpartum 433 Other uses of Ayurvedic medicine: contraception 433 Male contraceptives 434 Female contraceptives 434 Conclusion 434 Acknowledgments 435 References 435

22. Prenatal bonding: the importance of connecting with body and baby

439

Rita Kluny and Dana M. Dillard

21. Traditional Chinese medicine and Ayurvedic care during pregnancy

415

Diana Vaamonde, Paula Hernandez, Easter Bonnifield and Lara Rosenthal Introduction Part one: Traditional Chinese medicine Preparing the body for pregnancy General guidelines during pregnancy Common patterns of imbalance during pregnancy Issues relating to TCM research Safety considerations

415 415 415 416 419 420 420

Introduction Human stress responses Effects of stress, attachment, and love on physical, psychological, and social wellbeing Women of the childbearing age and preconception Fetal development Postpartum depression and infant wellbeing Promoting connections between parents and unborn children Connections before birth Examining myths of mindfulness practices Addressing mindfulness as a spiritual practice

439 439 440 440 441 442 442 443 444 444

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Contents

Addressing mindfulness as a required state of relaxation Addressing mindfulness’s contemporary popularity Take-home message Key points Conclusion References

23. Aromatherapy, music, and acupuncture for optimizing emotional wellbeing in pregnancy

444 445 445 445 445 446

449

Alys Einion, Shunji Suzuki and Chiharu Tobea Introduction Complementary medicine and pregnancy Aromatherapy for emotional wellbeing during pregnancy Enhancing emotional and psychological wellbeing during pregnancy using aromatherapy Combining aromatherapy with other therapies Using aromatherapy when medical complications affect the pregnancy experience Using sound and music for emotional wellbeing during pregnancy Acupuncture, moxibustion, and acupressure in traditional Asian treatments Conclusion References

449 450 452 452 454 454 455 458 461 462

24. Nutraceutical and phytotherapeutic support in pregnancy 465 Dana M. Dillard Supporting maternal wellbeing with nutraceuticals Chamomile Echinacea Garlic (Allium sativum L.) Ginger (Zingiber officinale Roscoe) Lavender (Lavandula angustifolia Mill.) Lemon balm (Melissa officinalis L.) Mallow (Malva sylvestris)

465 465 466 467 467 468 468 468

Marshmallow (Althaea officinalis) Mint Psyllium (Plantago ovata) Witch hazel (Hamamelis virginiana) Other herbs and phytotherapeutic plants Limitations, precautions, contraindications, and special considerations Summary of key points Conclusion References

25. Ethics in fertility and pregnancy management

468 469 469 470 470 470 471 473 474

479

Susan Gitlin and Alys Einion Ethics of fertility treatments Introduction on assisted reproductive techniques Principles of medical ethics Counseling and shared decision making Examples of fertility treatments for ethical considerations Fertility procedures Disclosure of success rates Ovulation induction cycles In vitro fertilization: number of embryos to transfer Futility continuing treatment In vitro fertilization: preimplantation genetic testing Embryo sex selection Donor gametes Posthumous reproduction Uterus transplantation Mitochondrial DNA transfer Germline gene editing Misconceptions Summary Ethics, informed consent, and childbearing References

26. Conclusion

479 479 479 480 480 480 480 481 481 481 481 482 483 483 484 484 484 484 485 485 489

493

Diana Vaamonde Index

495

List of contributors Carolina Algar-Santacruz, Department of Morphological Sciences, School of Medicine and Nursing, Universidad de Cordoba, Cordoba, Spain; Reina Sofia Hospital, Cordoba, Spain Pilar Aparicio Martı´nez, Nursing, Pharmacology and Physiotherapy Department, University of Cordoba, Spain Natalibeth Barrera, In vitro Fertilization Laboratory, Montevideo Sterility Center, Montevideo, Uruguay; Andrology Research Laboratory, Reprovita (Reproductive Medicine Division of Fertilab Laboratory), Montevideo, Uruguay Easter Bonnifield, Everyday Healing, Los Ranchos de Albuquerque, NM, United States Marı´lia Porto Bonow, Universidade Positivo, Curitiba, PR, Brazil Kelly Buckingham, Behavioral Healthcare, Volunteers of America Northern Rockies, Sheridan, WY, United States Blanca Chacon, Fı´sicoMed, Cordoba, Spain Jessica Clifton, Department of Medicine, Division of General Internal Medicine Research, Larner College of Medicine, University of Vermont, Burlington, VT, United States Joao Sabino Cunha Filho, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil Dana M. Dillard, Department of Behavioral Sciences, University of Arizona Global Campus, San Diego, CA, United States Alice D. Domar, Domar Centers for Mind/Body Health, Boston IVF, Harvard Medical School, Boston, MA, United States Stefan S Du Plessis, Basic Medical Sciences, College of Medicine, Mohammed Bin Rashid University of Medicine and Health Sciences, Dubai, United Arab Emirates; Medical Physiology, Faculty of Medicine and Health Sciences, Stellenbosch University, Tygerberg, South Africa Alys Einion, Faculty of Health and Life Science, Department of Interprofessional Health Studies, Swansea University, Swansea, United Kingdom

Cristiana Libardi Miranda Furtado, Drug Research and Development Center, Postgraduate Program in Medical and Surgical Sciences, Federal University of Ceara´, Fortaleza, Brazil Juan Manuel Garcia-Manso, INPEF—International Network on Physical Exercise and Fertility, Cordoba, Spain; Physical Education Department, School of Physical Activity and Sport Sciences, University of Las Palmas de Gran Canaria, Las Palmas de Gran Canaria, Spain Francisco Genil Marquez, MD General practitioner, Cordoba, Spain Fernando Gil Herna´ndez, Department of Legal Medicine, Toxicology and Physical Anthropology University of Granada, Spain Susan Gitlin, American Society for Reproductive Medicine (retired), Birmingham, AL, United States Lynn Gordon, Clinical/Training, Family and Personal Counseling Center / Family and Personal Wellness, Sheridan, WY, United States Anthony C. Hackney, INPEF—International Network on Physical Exercise and Fertility, Cordoba, Spain; Exercise and Sport Science, University of North Carolina, Chapel Hill, NC, United States Paula Hernandez, Department of Morphological Sciences, School of Medicine and Nursing, Universidad de Cordoba, Cordoba, Spain Johanna K. Ihalainen, Faculty of Sport and Health Sciences, University of Jyva¨skyla¨, Jyva¨skyla¨, Finland Rita Kluny, Seton Family of Hospitals Network, Austin, TX, United States Bo Liu, Anatomical Sciences, Liberty University College of Osteopathic Medicine, Lynchburg, VA, United States Dana M. Dillard, Department of Behavioral Sciences, University of Arizona Global Campus, San Diego, CA, United States Pilar Aparicio Martı´nez, Nursing, Pharmacology and Physiotherapy Department, University of Cordoba, Cordoba, Spain

xiii

xiv

List of contributors

Ciro Dresch Martinhago, Chromosome—GeneOne/ DASA, Sa˜o Paulo, Brazil Ritva S. Mikkonen, Sports Technology Unit, Faculty of Sport and Health Sciences, University of Jyva¨skyla¨, Vuokatti, Finland Guillermo Molina-Recio, Department of Nursing, University of Cordoba, Cordoba, Spain Sergio Oehninger, Department of Obstetrics and Gynaecology, Reproductive Biology Unit, University of Stellenbosch, Stellenbosch, South Africa Temidayo S Omolaoye, College of Medicine, Mohammed Bin Rashid. University of Medicine and Health Sciences, Dubai, United Arab Emirates Jennifer Pettit, Chandrakala Yoga, New York, NY, United States Juan Andre´s Ramı´rez-Gonza´lez, Morphology, Faculty of Health Sciences, University of Las Palmas de Gran Canaria, Spain Daiana P. Rodrigues-de-Souza, Department of Nursing, Pharmacology and Physical Therapy, Universidad de Cordoba, Cordoba, Spain Lara Rosenthal, Rosenthal Acupuncture, Women’s Health and Fertility, New York, NY, United States

Andrea Sansone, Endocrinology and Medical Sexology (ENDOSEX), Department of Systems Medicine, University of Rome Tor Vergata, Rome, Italy Alessandro Schuffner, Conceber Centro de Medicina Reprodutiva, Curitiba, Parana´, Brazil Shunji Suzuki, Department of Obstetrics and Gynecology, Japanese Red Cross, Katsushika Maternity Hospital, Katsushika-ku, Japan R. James Swanson, Anatomical Sciences, Liberty University College of Osteopathic Medicine, Lynchburg, VA, United States Rachel M.M. Tardin, Conceber Centro de Medicina Reprodutiva, Curitiba, Parana´, Brazil Chiharu Tobea, Department of Obstetrics and Gynecology, Japanese Red Cross, Katsushika Maternity Hospital, Katsushika-ku, Japan Diana Vaamonde, Morphological Sciences Department, School of Medicine and Nursing, Universidad de Cordoba, Cordoba, Spain; INPEF–International Network on Physical Exercise and Fertility, Cordoba, Spain Manuel Vaquero-Abellan, Department of Occupational Risk Prevention and Environmental Protection, University of Cordoba, Cordoba, Spain

About the editors Diana Vaamonde, PhD, is a Professor at the Department of Morphological Sciences of the School of Medicine and Nursing in the University of Cordoba (Cordoba, Spain). Dr. Vaamonde currently leads a PhD research group in the area of physical exercise and fertility, with several ongoing doctoral theses. She received her bachelors in biology from Washington and Lee University in 1998 and her masters in biology with emphasis on human reproduction from Old Dominion University/Jones Institute for Reproductive Medicine in 2004. She later completed a PhD program in Physical Activity and Sport Sciences at the University of Cordoba (Spain) where she brought together knowledge from the fields of reproductive medicine and sports medicine, having published numerous research articles, books, and book chapters. Her main expertise and research focus is the relationship between infertility and physical exercise. Most of her work has been on the effect of sports training, especially in elite athletes, on the male reproductive system. Her most recent work has included assessment of sperm DNA damage as a result of physical exercise as well as the use of antioxidant agents to revert exercise-associated damage in animal models. She has presented part of her work in prestigious international meetings [European Society for Human Reproduction and Embryology (ESHRE), American Society of Reproductive Medicine, European College of Sport Sciences]. She has been an invited speaker to several international meetings such as the plenary session on the topic of physical exercise and male reproduction at the ESHRE meeting of 2012, the Andrology pre-Congress course of the ESHRE and several meetings on Lifestyle and Human Reproduction. In 2016 she coordinated the Andrology pre-Congress course of ESHRE 2016, focused entirely on exercise and fertility. Dr. Vaamonde was funded by the Andalusian Government (Consejeria de Comercio, Turismo y Deporte) for studies on semen quality and endurance exercise. Since 2016 Dr. Vaamonde has also directed end of degree dissertations focused on lifestyle interventions and the use of complementary therapies to improve fertility and for supporting a healthy pregnancy. During the academic year 2018
19 she launched a postgraduate course in Yoga and Integral Health at the University of Cordoba.

Due to her expertise in the field of Exercise and Human Reproduction, she serves as a reviewer for a number of journals including Fertility and Sterility, Human Reproduction, and Asian Journal of Andrology, and she is part of the Scientific Committee and Editorial Board for several journals (Revista Andaluza de Medicina del Deporte, Histology Histopathology, etc.). She has also been an embryologist and head of research at several in vitro fertilization clinics such as the Reproductive Care Center (United States) as well as scientific consultant for FIV Marbella (Spain). She recently founded the International Network on Physical Exercise and Fertility (INPEF) along with Profs. Drs. Juan Manuel GarciaManso and Anthony C. Hackney, who have extensively studied the effect of physical exercise on the endocrine system and fertility. Anthony C. Hackney, PhD, DSc, is a Professor of Exercise Physiology and Nutrition at the University of North Carolina, Chapel Hill, North Carolina, United States. He is the editor for the Endocrinology of Physical Activity and Sport and Sex Hormones, Exercise & Women: Scientific and Clinical Aspects series books, as well as the author of the books Exercise, Sport, and Bioanalytical Chemistry: Principles and Practice and Doping, Performance-Enhancing Drugs, and Hormones in Sport: Mechanisms of Action and Methods of Detection. He is a former Fulbright Scholar of Medical Sciences having served in Eastern Europe. Juan Manuel Garcia-Manso, PhD, is a Professor at the Faculty of Physical Activity and Sports Sciences at the University of Las Palmas de Gran Canaria (Gran Canaria, Spain). Dr. Garcı´a Manso received his bachelor’s degree in CAFD from the National Institute of Physical Education of the Polytechnic University of Madrid in 1974. He completed his Master’s degrees in Nutrition and High Sports Performance and completed a doctoral program in Physical Activity and Sports Sciences at the University of Las Palmas de Gran Canaria (Spain). His main professional and research work is linked to the area of training high-performance athletes, and most of his work has been related to studying the effect of training on the improvement of sports performance, xv

xvi

About the editors

especially in relation to the adaptive response of the body to mechanical and physiological stress. He has extensive practical experience in the field of sports, having trained experienced athletes in national and international competitions (Olympic Games, World Championships, European Championships, Mediterranean Games, Ibero-American Games, and international tournaments and meetings). He has presented part of his academic work at numerous national and international conferences, having participated as a guest speaker in more than 200 of these held in 20 different countries. He has published 115 scientific articles in indexed journals (JCR and SJR). He works as a reviewer for

several journals, including PLoS One, Physica A, South African Journal of Science, Journal in Sport and Health Research, Athletic Training Journal, Archives de Medicina del Deporte, Apunts de Educacio´n Fı´sica, Revista Andaluza de Medicina del Deporte, and Revista Espan˜ola de Educacio´n Fı´sica y Deportes. He has also been part of the scientific committee and editorial committee of several magazines. He is part of the INPEF together with Profs. Drs. Diana Vaamonde and Anthony C. Hackney, who have extensively studied the effect of physical exercise on the endocrine system and fertility.

Preface Years ago, the seed of this book was engendered from dedicated practice and study in the fields of human fertility, human-assisted reproduction, and exercise sciences. After gaining understanding on the numerous factors that modulate fertility and the chances of conceiving and carrying a baby to term, I saw the unchartered space that deserved closer attention in matters of lifestyle interventions for improving pregnancy rates. In the beginning, my own beliefs were aligned to a more physical realm, focusing on exercise and nutrition, but as new experiences unfolded, I came to the realization that there was much more to reproduction than just a physicality. My focus and interest started shifting and embracing more integrative approaches due to a deeper understanding and empathy regarding how much pressure, stress, and anxiety subfertile couples could experience. Moreover, the so-called idiopathic infertility (unknown cause) has always been a challenge, given that, despite no apparent blockages to fertility, these couples do not achieve pregnancy. Over time, gradually, more studies began to add the scientific evidence of how the emotional status affects fertility and also how fertility issues affect the emotional landscape, what a vicious cycle that can

become! Having worked in clinical in vitro fertilization, I had witnessed many women challenging their physical, mental, emotional, and economic boundaries with the desire to have a baby. All expectations and efforts were put onto science, but amazingly few recognized they could help science by taking care of their body and mind. Accordingly, this book covers a large spectrum of topics from physiological to behavioral to psychological. It is essential to highlight and acknowledge the vital role of my coeditors during the writing process. Their belief in the project from its inception has been matched along the way with continuous support and invaluable insights. They have shared an incredible amount of experience in the field of sport sciences and incalculable wisdom from years of practice, thereby allowing this project to take shape. A practical and effective convergence of study, research, knowledge, practice, and many twists and turns of life have brought this book to fruition. This book, Fertility, Pregnancy, and Wellness has been created with much enthusiasm and purposefulness in hoping that the reader will greatly benefit from a vast amount of information and years of experience on a variety of related topics. Enjoy reading it!

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Chapter 1

The importance of healthy lifestyles in helping achieving wellbeing Manuel Vaquero-Abellan1, Francisco Genil Marquez2 and Pilar Aparicio Martı´nez3 1

Department of Occupational Risk Prevention and Environmental Protection, University of Cordoba, Cordoba, Spain, 2MD General practitioner,

Cordoba, Spain, 3Nursing, Pharmacology and Physiotherapy Department, University of Cordoba, Cordoba, Spain

Individual habits, behaviors, or conducts have been linked to health and illness since ancient times [1]. A clear example would be the humans’ ability to integrate previously unknown products into our body that were later incorporated as eating habits, such as grains [2]. In the case of grains, they have become the main energy source, unchanged in the last millennium, helping human beings to keep a healthy metabolic balance [3]. In a wide perspective, a healthy life implies a harmony between mind, body, thoughts and feelings. In this sense, as humans are part of a cohesive society, the health of the individual affects the rest of the population and vice-versa. From this point of view, the World Health Organization defines “health is a state of complete physical, mental and social wellbeing and not merely the absence of disease or infirmity” [2]. Nevertheless, a precarious lifestyle not only determines the short-term wellness but also can resulted in chronic conditions. Since the late fifties, the primary cause of death shifted from infections to chronic conditions, such as heart attacks [2]. From this point, research and government have promoted healthy lifestyles, based in lowering the risk of having a chronic disease [3]. In the primary investigations, certain types of behaviors or habits have been highlighted as factors that contribute to increases in noncommunicable disease and therefore early deaths [4]. Among these habits, smoking, consumption of alcohol, sedentarism (i.e., physical inactivity) and overeating have more relevance [5]. However, even though social and environmental factors are a key element in the structure of people’s health, the biological aspect, especially the DNA, codifies the behavior of the person, determining his or her health and disease. In fact, the field of study of the

interaction of genes and environment, called epigenetics, focuses on genetic alterations that are linked to environmental changes, such as the decrease of fertility linked to the increase of contamination [6]. In this sense, the contamination and less sustainability of the environment sum up with other individual factors, such as the age at fertilization, resulting in major pathologies that are passed onto future generations [7]. Nevertheless, when health professionals and researchers talk about healthy lifestyles, they do not only focus on the physical welfare but also on the mental and social wellbeing [6]. In this sense, a healthy lifestyle should include the familiar and social structure for promotion and maintenance of positive behavior [7]. This being said, achieving wellness is challenging since it is conceived as the most favorable health status in physical, mental, and social areas. This definition is complex, but some factors that contribute to wellbeing such as emotions or genes have not been included. The emotional area is one of the main fields that merits study. Evidence suggests that changes in emotions play a critical role in the biochemical balance of the human body. Emotions are linked to physical and mental health, regulated by social factors, which results in the increase of hormones associated to either the stress or relax systems. All these changes contribute to alter the homeostatic balance of each human coexisting with the environment, contributing to disease and degradation of the population’s health. Nevertheless, by all means the most common way to accomplish a healthy level is by modifying risk factors and reducing them as much as possible [8,9]. However, unhealthy lifestyles have been increasing especially during the past 20 years. One of the things that could be related is the change of the actual diet. Human’s previous diet was based on 33% proteins (75% animal origin), 32% fat (41% animal origin), and 45%

Fertility, Pregnancy, and Wellness. DOI: https://doi.org/10.1016/B978-0-12-818309-0.00020-4 © 2022 Elsevier Inc. All rights reserved.

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Relation between lifestyle and health

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Fertility, Pregnancy, and Wellness

carbohydrates (without saccharose and lactose). The current diet is based on 11% proteins (62% animal origin), 32% fat (75% animal origin), and 45% carbohydrates (27% saccharose and 27% lactose). In this sense, it could be argued that the human body is a precision machine in which the fuel is coming from the different foods. This machine starts with the inclusion of food, usually by consuming breakfast, resulting in the activation of the several systems dependent on energy. When the fuel of this machine is inadequate, the gears start to crumble and fail. An example would be the increase of the food prior to sleep time; dinner is essential, but too much food provokes side effects in the sleeping rhythm and metabolism. These alterations are reflected by the increased number of people dealing with obesity, pulmonary problems, hypertension, or diabetes [10]. For instance, the increase of overweight people has been linked to sedentarism and eating, although there are other factors related to this increase such as emotional wellbeing, stress, or sleep habits [11,12]. Therefore, the increase of unhealthy lifestyles depends on several individual, emotional or social factors. In this sense, emotional distress or social isolation related to unhealthy lifestyles have been also linked to social media, fast food or changes in the production of food [13,14]. Additionally, the public concern is that all of these unhealthy lifestyles increases the risk of having health issues, such as hypertension [1,15]. These health issues are even more disturbing in children and women, especially as the number of women or children with low levels of physical activity, disordered eating, or emotional issues is growing [16]. In the case of children, unhealthy habits during the first years determine the adulthood and aging process as well as emotional and social development. Meanwhile, women’s habits define their aging process, their reproductivity, and the future growth of their children [17,18]. Numerous studies have linked how the wellbeing of women is in line with the increase of infertility or difficulties during or after the pregnancy [19
22]. Moreover, previous researchers have pointed out how the maternal wellbeing determines the health and growth of the fetus and the development of the child [23
26]. One concerning habit increasing among teenagers and young adults is the use of recreative substances such as smoking tobacco or marihuana among others, which has been linked to health problems such as pulmonary distress, cancer, or even schizophrenia. In fact, studies proved how the exposure to toxics during the pregnancy increases the probability of using substances among descendants [27
29]. Nevertheless, it is important to highlight the relevance of the modulation carried out by the immune system, being different in some cell types, such as gametes. In the case of gametes, this modulation is intimately related to the process of maternal and paternal genetic exchange that takes place during fertilization. This fact could mean that

harmful epigenetic modifications of the cells as a result of unhealthy lifestyles are not corrected and are transmitted to the descendants or even future generations. Cholesterol, a basic pillar for hormone production, and, thus, of the correct functioning of the reproductive system, may contribute to such modulation. As the diet has been extensively modified, altering the levels of cholesterol, this could result in a reproductive system that works inappropriately, leading to fecundity problems and health problems in the children. The alterations of cholesterol and diet are clear examples of how the environmental factors disrupt the equilibrium of health and disease [30,31]. Thus, healthy lifestyles may result in improvements in the emotional, metabolic, physical, or social state depending on the individual. Based on this, a previous research was carried out in Scopus using the terms “healthy,” “lifestyle,” and “wellbeing.” Out of this initial research (784 documents), the more recently published papers in journals with high impact, showed an increase of publications focusing on lifestyles and wellbeing (Table 1.1). Table 1.1 summarizes some of the studies that have focused on lifestyle as factors related to health. This research is focused in the latest predominant journals in the health field such as PLoS One. In this sense, the results from this research have shown that the health and promotion thematic are based on healthy lifestyles to increase the wellbeing of the population. Moreover, the population most studied was women in their reproductive years (Fig. 1.1). All being said, the principal objective of this paper was to determine the tendency of publications focused on the heathy lifestyles related to the wellbeing during the past 40 years (from 1978 to 2018). Additionally, another objective in this research was to determine the tendency of publications focused on women. These purposes were stated in order to understand more widely the interaction of lifestyle in the wellbeing of the population, assisting in the decision-making of health care professionals.

The study of number of publications: bibliometric analysis The analysis of previous works is an essential step in the research in any field but especially in the health field since new pathways, treatments, or risk factors are constantly being discovered. Moreover, this type of analyses is a complementary tool that allows measuring of the quality of the latest scientific knowledge and the impact in the health of the population. In this sense, it is possible to access the scientific data as well as their study and sources. Bibliometric studies provide a wider perspective of the scientific data since both national and international research are studied. Additionally, these studies contextualize the modifications, changes, and presence of health research in past decades including health system,

The importance of healthy lifestyles in helping achieving wellbeing Chapter | 1

3

TABLE 1.1 Main areas of research on social networks related to health during the past 10 years. Year

Relation with health

Analysis

Area

Group

Field

Reference

2019

Health-intervention programs

Review

Obesity

Adolescents

Health

[32]

2019

Social determinants and healthier lifestyles

Crosssectional study

Chronic diseases

Young people

Health

[33]

2019

eHealth and promotion

Mixed method

Record data

Patients

Medical Informatics

[34]

2019

NHS and healthy lifestyle

Crosssectional study

Determine healthy lifestyles

Patients

Health Promotion

[35]

2018

Emotion distress, physical activity and healthy eating

Mixedmethods study

Physical activity and healthy eating

Women

Health

[36]

2018

eHealth and promotion

Validation

Screening

Workers

Medical Informatics

[37]

2018

Social media and diet

Frame study

Determine healthy food

Women

Sociology

[38]

2018

Emotional distress and lifestyle

Experimental

Healthintervention programs

Patients

Health Promotion

[39]

2018

Sexual wellbeing and lifestyle

Report

Side effect

Women

Psychology, Sociology and Sexuality

[40]

2017

Promotion of physical activity

Experimental

Physical activity

Mothers and their children

Health Promotion

[41]

2017

Determine lifestyles and wellbeing

Review

Factors related to health

General

Health Promotion

[42]

government, or health professional. All this information expedites the decision-making of the health professionals and aids in the future of research of healthy lifestyles. Elsevier’s database, known as Scopus, was used to carry out the analysis. The research was used to identify publications addressing the topic of this research from 1978 to 2018. Scopus is a scientific bibliographic database that collects items from scientific journals. This database has been described as “the largest index database,” including up to 65 million records and claims. This platform incorporates the health field with titles indexed including complete coverage of Medline, Embase, and Compendex. In addition to papers, this database includes series, conferences papers, books, and patents. The database time can be tracked back to 1823, although it was set up in 1996. Moreover, Scopus also provides data regarding the performance of a paper and author according to the citation received for each work [43,44]. For this research, the inclusion criteria was from 1978 to 2018 with a theme of healthy lifestyle and wellbeing.

The results from the research were studied. The analysis that was carried out focused on descriptive analysis, especially the frequencies. This descriptive analysis was used for the type of document, the language, trends in scientific publications, main sources, thematic areas or fields, scientific institutions, works between countries, main authors in the area, and the evolution of the keywords used. In the case of keywords, a normalization of the terms has been carried since many of the main keywords are found in both singular and plural. The keywords included in the manuscript were the author’s keywords, therefore the keywords used were not Medical Subject Headings (MeSH) terms, which are recognized as controlled vocabulary for the research in medical sciences and especially in the PubMed database. Another analysis utilized was the identification nets by using the VOSviewer software (http://www.vosviewer.com/). This open program was created for constructing and viewing bibliometric maps, importing the data from several sources including Web of Science or Scopus. The analysis

4

Fertility, Pregnancy, and Wellness

FIGURE 1.1 Methodology structure.

used to create the maps focused on a minimum of ten connections or concurrence, and each author must have a minimum of ten published in the area. Moreover, the map of concurrence of authors also included as exclusion criteria that each document could only have as a maximum of five authors. This strategy was followed for the concurrency of keywords and connections between authors and countries. The methodology followed was segmented in three steps (see Fig. 1.1): 1. Research of the information. The Scopus database was used using the following search fields: (TITLE ({lifestyle}) OR ABS ({lifestyle}) OR AUTHKEY ({lifestyle}) OR TITLE ({habits}) OR ABS ({habits}) OR AUTHKEY ({habits}) OR ({behavior}) OR ABS ({behavior}) OR AUTHKEY ({behavior})) AND (TITLE ({health}) OR ABS ({health}) OR AUTHKEY ({health}) OR TITLE ({healthy}) OR ABS ({healthy}) OR AUTHKEY ({healthy}) OR TITLE ({healthful}) OR ABS ({healthful}) OR AUTHKEY ({healthful}) OR TITLE ({adequate}) OR ABS ({adequate}) OR AUTHKEY ({adequate}) OR TITLE ({balance}) OR ABS ({balance}) OR AUTHKEY ({balance}))

AND (TITLE ({wellbeing}) OR ABS ({wellbeing}) OR AUTHKEY ({wellbeing}) OR TITLE ({welfare}) OR ABS ({welfare}) OR AUTHKEY ({welfare})). The data obtained was a .csv file that contained the following data: Authors, Title, Autor Ids, Year, Volume, Issue, Source title, Art. No, Number of pages, Cited by, DOI, Link, Document Type, Access Type, Source, EID. 2. Bibliometric data examination. Each item from the previous step was analyzed and studied separately. For instance, the number of documents per Country or the rate of publication of each author was examined. 3. Cluster determination. The cluster determination of the thematic collections was examined with the VOSviewer, resulting in diverse maps of global connections between authors and countries and the research tendencies by using the keywords.

Relationship between lifestyle, habits, and wellbeing Based on the importance of the bibliometric analysis, the analysis of the results from the research strategy showed

The importance of healthy lifestyles in helping achieving wellbeing Chapter | 1

that 15,297 documents were obtained about this topic of study from 1978 to 2018. The first analysis of the results showed that the main types of documents were scientific articles (71%) (Fig. 1.2). Furthermore, most of these

Document type distribuon Book Others 2% 2% Conference Paper 4% Book Chapter 5% Arcle 75%

Review 12%

FIGURE 1.2 Type of document.

5

articles were on quantitative research (82%), and these articles highlighted experimental and cross-sectional studies. The rest of the articles were qualitative studies (12%), based on structured or semistructured interviews. The second most common type of publication was reviews (12%), followed by book chapters (5%), and conference papers (4%). Other types of documents (2%), such as conference review (0.2%), were less common (Fig. 1.2). Almost of the publications were English (92.3%), although other languages were Spanish (1.5%) or German (1.5%). Additionally, other less used languages presented in the analysis were Polish (0.2%), Croatian (0.1%), Dutch (0.1%) or Hungarian (0.1%) (Fig. 1.3). Fig. 1.4 represents the frequency of academic publications focused on healthy lifestyles and wellbeing during the past 40 years. This figure suggests an upwards trend, implying that the number of annual outputs increased markedly from around 1983 to 2018. This exponential increase reflects the shift change to chronic diseases and the promotion of healthy lifestyles [45,46]. Additionally,

FIGURE 1.3 Frequency of languages of the articles.

FIGURE 1.4 Frequency of the number of publications focused on this topic.

Publicaon Trends Number of Publicaons

10000 1000 100 10 1 1978

1983

1988

1993

1998

Years

2003

2008

2013

2018

6

Fertility, Pregnancy, and Wellness

the tendency from the late 1990s to 2018 may be the result of the definition of healthy lifestyle and recommendations made by the World Health Organization [47]. These results are consistent with previous analyses showing increased research attention on lifestyle and wellbeing [48,49]. Based on this figure, the main observation is the continued increase of publications about this topic, which coincides with the increase of prevalence of chronic disease such as diabetes or obesity [50
52]. This tendency with obesity could be linked to the epidemic situation of consuming more fat and carbohydrates by eating ultra-processed food. Moreover, the polarization regarding eating behaviors has proven to result in an increase of eating disorders and obesity, both of them connected to diet, exercise, and stress. Also, different studies have linked unbalanced diets, with predominantly more excessive consumption of carbohydrates, to different health issues, such as atherosclerosis [53]. The relationship between obesity and physical activity or exercise is typically a negative one, implying that most people who suffering from obesity typically exercise little. The problem with obesity and physical activity is the mental equilibrium. Physical activity improves the emotional state and increases dopamine, improving self-perception; meanwhile, being overweight has negative social

connotation, resulting in insults or bullying. These stigma around weight also induces damage to mental health, with self-esteem and anxiety problems being more prevalent in this population [54
57]. Despite these previous statements, excessive exercise has also been determined to increase health problems, such as an increase of cell stress and increased infertility. Based on all these statements, it is important to achieve an equilibrium in both exercise and food intake. In this sense, it could be concluded that the key point to a healthy life is equilibrium in all areas. Fig. 1.5 shows the production of relevant articles per country between 1978 and 2018. Colors indicate the number of papers, from red (highest) to gray (no publications). The country with most publications was the United States (US) (4304), followed by the United Kingdom (UK) (2723), Australia (2011), Canada (895), and Germany (697). Within these countries, the publications about healthy lifestyles related to wellbeing with different populations have been potentiated by the governments and institutions to decrease chronic illness and improve patients’ health [53,58,59]. In the case of the US, the increase of publications about healthy lifestyles and wellbeing might be linked to the growth in environmental, social, and corporate governance performance arising since 2004 regarding food consumption and production [46].

FIGURE 1.5 Frequency of research published according to countries around the world.

The importance of healthy lifestyles in helping achieving wellbeing Chapter | 1

Fig. 1.6 shows the tendency of research publications on healthy lifestyle and wellbeing with the highest production in each of the five countries, revealing the greatest increase is in the US. Nevertheless, in the year 2018 there was decrease of 28 publications compared to the previous year. This decrease could be a reflection of the change in the policies of US governance [60]. The healthy lifestyle and wellbeing map shown in Fig. 1.7, illustrates the pattern of international collaboration

7

between study authors. This figure was obtained after applying the software VOSviewer v.1.6.11., to a .csv file of the data extracted from Scopus during the literature search. Three countries dominate in the six clusters seen in Fig. 1.7 and Table 1.2: UK, US, and Australia. The first cluster is composed of Eastern Europe and Nordic countries, led by Germany, which is in the fifth position concerning publications. The green cluster, which is the FIGURE 1.6 Healthy lifestyle and wellbeing publications of the five top countries: United States, United Kingdom, Australia, Canada, and Germany.

900 800 700 600 500 400 300 200 100 0

United States

United Kingdom

Australia

Canada

Germany

FIGURE 1.7 Collaboration among countries.

8

Fertility, Pregnancy, and Wellness

second most important cluster, is led by Canada. Canada is the node of this cluster because of the number of connections with other countries and number of publications. All of these countries from this cluster seem to be connected via economic and political relations [61]. The blue cluster is the third in importance led by the US, followed by Australia. The yellow cluster is led by Spain, with connections to Latin America and Western Europe. The purple is led by the Netherlands, which is connected to Africa. The pink cluster is linked to the UK and African countries. The last cluster is the orange led by South Korea, which is connected to different countries such as Nigeria or Cameroon. Based on these and previous results, it could be concluded that the US dominates in this research field with a comparatively higher number of publications and one of the highest number of connections. The important or significant role of the US is also shown by the connections between

authors and affiliations, most of which belong to the US (Table 1.3). All this being said, these results might be explained by the fact that there might be economic, historic, geographical or cultural influences between the groups, which can be applied to the six first clusters. Additionally, the connections between clusters might be related to the topic of this research especially in the case of countries with a more recent improvement of lifestyles and wellbeing. In Table 1.3, the top 10 organizations with higher rates of publications in the field of social networks related to the health of young people is represented. Additionally, the top three keywords used each of the previous institutions are included in this table. The University of Melbourne in the first position stands out with 298 documents. Following this institution is the University of Sydney in second position with 237 and the University of Queensland in third position with 206. In the fourth and fifth position are the Monash

TABLE 1.2 International collaborations in research on social media for health in young people. Cluster

Color

1

Red

2

Green

3

Blue

4

Countries

Geographic Area

%

Germany
The Netherlands
Sweden

East Europe
Nordic countries

23.8

Canada
Iran
Israel

Canada
Iran
Israel

22.5

United States
Australia
China
India

US
Australia
China

17.5

Yellow

Spain
France
Portugal
Argentina
Colombia

Europe
Latin America

15.0

5

Purple

The Netherlands
Ghana
South Africa

The Netherlands
Africa

10.0

6

Pink

United Kingdom
Zimbabwe

United Kingdom
Africa

6.3

7

Orange

South Korea
Cameroon

South Korea
Africa

5.0

TABLE 1.3 Publications and keywords utilized by the top 10 international institutions Affiliation

Country

Publications

Main keywords used 1

2

3

University of Melbourne

Australia

298

Human/s

Female

Article

The University of Sydney

Australia

237

Human/s

Female

Male

University of Queensland

Australia

206

Human/s

Female

Article

Monash University

Australia

186

Human/s

Female

Article

University College London

United Kingdom

183

Human/s

Female

Article

University of New South Wales UNSW Australia

Australia

177

Human/s

Female

Article

Deakin University

Australia

156

Human/s

Female

Article

Karolinska Institutet

Sweden

151

Human/s

Female

Male

University of Bristol

United Kingdom

143

Female

Human/s

Article

University of Toronto

Canada

142

Human/s

Female

Article

The importance of healthy lifestyles in helping achieving wellbeing Chapter | 1

University with 186 and the University College London (UCL) with 183. The University of New South Wales UNSW Australia with 177 is the sixth position. The Deakin University is the seventh position with 156 and in the eighth position is the Karolinska Institutet with 151 documents. The last two positions are taken by the University of Bristol with 143 documents and University of Toronto with 142 documents published. The increase of publications and ranking of the affiliations might be related to collaborations between authors. These collaborations have been previously studied, showing how, since 1997, there has been a 20% increase of collaborations between the US and Australia [62]. Additionally, the tendency of the production of each the five institutions with higher production was analyzed (Fig. 1.8). This analysis showed a difference of publications between 2008 to 2018, reflecting a growth. The University of Melbourne showed a difference of 42 publications, proving an increment of 92%. The University of Sydney also proved to have increased the production by 34 documents. Nevertheless, the University of Queensland showed a lower increase of the production, going from 1 document to 24.

9

Meanwhile, the Monash University and University College London also increased the number of publications with a difference of 32 and 22 documents, respectively. Regarding the type of study implemented by each institution according to Scopus, the results showed that the all the institutions focused on articles in the area of medicine, being followed by the area of social sciences. The main countries with higher numbers of these themed publications were the US, UK, and Canada. Finally, the author’s keywords used according to Scopus were “Human/s,” “Female,” “Articles,” “Male,” and “Wellbeing.” The frequency of publications by each thematic area was acquired after using the Scopus database. In Fig. 1.9, the distribution of the main thematic areas is represented. This figure shows that the area with the highest percentage of documents was medicine (46.6%), followed by social sciences (24.9%), agricultural and biological sciences (16%) and psychology (15%). Other areas of study such as computer sciences (1.3%) or pharmacology, toxicology and pharmaceutics (1.0%) were found less in the database. The “undefined” (0.3%) were included in unspecified areas (Table 1.4). 200

Publicaons according to affiliaon

180 160 140 Monash University

120 100

University of Queensland

80

The University of Sydney University of Melburne 2008

2009

2010

2011

2012

2013

2014

2015

2016

2017

60

Number of publicaons

UCL

40 20 0 2018

Years FIGURE 1.8 Tendency of publications of the top five institutions.

Documents Medicine 7130 Social Sciences 3804 Agricultural and Biological Sciences 2450 Psychology 2300 Bichemitry, Genecs and Molecula Biology 1092 Veterinary 1085 Nursing 1013 Arts and Umanies 842 Economics, Econometrics and Finance 674 Environmetal Sciences 648 Engineering 527 Business, Management and Accounng 521 Neurosciences 370 Health Professions 359 Computer Science 197

FIGURE 1.9 Distribution of scientific production according to the main thematic areas.

10

Fertility, Pregnancy, and Wellness

TABLE 1.4 Main thematic areas concerning the total of scientific production found from the analysis. Documents Medicine

7130

Social sciences

3804

Psychology

2300

Agricultural and biological sciences

2450

Biochemistry, genetics, and molecular biology

1092

Veterinary

1085

Nursing

1013

Arts and humanities

842

Economics, econometrics, and finance

674

Environmental sciences

648

Engineering

527

Business, management and accounting

521

Computer science

197

Neurosciences

370

Health professions

359

Pharmacology, toxicology, and pharmaceutics

154

Mathematics

127

Immunology and microbiology

112

Energy

87

Decision sciences

83

Earth and planetary sciences

74

Dentistry

50

Multidisciplinary

50

Physics and astronomy

49

Chemical engineering

43

Chemistry

42

Material science

32

Undefined

43

These results show the main two thematic areas: medicine and social sciences. Medicine is the main area of publication in the health field since medicine is one of the most ancient areas of research [63]. The same could apply for the social sciences since social structures and social behavior have been studied for centuries [64]. The first 11 journals that have published in the research field and the number of publications according to the Scopus database are shown in Table 1.5.

As it can be seen, most of the journals with higher documents published and a higher impact factor are from UK, Canada, and US. As previously stated, the possible explanation for the main journals from US, UK, and Canada is the interaction or collaborations between these countries and development in the health field carry out in these countries [65,66]. A further analysis was carried out based on the dominant authors in the field of healthy lifestyle and wellbeing. Table 1.6 represent the scientific production of the top five researchers focused on this subject during the past decade. von Keyserlingk, M.A.G. arises in this field with 27 documents during the past 10 years. Nevertheless, this author has an h-index of 48 lower than Weary, D.M. with an h-index of 62. The following authors according to h-index were Bambra, C. with 43 and Slade, T. with 43. The author with a lower h-index was Tuyttens, F.A.M. with 29. Nevertheless, possible key authors in the field of lifestyle and wellbeing, such as Eysenbach, G. with an h-index of 44, are not included, based on the number of documents on this topic. Table 1.7 was included to show the ten top authors regarding documents in this topic, h-index, citations, total of publications, and the year of the first publication. Table 1.7 shows how younger authors have a smaller number of publications, lower h-index, and fewer publications. This is important to highlight since the number of publications in the topic of lifestyle, wellbeing and chronic diseases, identified in the current chapter, is not fully representative of the relevance of the authors. As with any community, a scientific society is highly linked, creating a collaborating and active network. This type of group usually has a central nucleus cohesively associated to other elements from groups less represented. A scientific community is normally conformed by other clusters from other groups. Clustering is a significant issue in the current work. Recognizing these groups has relevant importance to the topic of study since determining them makes it possible to define the quantity and quality of the existing associations between the authors of different institutions and areas of knowledge. The finding of interactions between different thematic areas, such as medicine and engineering, has been established [67]. The algorithm mapping technique used by the software VOSviewer [68] was applied in order to identify and measure the association between authors. VOSviewer’s algorithm purposes focused on the detection of the items in a lowdimensional space so that the distance between two items is a precise pointer of their affinity. Fig. 1.10 reflects the detection of the cluster from the scientific communities of the authors. This figure displays

The importance of healthy lifestyles in helping achieving wellbeing Chapter | 1

11

TABLE 1.5 Quartile, SCImago Journal Rank (SJR), and impact factor of major worldwide journals. Source

Q1a

SJRb

JCRc

Total docs (2018)

Total doc (3 years)

Total ref.

Total cites (3 years)

Cites/docs (2 years)

Country

Social Science and Medicine

Q1

2.03

3.08

509

1599

44,305

18,063

3.71

UK

PLoS One

Q1

1.18

2.76

217,985

62,994

223,689

74,005

3.11

US

BMC Public Health

Q2

1.38

2.56

1.322

3650

58,519

3335

2.94

UK

Children And Youth Services Review

Q1

0.75

1.68

506

1224

8042

1950

2.04

US

Applied Animal Behaviour Science

Q1

0.86

1.82

190

566

9580

5378

2.1

The Netherlands

Animal Welfare

Q2

0.69

1.57

38

138

1491

2079

1.78

UK

Journal of Dairy Science

Q1

1.34

2.56

1023

2910

45,459

24,627

3.11

US

Child Abuse and Neglect

Q1

1.5

2.84

339

797

18,601

5810

2.65

US

International Journal of Environmental Research and Public Health

Q2

0.81

2.47

2929

5.720

144,270

52,508

2.81

Switzerland

Ageing and Society

Q2

0.84

1.89

198

289

6109

389

2.2

UK

a

Quartile represents the journal’s position based on the ranking of such journal in the specific area according to the impact factor. SCImago Journal Rank (SJR), developed by SCOPUS, is represents the relevance or impact of a journal in one area, based on the citations of publications and number of the journal in the scientific field. c Journal Citation Report (JCR), available in Web of Science and created by Clarivate Analytics, represents the impact factors of each indexed journal based on several measures like the number of publications and frequency of citations of each journal. It is the equivalent to the SJR. b

TABLE 1.6 Authors that have published a high number of studies focused on the topic of this chapter (like lifestyle, wellbeing, nutrition or chronic diseases) in the past decades according to publication number. von Keyserlingk, M.A.G.

Weary, D.M.

Tuyttens, F.A.M.

Bambra, C.

Slade, T.

Total

2008

0

0

3

3

0

6

2009

2

1

2

3

1

9

2010

1

1

0

2

1

5

2011

2

3

2

0

2

9

2012

0

0

2

1

1

4

2013

2

3

0

0

2

7

2014

1

1

2

3

4

11

2015

4

4

3

4

5

20

2016

2

5

4

3

1

15

2017

5

4

4

1

2

16

2018

8

4

1

2

0

15

TOTAL

27

26

23

22

19

117

the interactions between the key authors and the rest of the researchers in the field of social networks related to the health of young people. The cluster 1 (red) is the major one in terms of 20 authors, most of them Asian or

Canadian. The following cluster (green) is formed by 18 authors, with the top author Veissier, I. with 20 documents (Table 1.7). The top author in collaborations and publications is Landsverk, J. with 26 publications and 18

12

Fertility, Pregnancy, and Wellness

TABLE 1.7 Top 10 authors published in the topic discussed in this chapter (like lifestyle, wellbeing, nutrition or chronic diseases), h-index, citations, and total of publications. Authors

Publications

h-Index

Total citations

Total publications

First publication

Weary, D.M.

29

62

12005

317

1984

Landsverk, J.

27

64

12889

192

1983

Tuyttens, F.A.M.

27

29

3116

165

1976

von Keyserlingk, M.A.G.

27

48

7412

248

1993

Bambra, C.

22

43

5549

183

2003

Brooks-Gunn, J.

21

94

35870

474

1974

Barth, R.P.

20

51

8869

216

1979

Veissier, I.

20

39

4868

130

1988

Zubrick, S.R.

20

43

6350

209

1982

Broom, D.M.

19

49

6747

180

1968

FIGURE 1.10 Scientific clusters of researchers focused on social networks in health.

The importance of healthy lifestyles in helping achieving wellbeing Chapter | 1

TABLE 1.8 Forty key keywords used in publications Order

Keyword

Documents

%

1

Human/s

13,243

86.57

2

Female

5402

35.31

3

Male

2517

16.45

4

Adult

3577

23.38

5

Adolescent

2088

13.65

6

Wellbeing

2088

13.65

7

Middle Aged

1908

12.47

8

Controlled Study

1884

12.32

9

Mental Health

1837

12.01

10

Psychology

1735

11.34

11

Questionnaire

1911

12.49

12

Mental Health

1899

12.41

13

Review

1845

12.06

14

Young People

1623

10.61

15

Psychology

1595

10.43

16

Child

1351

8.83

17

Priority Journal

1317

8.61

18

Aged

1315

8.60

19

Major Clinical Study

1480

9.68

20

Questionnaire

1355

8.86

21

Animal Welfare

1220

7.98

22

Quality of Life

1183

7.73

23

Health Status

1178

7.70

24

Review

1166

7.62

25

Health

1154

7.54

26

Young Adult

1114

7.28

27

Animals

1109

7.25

28

Depression

1026

6.71

29

United States

943

6.16

30

Animal

935

6.11

31

Welfare

920

6.01

32

Child Welfare

906

5.92

33

Psychological Aspect

905

5.92

34

Animalia

850

5.56

35

Social Support

788

5.15

36

Socioeconomics

774

5.06

37

Physiology

761

4.97

38

Risk Factor

759

4.96

39

Socioeconomic Factors

738

4.82

40

Australia

736

4.81

13

collaborators. In this sense, the second author is Weary, D.M. with 29 publications and 9 collaborators. Another analysis was carried out to determine the keywords used in the publications in this field. During the past four decades, out of the 15,297 documents found, the author’s keywords were human/s, used in 13,243 items, followed by female (5402 items) and male (2517 items). Table 1.8 illustrates the 40 most important keywords used in the publications observed through the bibliometric analysis during the past four decades. The analysis of the authors’ keywords used showed that most keywords are usually utilized on this topic. Nevertheless, it is important to highlight that the term “Human/s” was probably used to differentiate it from animal research, more than for relevant significance for the topic. Based on these keywords, the results might be implying the transversal inclusion of a healthy lifestyle in the wellbeing, as mental health is to diabetes. It is important to highlight that keywords and the topics of the studies also presented different points of view, such as side effects of unhealthy diets. All being said, the study of the keywords in scientific works is highly relevant since this determines the trends and follow-up of these publications. In Fig. 1.11, a cloud of words, where the dimension of each word represents the significance of the keywords related to the number of documents in which it used, is represented. The growth of other words such as “Health” or “Health Status” might be related with the development of telemedicine and the studies focused on new technologies and healing [69
71]. Fig. 1.12 displays the map of cooccurring keywords selected by the researchers from the documents found that focused on social networks in the health of young people. The software VOSviewer with the VOS mapping technique was used in order to acquire Fig. 1.12. Each color symbolizes the separation between keywords concerning the thematic areas. Also, the dimension of the circles displays the frequency (use) of each word, and the lines linking each circle show the association among the different keywords used in the publications.

FIGURE 1.11 Cloud of words corresponding to keywords used by authors.

14

Fertility, Pregnancy, and Wellness

FIGURE 1.12 Map of cooccurring keywords.

In this analysis, “Human,” “Female,” and “Article” are the words most usually used. Table 1.8 shows the top keywords used by the five top groups identified in the subject of healthy lifestyle and wellbeing [72
74].

Role of wellbeing and lifestyles in women’s reproduction Form the previous data, it could be concluded that healthy lifestyles may be linked to the decrease of multiple diseases, such as infertility, and prevention of risky behaviors, such as mental distress, anxiety, or depression. Regarding fertility, Fig. 1.13 and Table 1.9 shows how among the six clusters that the most important, the red

cluster, focused on healthy lifestyles related to age, pregnancy, public health, social behavior, and organization and management. In this sense, out of the 15,306 documents found, 1550 were related to pregnancy and 867 focused on fertility, reflecting the relationship of the pregnancy with public health, organization and management or age of the population. This cluster matches previous results that stated the importance of lifestyle, social behavior, age, and organization and management related to pregnancy. An example of this is the late age of the mothers carrying child due to economic or social issues, such as job instability or internal promotions [75
78]. Moreover, most of these authors estimate that up to 17% of women suffered from infertility, being the most preventive keystone focused on healthy lifestyle policies to increase natality and decrease neonatal diseases [75
78].

The importance of healthy lifestyles in helping achieving wellbeing Chapter | 1

15

FIGURE 1.13 Coocurring of the keywords in the analysis of fertility and wellbeing.

TABLE 1.9 Keywords most utilized by the six top communities identified in the topic to wellbeing and fertility. Cluster

Color

Main keywords

Topic

%

1

Red

Public health
Pregnancy
Organization and management
Social behavior
Health education
Age

Public health
Pregnancy
Education
Age

22.8

2

Green

Adult
Aged
Depression
Major clinical study
Quality of life
Exercise

Adults
Mental health
Exercise

22.0

3

Blue

Male
Middle-Aged
Young adult
Health status
Socioeconomics

Men
Aged
Health

14.6

4

Yellow

Animal production
Diet
Weight

Animal production

14.5

5

Purple

Family
Child welfare
Consumption

Child wellbeing
Consumption

13.4

6

Orange

Psychology
Wellbeing
Emotional distress
Working

Psychology
Working

12.7

The green cluster focuses on lifestyles and mental health, especially including the quality of life of aged adults and the role of exercise. This cluster matches the last works focused on preventing and improving the mental state by including exercise or physical activity. The third cluster focuses on young adults or middle-aged men whose health status is linked to their lifestyle and the socioeconomical status. This cluster presents the current public health issue regarding the increasing number of young or middleaged men with unhealthy lifestyles which is linked to their

health and their incomes [79
81]. The fourth cluster points out the relationship between animal production, dieting, and weight. In this sense, recent studies have pointed out the increase of individuals who follow a vegetarian or vegan diet based on their weight or moral views on animal protection [82,83]. Nevertheless, it is important to have in mind the repercussion that dietary changes induce in the health of the person. One major impact is on the intestinal flora, or microbiota, which may result in different alterations, and even immunity-related diseases. The microbiota,

16

Fertility, Pregnancy, and Wellness

which is the entire population of the microorganisms mainly located in the intestine, is linked to metabolic, immunological and gut protective functions in the healthy individual. In fact, the intestine is called the second brain because of its relationship and equilibrium with the rest of body, which is based on its extensive metabolic ability and considerable purposeful plasticity [84]. Moreover, the intestine is the main channel for incorporating vitamins and nutrients through the bloodstream. The problem arises when a toxic or unknown compound reaches the bloodstream through the intestine and is incorporated into the body of the individual or even the fetus. The purple cluster is focused on child welfare and the relation of family and the consumption of substances or unhealthy diets. The family influence has been studied as a major negative and positive factor related to the mother’s health, behavior, and lifestyle [85,86]. In this sense, an environment where the child feels protected and laughter and happiness are promoted may reduce the risk of developing health issues. In fact, many health therapies based on laughter have been created to improve the immune system and emotional distress in children during hospitalization and especially in children with cancer. These therapies are based on the biochemical responses, with oxytocin as one of the main compounds released. This hormone mediates in the liberation of endorphins, which decrease pain and relax the system. Therefore, laughter is a resourceful tool to improve the health of children and adults alike and currently used in hospitals around the world [87,88]. The last cluster, the orange, focused on the relationship between lifestyle, wellbeing, psychology, and workplace. This result matches with previous analysis that have stated the relationship between unhealthy lifestyle and stress in the workplace [89
91].

Conclusions Although there was a growth in research topics, the major increase was observed from 2004 to 2018. This growth matches with the increase inclusion of new policies and recommendations from the organizations [47]. Also, during that time the increase of chronic diseases and the role that lifestyles play have been further studied [46,92]. In this sense, the inclusion on the prevention of unhealthy behaviors and healthy lifestyles have been included in the national health systems and companies [13,53]. The previous overview of the topic using bibliometrics analysis has shown that collaboration between authors and countries seems to be led by the US as the common connection. Based on this, countries with further number of institutions with a high interest in health prevention measures and health systems, and a higher number of inhabitants, like the US, could explain the rate of studies focused on healthy

lifestyles and wellbeing [72,93,94]. Nevertheless, the most common journals were from Australia, UK, or European countries. The connections are formed by those countries with traditional political, historic, and economic relationship. In general, therefore, it seems that the relationship between healthy lifestyles and wellbeing, especially focused on women and pregnancy, continues to grow to understand and determine factors connected to chronic disease and to decrease them. In this sense, the recommendation should focus on preventing risk factors and promoting balanced coexistence between the individual’s behavior and the environment. Moreover, healthy lifestyles that are more natural, provoking little stress-related changes in the humans’ genetic background, should be promoted. In this sense, food consumption and exercise seem to be a breakpoint for the health of the population. All being said, these findings have significant implications for the understanding of how the future of health care may lead to the role of lifestyle in the wellbeing of education and communication with patients.

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PART I

Background

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Chapter 2

Male reproductive system Juan Andre´s Ramı´rez-Gonza´lez1 and Andrea Sansone2 1

Morphology, Faculty of Health Sciences, University of Las Palmas de Gran Canaria, Spain, 2Endocrinology and Medical Sexology (ENDOSEX),

Department of Systems Medicine, University of Rome Tor Vergata, Rome, Italy

Anatomy of the male reproductive system The male genital system consists of internal organs (testis, epididymis, ductus deferens, seminal vesicle, ejaculatory duct, prostate, and bulbourethral gland) and external organs (penis and scrotum) [1,2] (Fig. 2.1).

Testis and epididymis The testis, or male gonad, is a paired structure that produces sperm and the hormone testosterone. Sperm is temporarily stored for maturation in the epididymis, where it acquires mobility and fertilization capacity. Together, the testis and the epididymis weigh about 30 g [1
3]. The testis is an ovoid organ measuring 40
50 mm in length, 25 mm in width, and 20 mm in thickness. Each testis has a medial surface and a lateral surface, separated by an anterior and posterior border, as well as an upper and a lower pole. Each testis has a thick capsule of white connective tissue, the tunica albuginea, which thickens at the posterior edge to form the mediastinum of testis. From the mediastinum multiple partitions are detached; the septula testis penetrates into its interior and divides it into lobules of testis. Located inside the lobules is the parenchyma of testis formed by seminiferous tubules, contoured tubes of very small diameter, in whose walls spermatozoa are produced (spermatogenesis). Such tubules are surrounded by testosterone-producing cells (Leydig cells). The ends of the tubules are rectified and form the straight tubules, which converge towards the testicular mediastinum and form a network, the rete testis, from which 8
12 efferent ductules are detached, leaving the testis and flowing into the anterior face of the epididymis. The vascular layer covers the tunica albuginea inside and carries the vessels that irrigate the testicular parenchyma [1,4
6]. The epididymis, a paired structure, is about 6 cm long, and 5
10 mm thick. It has the shape of a comma, with Fertility, Pregnancy, and Wellness. DOI: https://doi.org/10.1016/B978-0-12-818309-0.00006-X © 2022 Elsevier Inc. All rights reserved.

several differentiated regions (head, body, and tail). It is connected by the superior and inferior ligaments of the epididymis to the surface of the posterior edge of the testis, with which it forms an indissoluble functional unit. Structurally, the epididymis is a very complex tubular system, in which the efferent ductules, straight during their passage through the mediastinum, undergo an important winding and anastomose to give rise to the lobules of epididymis, which form the head of the epididymis. The duct of epididymis runs longitudinally inside the body and the tail of the epididymis. Distally, the epididymis continues with the ductus deferens [3,4]. Coverings of the testis and epididymis: The testis and epididymis are invested by a double serous leaf, derived from an embryonic evagination of the abdominal peritoneum, called the tunica vaginalis. The inner layer, defined the visceral layer, intimately adheres to the surface of the testis and epididymis, while the outer layer, or the parietal layer, adheres to the inner surface of the internal spermatic fascia. The two layers continue, one with the other, at the level of a vestige of processus vaginalis, located at the posterior edge of the testis: a permeable vaginal process allows the passage of liquid from the peritoneal cavity to the interior of the space located between the two layers and its accumulation inside, giving rise to the appearance of hydrocele. This situation can be reversed with decubitus. Outside the tunica vaginalis, the testis is surrounded by the expansions of the covers of the spermatic cord and the scrotum [3,4,7]. Blood and lymphatic vessels: The arterial vascularization of the testis and epididymis comes mainly from the testicular artery. This long and narrow artery originates at the anterior face of the aorta at the level of the LII vertebra, caudally to the renal artery, and descends across in front of the structures of the posterior wall of the abdomen (inferior cava vein and ureter on the right; only ureter on the left) to reach the inguinal canal and join the spermatic cord. When it reaches the testis, it is divided 23

24

PART | I Background

FIGURE 2.1 Diagram male genital system.

into anterior and posterior testicular (these are perforating branches distributed through the vascular layer), epididymal and cremasteric branches. It ends in anastomoses with branches from the artery of the ductus deferens and the cremasteric artery, arteries that form part of the spermatic cord. The testicular veins are numerous; they leave the testis crossing the mediastinum and are anastomosed abundantly with other veins from neighboring territories (deferential and cremasteric territories) to form the pampiniform plexus. The pampiniform plexus occupies the anterior part of the spermatic cord and ascends, surrounding the ductus deferens and the testicular artery. Once in the abdominal cavity, the plexus continues as testicular vein which, in a retroperitoneal situation, goes up and back to drain into the inferior vena cava (the right vein) or else into the left renal vein (the left vein) [3,4,8,9]. Dilatation of the testicular venous system can condition the appearance of a varicocele, more frequent on the left instead of the right vein. This anomaly causes the normal blood circulation of the testis to be altered with an increase in the local temperature, which might possibly lead to a decrease in the formation of spermatozoa. Additionally, varicocele might influence testicular development, resulting in impaired testicular growth.

of

the

The lymphatic vessels from the testis and epididymis ascend accompanying the ductus deferens, along with the one running through the inguinal canal. In the abdominal cavity they follow the testicular artery and end up draining mostly in the (periaortic) lumbar nodes. Nerves: The testis is an organ extraordinarily sensitive to pressure, a circumstance that must be taken into account during the physical examination. The testis and epididymis are innervated by the testicular plexus, which predominantly carries sympathetic postganglionic fibers from the abdominal aortic plexus to the smooth musculature of the blood vessel wall and the epididymis. Also contains sensitive fibers to transmit the painful sensation through the T10
T11 spinal ganglia [7,10].

Spermatic cord The spermatic cord is a paired structure whose main function is to “suspend the testis inside the scrotum” [7,8,10]. It consists of the structures that allow the testis to function normally and includes the ductus deferens, arterial and venous blood vessels, lymphatic vessels, and nerves for the testis and its covers. The spermatic cord starts at the level of the deep inguinal ring, laterally to the inferior

Male reproductive system Chapter | 2

epigastric artery, then goes through the inguinal canal and reaches the outside of the abdominal cavity through the superficial inguinal ring. From there, it descends into the scrotum to reach the testis. In its extra-abdominal path, the spermatic cord is surrounded by its protective covers derived from the anterior wall of the abdomen, which were dragged into the scrotum by the testis and the vaginal process. The covers of the spermatic cord are located deep in the skin that covers the pubic and scrotal regions. From deep to superficial, the internal spermatic fascia, the cremasteric fascia, and the external spermatic fascia are arranged [4,7,11]. The internal spermatic fascia is the thinnest and deepest layer surrounding the spermatic cord. It was formed by the drag of a thin layer of the transversalis fascia. The cremasteric fascia forms after the dragging of some of the fibers of the cremaster muscle, caudally located at the lower edge of the internal oblique muscle. It is closely related to the genital branch of the genitofemoral nerve. Physical stimuli, such as scratching and cold, cause the spermatic fascia to contract and elevate the testicle within the scrotum, a reaction defined as cremasteric reflex. The external spermatic fascia is the most superficial cover of the spermatic cord. It corresponds to a dragged extension of the aponeurosis of the external oblique muscle. It is related to the genital branch of the ilioinguinal nerve. Located on its surface are the dartos muscle and the skin. Blood and lymphatic vessels: The arteries of the spermatic cord are of multiple origins and usually anastomosed to form an arterial network around the testis and epididymis. In addition to the testicular artery, the spermatic cord contains the artery of ductus deferens, usually a branch of the umbilical artery, and the cremasteric artery, a small branch of the inferior epigastric artery that supplies the cremaster muscle and other components of the coverings of the spermatic cord. The veins of the spermatic cord are part of the pampiniform plexus. The lymphatic vessels pass through the inguinal canal to drain into the (periaortic) lumbar nodes. Nerves: Inside the spermatic cord there are sympathetic fibers, which surround the arteries, and sympathetic and parasympathetic fibers that surround the ductus deferens. The genital branch of the genitofemoral nerve, at a deeper level, reaches the spermatic cord to innervate the cremaster muscle; the genital branch of the ilioinguinal nerve, more superficial, provides sensory innervation.

Ductus deferens The ductus deferens, a paired structure, is a long, thickwalled tube that carries sperm from the testis to the urethra. It is 40
45 cm long and 3 mm in diameter. This organ, which has a highly developed muscular layer and a

25

stratified epithelium in its wall, begins as a direct continuation of the tail of the epididymis (scrotal portion) and ascends inside the spermatic cord (funicular portion), in the direction of the inguinal canal (inguinal portion). Once it passes through the duct, it crosses ventrally to the external iliac vessels, enters the lesser pelvis, and runs along its lateral wall, located medially to the vessels and nerves in a retroperitoneal situation (pelvic portion). Then, and always outside the peritoneum, to which it is attached, it changes its direction and runs downward, inward, and forward to cross over the ureter (near the posterolateral angle of the bladder) and reach the posterior face of the urinary bladder, where it comes into contact to the seminal vesicle. Initially, the ductus deferens are above and outside the seminal vesicle and the ureter; then, on descending, is medially situated to these structures. When located on the posterior face to the bladder, the ductus dilates forms the ampulla of ductus deferens and then joins the excretory duct of seminal vesicle to form the ejaculatory duct, which passes through the prostate [3,4,10]. Blood and lymphatic vessels: The artery of the ductus deferens comes from the internal iliac artery, either directly or through the umbilical artery, or the inferior vesical artery. Small in caliber and closely applied to the surface of the duct, the artery ends up being anastomosed distally with branches of the testicular artery. The veins of the ductus deferens follow the artery along its path and end up forming part of the pampiniform plexus, on the one hand, and draining into the vesical venous plexus, on the other. The lymph is drained to the internal and external iliac nodes. Nerves: The ductus deferens receives abundant innervation through sympathetic fibers, coming from the inferior hypogastric plexus, which facilitates its rapid and energetic contraction for the expulsion of sperm during ejaculation. It also receives parasympathetic fibers that control the secretion of the epithelium (Fig. 2.2).

Seminal vesicle and ejaculatory duct The seminal vesicle, is a thin-walled paired structure measuring about 5
10 cm in length and 3
5 cm diameter. Despite its oval macroscopic appearance, the seminal vesicle is actually a blind tube, about 10
15 cm long, and 1 cm in diameter. Rolled up on itself and surrounded by a connective tissue capsule, it takes on an appearance similar to a bag. Distally, the excretory duct of the seminal vesicle, the neck of the seminal vesicle, joins to the corresponding ductus deferens to form the ejaculatory duct [4,7]. The seminal vesicle is located behind the urinary bladder, between the bottom of the bladder and the rectum. The upper ends of both seminal vesicles are covered by the peritoneum, which at this level forms the bottom

26

PART | I Background

FIGURE 2.2 Sagittal section view of pelvic viscera and perineum in men.

of the rectovesical pouch, a recess that separates them from the anterior surface of the rectum. Their lower extremities are closely related to the ureter and the rectum, from which they are separated only by the rectovesical septum. The wall of the seminal vesicles has three layers: an internal layer of pseudostratified columnar epithelium, with goblet cells and a lamina propria (mucosa);

an intermediate layer of smooth muscular tissue (muscular); and an external layer composed of areolar tissue (adventitia). Seminal vesicles do not store sperm; on the contrary, they secrete a thick alkaline liquid that mixes with sperm as they pass through the ejaculatory ducts and urethra, providing most of the semen volume.

Male reproductive system Chapter | 2

The ejaculatory duct, a paired structure, is about 2.5 cm long and 2.3 mm in diameter. It is the result of the join, near the neck of the urinary bladder, of the neck of the seminal vesicle with the terminal portion of the ductus deferens. Immediately after its formation, it penetrates the thickness of the prostate and goes downward, forward, and inward, to flow into the posterior wall of the prostatic segment of the urethra, at the seminal colliculus, where its drainage hole takes the form of a cleft located laterally to the prostatic utricle [3,4,11,12]. Blood and lymphatic vessels: The arteries of the seminal vesicle come from the inferior vesical artery, the artery of the ductus deferens and the middle rectal artery. The veins accompany the arteries and end up draining into the vesical and prostatic vein plexuses. Lymph from the vesicles is mainly drained to the lymph nodes of the iliac chains, especially to the internal iliac. Nerves: The walls of the seminal vesicles contain a plexus of nerve fibers and some sympathetic nodes. The sympathetic fibers are preganglionic and come from the inferior hypogastric plexus, through the prostatic and vesical plexuses: they produce the contraction of the muscular fibers of the wall of the vessels and of the vesicle itself, facilitating the expulsion of their contents during ejaculation. Parasympathetic fibers come from the pelvic splanchnic nerves and promote glandular secretion [4,10,13].

Prostate The prostate is the largest accessory gland of the male genital system. It has the shape and size of a chestnut (30 mm long, 40 mm wide, and 40 mm thick) and weighs about 25
30 g. Structurally, the prostate is a compact organ, partly glandular and partly fibromuscular, surrounded by its own fibrous capsule—thin and dense, the capsule of prostate—which separates it from the fascia of prostate, a dependence of the endopelvic fascia. The parenchyma of prostate consists in 20
30 glands whose ducts open mainly in the prostatic sinuses, depressions located in the posterior wall of the prostatic urethra, on each side of the urethral crest. The prostatic secretion is a liquid, milky appearance, which provides about 20% of the volume of ejaculated semen and plays a determining role in protecting the viability of sperm and their ability to fertilize. The fascia of prostate surrounds the gland. Laterally, it continues with the fascia of the levator ani muscle. Behind it, it forms part of the rectovesical septum, a thickening of the connective tissue that separates the urinary bladder, the seminal vesicles, and the prostate from the anterior surface of the rectum [4,8,14,15]. Macroscopically, the prostate describes a base, an apex, and four surfaces: anterior, posterior, and inferolateral surfaces.

27

The base of the prostate is directed upward and slightly backward. A small transverse elevation divides it into two fields, urinary field (anterior) and seminal field (posterior). The urinary field is closely related to the neck of the urinary bladder (bladder face), so that the prostatic urethra penetrates through the central region of the base, very close to its anterior face. The seminal field takes the form of a transverse groove that allows the ejaculatory ducts to enter through it. In front of and behind the groove, the pre-spermatic and retro-spermatic areas are located. The apex of prostate is lower and directly related to the floor of the pelvis, resting on the external sphincter muscle of the urethra, medially, and the medial edges of the levator ani muscles, laterally. The anterior face is convex and narrow. It extends from the base to the apex and looks towards the retropubic space, where an extensive preprostatic venous plexus is located. This face is surrounded by deep fibers from the urethral sphincter muscle. The inferolateral surfaces are also convex and narrow. They rest directly on the fascia that covers the levator ani muscles. The posterior face of the prostate is triangular and flattened in a transverse direction. Directed backwards and slightly downwards, towards the pelvic floor, this face rests on the anterior surface of the rectal ampulla, allowing it to be palpated from the rectum, about 4 cm from the anus. In general, the posterior surface presents a shallow middle groove, which indicates the medial boundary of the lateral lobes (often the lateral lobes are fused together, making it possible to refer to them, as a whole, as the posterior lobe) [3,4,8,10]. Structurally, there are two lobes in the prostate (right and left); they are joined by the isthmus of prostate, which covers the anterior face of the urethra. Each lobe is divided into four lobules: the anteromedial and superomedial lobules, derived from the middle embryonic lobe, that occupy a high and central position, surrounding the proximal prostatic urethra and the ejaculatory ducts and the inferoposterior and inferolateral lobules, which occupy a lower and lateral position, related to the distal prostatic urethra. Likewise, from a clinical
histological point of view, in the prostate it is possible to distinguish different glandular zones: the periurethral covering zone, which surrounds the proximal urethral, is flanked by the transitional zone, in which the prostatic glandular mass is scarce (5%). The central zone, located behind the periurethral and transitional zones, forms the base of the gland surrounding the ejaculatory ducts and comprises 25% of the glandular mass, while the peripheral zone, which is the widest zone (70% of the glands), surrounds most of the central zone and partially the distal part of the prostatic urethra. There is also an anterior nonglandular area,

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PART | I Background

consisting mainly of muscle and fibrous tissue, located at the apex of the prostate [7,8,12,16]. Blood and lymphatic vessels: The prostatic arteries derive mainly from the inferior vesical, middle rectal, and internal pudendal arteries, branches of the internal iliac artery. The prostatic artery branches inside the gland and emits two types of branches: urethral branches, which irrigate the central portion of the prostate, and capsular branches, for irrigation of the peripheral portion of the organ. The veins of the prostate form the prostatic venous plexus, located between the prostate capsule and its fascia. Surrounding the prostate all sides, including the base, this plexus communicates with the vesical venous plexus, from above, with the rectal venous plexus and, at a greater distance, with the vertebral venous plexus from behind. Most of the venous blood ends up being drained into the internal iliac veins. The prostatic lymphatic

FIGURE 2.3 Posterior view of bladder and prostrate.

vessels lead the lymph to the obturator, common and internal iliac nodes, and sacral nodes, although some vessels from the posterior side join the lymphatic vessels of the bladder and drain into the external iliac nodes. Nerves: The sympathetic fibers come from the inferior hypogastric plexuses, originate the prostatic plexus and, following a perivascular trajectory, reach the gland, where they promote the contraction of the smooth muscle of the capsule and the glandular stroma, an action that makes it possible to empty the gland. Parasympathetic fibers come from the pelvic splanchnic nerves (S2
S4): they control the secretion of prostatic acini (Fig. 2.3).

Bulbourethral glands The bulbourethral glands are two small, round, yellowish pea-sized organs (1 cm in diameter), located behind and

Male reproductive system Chapter | 2

outside the membranous urethra, surrounded by fibers of the external urethral sphincter. The duct of secretion of the bulbourethral gland is relatively long (3
5 cm) and runs through the perineal membrane, accompanying the urethra. It opens through a tiny hole in the proximal part of the spongy urethra of the penis where it pours its secretion, which intervenes in the lubrication of the urethra. The bulbourethral gland shares vascularization and innervation with the corpus spongiosum of penis [7,10,12,15].

Penis The penis is the male organ of copulation. It constitutes the common route for elimination of urine and semen to the outside. The skin of the penis is very thin, dark in color, and usually has very little adherence to deep planes. This is followed by the skin of the anterior wall of the abdomen (dorsal surface), and the skin of the perineum and scrotum (ventral surface). Structurally, the penis consists of three erectile bodies: the paired corpus cavernosum penis, dorsally situated; and the corpus spongiosum penis, ventrally situated. Three parts are described in the penis: the root, the body, and the glans. The root of penis is the fixed portion. It is situated in the superficial perineal pouch, between the perineal membrane (deeply) and the perineal fascia (superficially). It is formed by the crura of penis and the bulb of penis. The crus of penis, located laterally, constitute the proximal end of the corpus cavernous; it is solidly anchored to the ischiopubic ramus and covered by the ischiocavernosus muscle. The bulb of penis belongs to the corpus spongiosum penis, which has a central placement and receives, on its dorsal side, the spongy urethra and, on each side, the ducts of the bulbourethral glands. It is covered by the bulbospongiosus muscle [3,4,11,14,17]. The body of the penis is the free part of the penis and is surrounded by skin. Cylindrical in form, it consists of the central portion of the corpora cavernosa and the corpus spongiosum. The corpora cavernosa extend along the ischiopubic rami, to which they are intimately attached, converge towards the midline—in front of and below the pubic symphysis—and are parallel to each other on the dorsal face of the penis body. The corpus spongiosum occupies the space situated ventrally to the corpora cavernosa and carries the spongy urethra in its interior. Each erectile element has its own fibrous envelope, the tunica albuginea of corpora cavernosa and the corpus spongiosum [3,4,6]. The albuginea of corpora cavernosa is attached to the midline and forms the septum penis, a structure that connects them intimately and allows them to function together, dragging the corpus spongiosum dorsally during erection. Superficially, the fascia of the penis surrounds all the erectile structures. Except of some fibers from the anterior parts of the ischiocavernosus and

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bulbospongiosus muscles, located very close to the root, the body of the penis does not contain muscles. The weight of the body of the penis is aided to support by two ligaments: the suspensory ligament of penis (deep) and the fundiform ligament of penis (superficial), which detach from the anterior abdominal wall. The glans of the penis is the dilated expansion of the anterior end of the corpus spongiosum. Projected distally to the end of the corpora cavernosa, which it covers, the glans has a conical appearance with a peripheral edge directed downwards and forwards called corona of glans. In the base of the corona are located abundant glands that secrete the smegma. The external urethral orifice has the appearance of an open vertical cleft at the apex of the glans. Structurally, the glans has a higher concentration of sensory nerve endings than the rest of the body of the penis, making it especially sensitive to physical stimulation [3,8,18]. The prepuce of penis is the part of the skin that covers the glans in a variable extension. It consists of two layers, superficial and deep, which continue at the level of the orifice of the foreskin. The deep layer has a mucous aspect; it is inserted in the internal rim of the base of the glans (neck of glans) and, from it, a sagittal septum is detached, the frenulum, which is inserted ventrally and immediately below the external orifice of the urethra. It controls the displacement of the prepuce. Blood and lymphatic vessels: The penis is vascularized by the internal pudendal artery through its different penile branches. These include dorsal branches, deep branches, and bulbo-uretral branches. The dorsal arteries of the penis run longitudinally along the dorsal face of the corpora cavernosa, on either side of the deep dorsal vein of penis, located deep in the fascia of the penis. They emit collaterals that irrigate the superficial part of the corpora cavernosa and end up in an anastomosis at the base of the glans. The deep arteries of the penis are destined for the corpora cavernosa; they approach, from behind and above, the crus of penis and introduce into each of the corpora cavernosa, to which they run from behind forward. They are the main vessels of the erectile tissue and emit numerous branches that communicate directly with the cavernous spaces. When the penis is flaccid, these branches are rolled up and shortened, adopting a spiral arrangement (helicine arteries). The artery of the bulb of the penis penetrates on each side and assumes the irrigation of the erectile tissue of the corpus spongiosum, including the glans, and the spongy urethra. On the other hand, the perineal arteries contribute to vascularization of the root of the penis and the adnexa musculature (bulbospongiosus and ischiocavernosus muscles). The blood coming from the cavernous spaces and from the bulb of corpus spongiosum is drained by a complex peri-penile venous plexus, located below the fascia of the penis. This

30

PART | I Background

FIGURE 2.4 Cross-section view of the penis.

plexus, after encircling each side of the body of the penis, joins the deep dorsal vein of penis, located in the dorsal face, deeply to the fascia. This vein enters the pelvis, between the arched ligament of the pubis, located in front and above, and the transverse ligament of the perineum, behind and below, and is incorporated into the prostatic venous plexus, through which it drains mostly into the internal iliac vein. Blood from the skin of the penis is drained through the superficial dorsal vein of penis, a subcutaneous vein that drains into the great saphenous vein, through the external pudendal veins. Although most of the lymphatic vessels in the penis drain the superficial inguinal nodes, those coming from the glans usually drain the deep inguinal nodes [7,10,18]. Nerves: The dorsal nerve of the penis, the terminal branch of the pudendal nerve, carries the sensitive innervation of the penis. This nerve originates inside the pudendal duct and goes forward through the deep perineal space to reach the dorsum of penis, where it is located laterally to the dorsal arteries. It supplies the glans and the corpora cavernosa, as well as the skin of the penis. The sympathetic fibers come from the inferior hypogastric plexus, cross the urogenital diaphragm to reach the penis (they form a plexus around the deep artery of the penis) and participate in controlling the blood flow to the corpora cavernosa (vasoconstriction). On the other hand, the parasympathetic fibers relax the arterial wall, making it possible for blood to enter the cavernous spaces. The skin covering the root of penis is innervated by the ilioinguinal nerve and posterior scrotal branches of the perineal nerve [3,4,7,12] (Fig. 2.4).

Urethra The male urethra is a tubular structure that carries urine from the urinary bladder to the outside. it is 15
20 cm long and extends from the internal urethral orifice

(located at the neck of bladder) to the apex of glans, where the external urethral orifice is located. Topographically, the urethra can be divided into four parts, or segments: intramural, prostatic, intermediate, and spongy. The intramural portion, or preprostatic portion, is only 1.5 cm long. It extends from the internal urethral orifice to the upper face of the prostate. Its mucosa is folded, so it has a starry light. The smooth muscle that surrounds the neck of the bladder and the preprostatic urethra adopts a circular arrangement and is known as the internal urethral sphincter, or supracollicular sphincter. it is innervated by the hypogastric plexus. The prostatic portion is 3
4 cm long and crosses the prostate from its base to the apex. In the midline of its posterior wall, it presents a longitudinal elevation of the mucosa, called the urethral crest. On each side of the crest, there is a depression, a vertical groove, known as the prostatic sinus, in which the orifices of the ducts of the prostate glands open. Halfway up the urethral crest, there is an elevation known as the seminal colliculus, which divides the prostatic urethra into two segments: proximal, or supracollicular, and distal, or infracollicular. The prostatic utricle opens on the collicular surface. The orifices of the ejaculatory ducts are located on both sides of the prostatic utricle orifice. The intermediate portion, or membranous urethra, passes through the urogenital diaphragm. It is about 2 cm long and is the least dilatable part of the urethra. At this level the external urethral sphincter is located. It is innervated directly by the pudendal nerve. The spongy portion is contained in the thickness of the spongy body of the penis. It has two dilations: proximal and distal. The proximal dilation is located inside the bulb of the corpus spongiosum: at this level the ducts of the bulbourethral glands open. The distal dilation is located inside the glans of penis and is known as the

Male reproductive system Chapter | 2

navicular fossa. Mucous membrane of the spongy urethra presents the urethral lacunae, slight depressions in which the ducts of the urethral glands open. Blood and lymphatic vessels: The vascularization of the urethra is segmental, so that the prostatic portion is irrigated by the prostatic branches of the inferior vesical arteries. The intermediate portion receives branches from the inferior vesical and middle rectal arteries; and the spongy portion receives branches from the artery of the bulb of penis, as well as from the deep and dorsal arteries of the penis (branches of the internal pudendal artery). The veins of the urethra are associated with the veins of the free portion of the penis, which drain into the prostatic plexuses. The lymphatic vessels from the prostatic portion intermingle with the vessels of the prostate itself. Those from the membranous portion drain into the external iliac lymph nodes; and those from the spongy portion drain into the inguinal and external iliac lymph nodes. Nerves: The nerves of the urethra come from the lower hypogastric plexus, directly (prostatic and intermediate portions) or through the nerves of the penis (spongy portion) [7,10,18].

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testicular temperature stability, a situation necessary for normal spermatogenesis. Blood and lymphatic vessels: The external pudendal arteries, consisting of branches of the femoral artery, assume the irrigation of the anterior face of the scrotum; the internal pudendal arteries, branches of the internal iliac artery, assume that of the posterior face. The scrotum also receives some collateral branches from the testicular artery and the cremasteric artery. All of them contribute to the formation of a rich subcutaneous arterial plexus, in which arteriovenous anastomosis are abundant. The veins of the scrotum accompany the arteries: the anterior group drain into the femoral vein, through the external pudendal veins and the great saphenous vein; the posterior veins drain into the pudendal vein. The lymphatic vessels of the scrotum drain into the superficial inguinal nodes. Nerves: Sensitive innervation of the skin anterior part of the scrotum is provided by the inguinal branches of the ilioinguinal and genitofemoral nerves. Branches from the superficial perineal nerves assume the innervation of the posterior part [7,10] (Fig. 2.5).

Perineum Scrotum The scrotum is a pouch developed from the skin of the anterior wall of the abdomen. Structurally, the scrotum is a fibromuscular, cutaneous sac, located below the pubic symphysis, behind and below the penis. The scrotal ligament, a remainder of the embryonic gubernaculum testis, join the inferior pole of testis and the tail of epididymis to the inner surface of the scrotum [7,10]. In the scrotum, skin and dartos muscle and fascia are distinguished. The skin of the scrotum is darker in color than the skin of neighboring regions; the hair is long and thin [3,5,8]. The dartos is a muscular layer of smooth fiber which, in the midline, is reflected dorsally to form the scrotal septum, dividing the scrotal cavity and separating both testes: this division is reflected externally by the presence of the raphe of scrotum, which is elongated by the ventral surface of the penis (raphe of penis) and backward, along the midline of the perineum (perineal raphe). Firmly attached to the skin, the dartos muscle contracts under the influence of cold, exercise, and sexual stimulation. In older men, the dartos loses muscle tone and the scrotum tends to be softer and hangs wider. When it is cold, the contraction of the dartos causes the scrotal pouch to stick to the testes, dragging them towards the body. On the other hand, when it is hot, the dartos relaxes and the scrotum separates from the testes, allowing them to hang freely outside the body cavity, also providing a larger skin surface for heat dissipation. The scrotal reflex, in response to temperature changes, helps maintain

The perineum consists of soft tissues, located superficially to the pelvic diaphragm that contributes to close the inferior opening of the pelvis. The external configuration of the perineum varies according to the position of the person. 1. When standing, the perineum takes the form of a sagittal cleft between the proximal ends of both thighs. Narrow and hidden, this space extends backwards with the gluteal region and forwards with the pubic region. 2. In decubitus (dorsal lithotomy position), the perineum adopts a rhomboidal shape, with an anteroposterior major axis; the anterior vertex is formed by the inferior edge of the pubic symphysis (laterally prolonged by the ischiopubic rami). The posterior vertex is indicated by the apex of coccyx (laterally prolonged by the sacrotuberous ligaments). The ischial tuberosities constitute the lateral vertices. A transverse line, drawn between the ischial tuberosities, divides the perineal surface into two triangular regions: anterior (urogenital perineum) and posterior (anal perineum). While the posterior region is the same in both sexes, the anterior region presents important differences between men and women: in men, it is crossed exclusively by the urethra; in women, it is crossed by the urethra and vagina. The urogenital perineum is a thin layer of striated muscle and fascia extended between both sides of the pubic arch and ischiatic rami. It covers the anterior

32

PART | I Background

FIGURE 2.5 Side view of the interior of a testis.

segment of the inferior pelvic opening. The layered arrangement of the perineum allows the description of a deep perineal pouch and a superficial perineal pouch, which precise limits and contents. The deep perineal pouch is delimited by the perineal membrane. It contains the transversus perinei profundus muscle and external urethral sphincter, the membranous urethra and the bulbourethral glands. In addition, deep perineal vessels and nerves are arranged at this level. At the midline, between the transverse ligament, behind, and the arched pubic ligament, in front, a subpubic orifice is formed which is passed through the deep dorsal vein of penis to enter the pelvic cavity and reach the prostatic venous plexus. The superficial perineal pouch is located between the perineal membrane and perineal fascia. The contents of the superficial perineal space include the root of the penis, the proximal part of the spongy urethra, and the superficial perineal muscles. At this level the superficial perineal vessels and nerves are arranged.

Physiology of the male reproductive system Endocrine regulation of the reproductive tract The fine regulation of the male reproductive system is largely attributed to endocrine factors, which are responsible for the onset of puberty, induction of spermatogenesis, and maintenance of secondary sexual characteristics throughout adulthood. The integrity of

the hypothalamic
pituitary
gonadal (HPG) axis is of paramount importance for both spermatogenesis and hormone production by the testis (Fig. 2.6). Disturbances occurring at every level can lead to changes in both fertility and general wellness, occurring either suddenly or over months, or even years. Gonadotropin-releasing hormone (GnRH) is a peptide hormone released by a small population of about 1500 GnRH-secreting neurons located in the hypothalamus [19]. GnRH binds to its receptor on pituitary gonadotropic cells [20], inducing the release of luteinizing hormone (LH) and follicle-stimulating hormone (FSH). The response of the pituitary to GnRH is influenced by the rhythm of GnRH secretion: low pulse frequency stimulates FSH secretion, whereas high frequency induces LH release. The reason and mechanisms for pulsatile GnRH release are still only partly understood. Episodic release of neuropeptides is an intrinsic property of GnRHsecreting neurons, and an autocrine interaction at the hypothalamic level has been suggested [21]. GnRH acts as the central regulator for the onset of puberty. Initial GnRH activation is likely triggered by kisspeptin release by neurons in the hypothalamic arcuate nucleus [22]: neurokinin B and dynorphin are also involved in the regulation of GnRH release, with stimulating and inhibiting roles, respectively [23]. Additionally, several other players are supposedly involved in GnRH regulation, such as γ-aminobutyric acid and neuropeptide Y, as well as environmental and epigenetic factors; their number is constantly increasing as new “gate-keepers” are discovered [23]. GnRH release peaks every 90 minutes at

Male reproductive system Chapter | 2

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secreted by the gonads inhibit the secretion of gonadotropins and GnRH. Testosterone inhibits LH secretion, whereas FSH release is controlled by a different peptide, inhibin B, secreted by Sertoli cells following FSH stimulus. Inhibin B levels increase during puberty, closely mirroring FSH [25]. In these regards, Inhibin B levels also provide useful insight concerning testicular maturity. Several other hormones, such as INSL-3 (insulin-like 3) and AMH (anti-Mu¨llerian hormone), can provide additional information concerning the different stages of testicular development, as well as providing a rationale for selected conditions, such as testicular maldescent [26]. Generally speaking, any disturbances in either testicular or pituitary or hypothalamic function have the potential to impair spermatogenesis and/or endocrine function of the testis.

Spermatogenesis and semen

FIGURE 2.6 The hypothalamic
pituitary
gonadal axis.

the onset of puberty, and stimulates the release of FSH and LH from gonadotropic cells by binding to its receptor and activating a protein kinase C-mediated pathway. FSH and LH are glycoproteins with two subunits, namely, the α and β subunit. While the α subunit is identical for both gonadotropins, the β subunit is different and determines the binding to the receptor. In human testis, the LH receptor (LHR) is mainly expressed on interstitial Leydig cells, whereas the FSH receptor (FSHR) is expressed on the Sertoli cells. The two cell populations have different roles in the regulation of the reproductive system, as Leydig cells secrete testosterone and Sertoli cells provide support for spermatogenesis. More in detail, the binding of LH to its receptor increases activity of the desmolase enzyme in Leydig cells, therefore inducing testosterone production. A fine interplay exists between Sertoli and Leydig cells, as proven by the presence of androgen receptors on Sertoli cells; adequate intratesticular androgen levels are required in order to allow successful spermatogenesis [24]. The role of FSH in human spermatogenesis is currently at the center of debate: FSHR is expressed by Sertoli cells only, with no direct action on germ cells, and FSH is seemingly acting as an antiapoptotic factor, rather than a proliferative signal for Sertoli cells [24]. The fine regulation of the HPG axis also involves a negative feedback mechanism, in which hormones

Spermatogenesis is a complex biological mechanism through which the male haploid gametes are produced from diploid precursor cells named spermatogonia in the seminiferous tubules of the testis. In healthy subjects, spermatozoa are continuously produced throughout all adult life; however, spermatogenesis requires a highly organized process which depends on the endocrine milieu of the testis, as well as on the integrity of the Sertoli cell population. When approaching puberty, Sertoli cells become progressively more sensible to intratesticular androgens, develop tight junctions and shift their pattern of protein expression [26]. Testosterone production is under strict control of the pituitary through LH levels (and, consequently, through hypothalamic GnRH secretion), and given the support role for FSH on the Sertoli cells, it is clear that the whole process is finely tuned by the HPG axis. Spermatogenesis is a long biological process which requires between 64 and 74 days, which are commonly distinguished in three phases: 1. Spermatocytogenesis, involving both mitosis (in utero) and meiosis (following the onset of puberty). Diploid Type Ad (“A dark”) spermatogonia maintain the stem cell supply for subsequent phases of spermatogenesis: by mitosis, each Ad divides into another Ad spermatogonium and into one Ap (“A pale”) spermatogonium. Ap spermatogonia undergo further differentiation into Type B spermatogonia, which then undergo mitosis to produce primary spermatocytes. Primary spermatocytes are the last diploid cells, as the next step in differentiation is the first meiosis (Meiosis I) which divides each diploid primary spermatocyte in two haploid secondary spermatocytes. Up to this point, spermatocytogenesis is a long and complex phase of

34

PART | I Background

differentiation, lasting almost 40 days and involving migration from the basal to the adluminal compartment. The last part of spermatocytogenesis, on the contrary, is a relatively short phase, lasting just a few hours: in this phase, Meiosis II occurs and each secondary spermatocyte is divided in two (haploid) spermatids. 2. Spermiogenesis is the subsequent phase of differentiation. If intratesticular concentrations of testosterone are adequate, spermatids undergo several changes, including the formation of the tail and of the acrosome, which are all required in order to create fully functional spermatozoa. During spermiogenesis, DNA is tightly condensed. 3. Spermiation is the last phase of spermatogenesis, taking place at the apical part of the seminiferous tubule, and shortly defined as the process of sperm release [27]. Through a series of subsequent steps, spermatids become “mature” spermatozoa and are finally released from the Sertoli cells; however, these spermatozoa lack motility, and are therefore unable to penetrate the oocyte. Another classification refers to the three stages of spermatogenesis as spermatogoniogenesis (spermocytogenesis), meiosis, and spermiogenesis (which includes spermiation as its final stage). Following the end of spermatogenesis, spermatozoa are carried via the testicular fluid through the seminiferous tubules to the epididymis, where they finally acquire motility. This phenomenon is achieved through tubular peristalsis, peripheral muscle contraction, and the rhythmic movement of cilia of ductal cells. When their journey through the epididymis is over, about 12 days later, spermatozoa are stored in the last portion of the epididymis (the cauda epididymis). The epididymal fluid provides all necessary support for mature spermatozoa, including substances (such as L-carnitine, sialic acid and inositol) which are often used in the nonhormonal treatment of male infertility. During ejaculation, spermatozoa are expelled through the urethra together with fluids produced by the prostate, the seminal vesicles, and the bulbourethral glands (commonly described as “accessory reproductive glands”), which make up 99% of the volume of the ejaculate. The resulting mixture is called semen; fluids from accessory glands provide nourishment as well as protection to spermatozoa, but several enzymes, such as prostate-specific antigen, are also involved in the physiology of sperm function.

Erectile and ejaculatory functions Sexual functions include both erection and ejaculation; barring assisted reproduction techniques, both erection and ejaculation are mandatory for male fertility.

Erection is defined as the physiologic mechanisms through which the penis becomes rigid: this phenomenon occurs following parasympathetic stimuli, which stimulate release of acetylcholine from nerve branches and, in turn, nitric oxide (NO) from the endothelial lining of penile trabecular arteries. Under the effects of NO, arteries in the corpora cavernosa dilate and become engorged with blood; veins in the penis become compressed, and as a result venous leak is diminished, further promoting erection. When the sexual stimuli are discontinued, or at the end of intercourse, erection physiologically ends. Several factors influence the firmness and rigidity of the erect penis, and many conditions can reduce the ability to achieve and/or maintain an erection which is adequate for intercourse. Testosterone, the main circulating androgen, is involved in both sexual desire and erectile function [28,29], and is therefore a key regulating element in sexual function. Several other endocrine factors, such as thyroid hormones and prolactin, are involved in the cycle of sexual response. Several endocrinopathies can therefore lead to sexual dysfunctions, as commonly observed in cases of severe hypothyroidism or hyperprolactinemia [30]. Vascular factors are involved as well. When endothelial function is impaired, or following administration of vasodilating drugs, erection is often compromised. It is therefore unsurprising that erectile function has an almost linear relationship with ageing. Data from large population studies, starting from the Massachusetts’ Male Aging Study [31], have confirmed that erectile function is progressively impaired with aging. Besides all organic factors, it’s worth remembering that erectile function is also strictly associated with psychological well-being, and in younger subjects with no apparent causes, psychological assessment can often suggest the underlying cause of sexual dysfunction. After an adequate level of stimulation has been obtained during sexual activity, sperm is moved from the vas deferens into the urethra, where it becomes mixed with secretion from accessory glands in a phase called emission. Shortly after, the rhythmic contractions of the urethra propel semen forward: this is called ejaculation and is generally accompanied by an extremely pleasurable sensation (orgasm). During erection, the corpora cavernosa compress the tunica albuginea; however, the penile urethra does not become fully compressed, and as such sperm can transit through it during ejaculation. During ejaculation, several pulses (10
15) of semen flow through the urethra. Ejaculation is a completely involuntary process once the first pulse has started, and as such there is no way to stop subsequent pulses. Following the end of ejaculation, most men undergo quick penile detumescence and can’t achieve another erection for some time; this is called the refractory period.

Male reproductive system Chapter | 2

Semen analysis: a short primer For men having fertility issues, there is undoubtedly no other exam providing as much information as semen analysis. This exam should be performed following recommendations from the World Health Organization (WHO) [32], which guide all phases of semen collection and analysis. Samples should always be collected by masturbation in a sterile container following an adequate period of abstinence; external factors likely to influence semen analysis, such as fever or administration of antibiotics, should be considered before starting semen analysis. In short, semen analysis provides information on two “major quantifiable attributes”—semen volume and sperm concentration—although three other parameters, namely sperm vitality, motility, and morphology, also provide useful clinical insight. Since the introduction of the 2010 manual, a “normal” sample is defined based on statistics, using the 5th centile of the reference ranges as the lower reference limit; these reference ranges have been calculated based on 400
1900 semen samples collected from recent fathers. According to reference values reported in the 6th Edition of the Manual, published in July 2021, a “normal” sample should have at least: 1.4 mL volume, 39 million sperm per ejaculate, 16 million sperm per mL, 42% total motility, 30% progressive motility, 54% vitality and 4% normal forms. While conventional semen analysis does not routinely assess DNA integrity or chromatin organization, it seems likely that these parameters could somehow be useful in identifying “at-risk” subjects. Semen analysis is the first step for infertility evaluation and should be performed in all couples seeking medical help. However, if the patient presented one altered sample, it should be repeated; WHO guidelines suggest that at least two semen analyses are needed for adequate diagnosis. Moreover, physical examination, anamnesis, and several other complementary exams could be demanded for male fertility investigation.

References [1] Federative Committee of Anatomical Terminology (FCAT). Sociedad anato´mica espan˜ola. terminologı´a anato´mica. 1a ed. Madrid: Editorial Me´dica Panamericana; 2001. [2] Dauber WF. Nomenclatura anato´mica ilustrada. 5a ed. Barcelona: Masson; 2006. [3] Drake RL, Vogl AW, Mitchell AWL. Gray’s anatomy for students. Fourth ed. New York: Elsevier Churchill-Livingstone; 2020. [4] Moore KL, Dalley AF. Anatomı´a con orientacio´n clı´nica. 7a ed. Barcelona: Wolters Kluver; 2013. [5] Ross M, Romrell L, Kaye G. Histologia. 3a ed. Interamericana; 1998.

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[6] Gartner LP. Color textbook of histology. Philadelphia, PA: W. B. Saunders Company; 1997. [7] Orts-Llorca F. Anatomı´a humana. 4a ed. Barcelona: Editorial Cientı´fico-Me´dica; 1972. [8] Standring S. Anatomy. The anatomical basis of clinical practice. 41st ed. New York: Elsevier; 2016. [9] Jime´nez-Reina L, Johann Maartens P, Jimena-Medina I, Agarwal A, du Plessis S. Overview of the male reproductive system. In: Vaamonde Diana, du Plessis Stefan S, Agarwal Ashok, editors. Exercise and human induced fertility disorders and possible therapies. New York: Springer; 2016, p. 1
11. [10] Testut J, Latarjet A. Anatomı´a humana. 9a ed. Barcelona: Salvat % Editores; 1982. [11] Paulsen F, Waschke JS. Atlas de anatomı´a humana. 23a ed. Barcelona: Elsevier-Espan˜a; 2012. [12] Anastasi G, Gaudio E, Tachetti C. Anatomı´a humana. Atlas. 1a ed. Madrid: Ermes-Ergon, Mila´n; 2018. [13] Ross M, Pawlina W. Histology: interactive atlas to accompany histology: a text and atlas; with correlated cell and molecular biology. Baltimore: Lippincott, Williams & Wilkins; 2007. [14] Schunke M, Schultze E, Schumacher U. Prometheus. texto y atlas de anatomı´a. 3a ed. Buenos Aires, Madrid: Editorial Me´dica Panamericana; 2014. [15] Rohen JW, Lu¨tjen-Drecoll E, Yokochi EC. Atlas de anatomı´a humana. Estudio fotogra´fico del cuerpo humano. 7a ed. Barcelona: Elsevier; 2007. [16] Sternberg SS. Histology for pathologists. Raven Press; 1992. [17] Netter FH. Atlas de anatomı´a humana. 7a ed. Barcelona: ElsevierMasson; 2007. [18] Platzer W. Atlas de anatomı´a con correlacio´n clı´nica. 9a ed. Madrid: Editorial Me´dica Panamericana; 2008. [19] Wray S. Development of luteinizing hormone releasing hormone neurones. J Neuroendocrinol 2001;13(1):3
11. [20] Stamatiades GA, Kaiser UB. Gonadotropin regulation by pulsatile GnRH: Signaling and gene expression. Mol Cell Endocrinol 2018;463:131
41. [21] Krsmanovic LZ, Hu L, Leung PK, Feng H, Catt KJ. The hypothalamic GnRH pulse generator: multiple regulatory mechanisms. Trends Endocrinol Metab 2009;20(8):402
8. [22] Uenoyama Y, Inoue N, Nakamura S, Tsukamura H. Central mechanism controlling pubertal onset in mammals: a triggering role of kisspeptin. Front Endocrinol 2019;10. [23] Leka-Emiri S, Chrousos GP, Kanaka-Gantenbein C. The mystery of puberty initiation: genetics and epigenetics of idiopathic central precocious puberty (ICPP). J Endocrinol Investig 2017;40(8): 789
802. [24] Huhtaniemi I. A short evolutionary history of FSH-stimulated spermatogenesis. Hormones 2015. [25] Edelsztein NY, Grinspon RP, Schteingart HF, Rey RA. Anti-Mu¨ llerian hormone as a marker of steroid and gonadotropin action in the testis of children and adolescents with disorders of the gonadal axis. Int J Pediatric Endocrinol 2016;2016(1). [26] Sansone A, Kliesch S, Isidori AM, Schlatt S. AMH and INSL3 in testicular and extragonadal pathophysiology: what do we know? Andrology 2019;7(2):131
8.

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PART | I Background

[27] O’Donnell L, Nicholls PK, O’Bryan MK, McLachlan RI, Stanton PG. Spermiation: the process of sperm release. Spermatogenesis. 2011;1(1):14
35. [28] Corona G, Maggi M. The role of testosterone in erectile dysfunction. Nat Rev Urol 2009;7(1):46
56. [29] Isidori AM, Buvat J, Corona G, Goldstein I, Jannini EA, Lenzi A, et al. A critical analysis of the role of testosterone in erectile function: from pathophysiology to treatment—a systematic review. Eur Urol 2014;65(1):99
112.

[30] Sansone A, Romanelli F, Gianfrilli D, Lenzi A. Endocrine evaluation of erectile dysfunction. Endocrine 2014;46(3):423
30. [31] Feldman HA, Goldstein I, Hatzichristou DG, Krane RJ, Mckinlay JB. Impotence and its medical and psychosocial correlates: results of the Massachusetts male aging study. J Urol 1994;151(1): 54
61. [32] World Health Organization. WHO laboratory manual for the examination and processing of human semen. 6th ed. Geneva: World Health Organization; 2021.

Chapter 3

Female reproductive system Joao Sabino Cunha Filho1, R. James Swanson2, Bo Liu2 and Sergio Oehninger3 1

Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil, 2Anatomical Sciences, Liberty University College of Osteopathic Medicine,

Lynchburg, VA, United States, 3Department of Obstetrics and Gynaecology, Reproductive Biology Unit, University of Stellenbosch, Stellenbosch, South Africa

Female anatomy and introduction to physiology for the female reproductive system Located in the pelvic cavity, inferior to the abdominal cavity, the female reproductive system consists of the glandular part, the ovary, where the oocytes (eggs) are formed and steroid hormones synthesized, and an extended ductal system. These ducts are the uterine or fallopian tubes (also called oviducts), uterus, and vagina, pictured in Figs. 3.1
3.4. As shown in these figures, the uterus and vagina are single midline structures while the fallopian tubes and ovaries are bilateral. These tubes serve as a conduit, providing a channel for ejaculated sperm to swim up into the peritoneal cavity (rectouterine space) and to bring either a fertilized or unfertilized egg down to the uterus and eventually out of the body at parturition (a baby) or menses (shedding of the endometrial lining of

the uterus). The uterine tube is a simple duct that collects the oocyte from the ovarian surface at ovulation and transports it to the uterus. If the egg is not fertilized, the uterus allows it to pass out with the menstrual flow. If the egg is fertilized, the uterus is responsible for housing and nurturing the fertilized egg during its development and, once it is mature, expelling the fetus through the vagina and outside the female body. The vagina is the passageway of movement for the fetus and placenta during labor, but is also an organ of copulation, which receives the penis and semen during intercourse. The inferior vagina ends in the vulva, which is formed by bodily tissues, some of which are dermal (skin) and some of which are erectile, finally opening external to the body. Many surrounding tissues (an adnexal system) are adjoined to the ovary, ducts, and vulva. This adnexa consists of smooth muscle surrounding the tubes, various glands at the lower end of the vagina and urethra, and skeletal muscle, FIGURE 3.1 Superior view into a plastinated female cadaver’s pelvis showing the posteriorly situated rectum, the anteriorly situated urinary bladder, with the uterus and its associated structures in between (LUCOM model). Copyright by R James Swanson. Used with permission.

Fertility, Pregnancy, and Wellness. DOI: https://doi.org/10.1016/B978-0-12-818309-0.00007-1 © 2022 Elsevier Inc. All rights reserved.

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38

PART | I Background

FIGURE 3.2 Superior view into a traditionally embalmed female cadaver’s pelvis showing the posteriorly situated rectum, the anteriorly situated urinary bladder, and the uterus and its associated structures, the fallopian tubes and ovaries, in between (Swanson dissection). Copyright by R James Swanson. Used with permission.

oocytes. These hormones will be discussed further in the fertilization section. The pubertal ovary is oval-shaped, creamy pink, and measures 2
3 3 1.5
2, 3 1
1.5 cm. After puberty, repeated ovulations make the ovarian surface rough and irregular. An ovarian artery branches from the abdominal aorta and connects with a uterine artery and a lower vaginal artery on either side of the body. Thus, the ovaries are actually supplied from three different vessels that join from above and below the ovary. The innervation of the ovary comes from autonomic (not conscious level of the brain) nerves, called the ovarian plexus. This autonomic innervation not only supplies the ovaries, but also the uterine tubes, and broad ligaments of the uterus. In addition, the ovary receives one or two branches from the pelvic hypogastric plexus. These nerves function primarily in control of blood flow to these organs. As mentioned above, pain can sometimes be perceived from the swelling follicle which can reach over five mL in volume. FIGURE 3.3 Plastinated pro-section of an isolated female cadaver’s internal reproductive tract with structures from an anterior view with the urinary bladder and rectum in place on either side, anterior and posterior, of the uterus to which are attached the bilateral fallopian tubes and ovaries (LUCOM model). Copyright by R James Swanson. Used with permission.

membranes, and fascia comprising the perineum, a fancy name for the bottom of the pelvic cavity which is the thin area between the thighs.

Internal organs Ovary The ovaries contain the oocytes (eggs) and produce the two major sex hormones from cells that surround the

Female reproductive tract Fallopian tube The uterine tube is a muscular structure that reaches from the ovary to the uterus where it opens into the uterine cavity through the muscular and endometrial layers. The oviduct’s lateral or distal end opens directly into the peritoneal cavity near the ovary. The oviduct therefore has a direct communication from the peritoneal (abdominal) cavity to the exterior of the body, a remarkable anatomical feature. The fallopian tube measures approximately 10 cm in length and 0.5
1 cm in diameter along its length. The ovulated ovum may be fertilized at the ovarian surface as it leaves the site of ovulation if sperm are in the rectouterine recess within the peritoneum. If sperm are not

Female reproductive system Chapter | 3

39

FIGURE 3.4 An externalized rabbit ovary with a glass microinjection needle in the mature follicle. The follicle, containing one ovum, looks like a blister on the ovary’s surface. The ovary is surrounded by fat with the reddish fallopian tube embedded to the right of the fat (Swanson research). Copyright by R James Swanson. Used with permission.

present in the abdominal cavity, they can fertilize the ovum in the fimbriated end of the fallopian tube, called the infundibulum. The fallopian tube subsequently transfers the zygote (fertilized ovum) to the uterine cavity by ciliary and peristaltic action as estradiol increases. The uterine tube has four parts beginning with the most medial part in the uterine wall, the intrauterine part, then isthmus, ampulla, and ending in the open infundibulum, mentioned above. After ovulation, the fimbriae, at the ovarian end of the oviduct, draw the oocyte, or zygote if fertilized, in with ciliary action and brings it into the uterus over 31/2 days of transport. The inner surface of the uterine tube has numerous, intricate, longitudinal mucous-lined folds that give it a labyrinthine feature. Uterus ’In the prepubertal and nonpregnant woman, the uterus is a hollow, muscular, thick-walled, inverted pear-shaped organ that, in the immature girl, is about one third the size of the mature woman. This muscular organ is where the fertilized oocyte develops. The nonpregnant uterus measures about 6.5
7.5 cm 3 4.5
5.5 3 2.5
3 cm. During pregnancy, the uterus enlarges to accommodate the initial embryo and eventual fetus. The term embryo applies to the developing baby until all of the organ systems are in place. The term fetus applies to the time when all of the organ systems grow to maturity to be ready for parturition (birth). The uterus lies between the urinary bladder, anteriorly, and the rectum, posteriorly. The uterine body is made up of three parts: the fundus, which is the part that is superior to the fallopian tubes, the body, which is the main part, and the cervix, which is the narrowing neck that opens into the vagina. The uterine body and fundus form the superior two-thirds of the uterus. The uterine blood supply comes

directly from a branch of the internal iliac artery and indirectly from the ovarian artery above and the vaginal artery below. Vagina The vagina is the lowest portion of the internal female reproductive tract, receiving the penis during intercourse. The vagina is also the route for elimination of uterine secretions throughout the female cycle and passage of the fetus and annexes (non-fetal birthing products) during parturition. The vagina is a fibromuscular tube 7
10 cm long by 2.5
3 cm in diameter. The inner surface is rough (vaginal rugae) with superior-to-inferior longitudinal folds called rough anterior and posterior columns. The anterior columns contain horizontal (medial to lateral) folds along the inferior 2 cm length. These horizontal folds respond to sexual stimulation and, along with clitoral stimulation, produce the female orgasm. The vagina stretches from the uterine cervix to the vestibule where it opens between the labia minora and majora. The entire vagina is inferior to the uterus, posterior to the urinary bladder and urethra, and anterior to the rectum. The vagina passes between the medial borders of the levator ani muscle (the muscular diaphragm forming the floor of the pelvic cavity) to pierce the urogenital diaphragm in conjunction with the urethra. Both tubular structures are surrounded by sphincters that are derived from the levator ani muscle.

External organs Perineum The perineum is formed by the soft tissues inferior to the pelvic diaphragm (levator ani muscles) ending with the skin. The external configuration of the perineum varies

40

PART | I Background

from individual to individual depending somewhat on their position: When standing, the perineum takes the form of a sagittal cleft between the proximal ends of both thighs. Narrow and hidden, this space broadens anteriorly to end at the pubic symphysis in front and broadens posteriorly to end at the coccyx. In dorsal decubitus (gynecologic or lithotomy) position, with the thighs spread, the perineum forms a rhomboid- or diamond-shaped area with an anteroposterior axis. The anterior apex is the inferior symphysis pubis; the posterior apex is the coccyx; the lateral apices are the bony ischial tuberosities. The diamond-shaped perineum has the urogenital diaphragm (the triangular layer from the symphysis pubis in the front and then left and right ischial tuberosities, the two bony lumps on which we sit) as its roof and the skin as its floor. In common language, the perineum would be a person’s bottom. This area contains a significant amount of adipose tissue with nerves, vessels, erectile tissue, and a few very small muscles within it. From posterior to anterior, the three openings of anus, vagina, and urethra also pierce the perineum. For a more detailed description of the perineum, several biological and medical textbooks are available through any library or online.

Vulva The vulva contains the external female genitalia and the urethral os or opening. The vulva’s skin is ovoid shaped with an anteroposterior orientation. Anteriorly, the vulva extends from the wall of the abdomen at the symphysis pubis, posteriorly to the anus. Laterally, the vulva joins the medial sides of the thighs at the genitofemoral folds. When the thighs are abducted (opened laterally), the cleft opens exposing the mons pubis in the front, the labia majora and minora behind the mons pubis, and the vestibule between the two labia minora. The bulbs, with their glands of vestibule and the clitoris, which are also part of the vulva, are located deep to the labial formations between the urethral and vaginal openings. The bulbourethral glands secrete mucus to moisten the labia minora and the vestibule. The bulbs of vestibule and the clitoris are erectile tissue located deep to the labial formations that surround the vestibule. The paired bulbs of the vestibule are 3
3.5 cm long and 1.2
1.5 cm wide located on the side of the urethral and vaginal orifices, surrounded by the bulbospongiosus muscles. They are homologs to the singular bulb of the corpus spongiosum of the penis, but unlike the single structure in male anatomy, the bulbs of vestibule in the female are separated by the vestibule of the vagina. Their anterior ends are joined at the urethral orifice to form a commissure and they attach to the body of the clitoris. The clitoris is 0.5
0.7 cm long and 0.6
0.8 cm wide located deep to the prepuce (analogous to the foreskin of the

penis) and anterior labial commissure. When flaccid, the clitoris is hidden by the labia majora. The clitoris divides superolaterally into a pair of 3
3.5 cm long crura, tail-like structures. Each crus of the clitoris is anchored to the ischiopubic ramus and covered by a thin ischiopubic muscle. The glans clitoris is the counterpart of the glans penis but does not contain the urethra, so it has no corpus spongiosum. The clitoris plays a fundamental role in female sexual arousal and enters erection through tactile stimulation, although it does not normally lengthen or swell significantly.

Detailed physiological aspects of the hypothalamic
pituitary
ovarian
endometrial axis Introduction The female reproductive system has a very complex relationship between a number of body tissues that work together in a tightly controlled manner referred to as an axis. This axis requires neuronal tissue from an area deep in the unconscious area of the brain called the hypothalamus. The hypothalamus is connected by a 3
4 mm long stalk to a pea-sized endocrine gland called the hypophysis or pituitary gland. The pituitary gland releases hormones that have effects mainly on the ovaries. Finally, the ovaries produce several steroid hormones that direct the development and function of the reproductive tissues and also provide negative feedback on the hypothalamic and hypophyseal hormones. Thus the complexity that we see in the male system in Chapter 2, Male Reproductive System, and what we will see next in Chapter 4, Conception and Pregnancy, on conception and pregnancy, is also abundantly evident in studying the female reproductive system (Fig 3.5).

Hypothalamus and pituitary gland The hypothalamus is the central processing unit of the female and male reproductive systems. It is located at the base of the brain above the pituitary gland. Hypothalamic neurons secrete hormones that reach the anterior pituitary to regulate the function of various cell types, via the portal vessels system of communication (non-neuronal pathway). These specialized neural networks act as a neuroendocrine system, where neurons have acquired a quasiendocrine function, releasing hormones that reach the anterior hypophysis via vascular channels. This portal system originates as a capillary network surrounding the hypothalamic nuclei and then forms long portal vessels that run down the pituitary stalk to terminate in capillaries in the anterior lobe of the hypophysis. Menstrual cyclicity and timely ovulation depend upon the integrity of three processes: the activity of a hypothalamic gonadotropin releasing hormone (GnRH) pulse generator, the pituitary secretion of gonadotropins (FSH and

Female reproductive system Chapter | 3

Hypothalamus Hypothalamus GnRH secreon

Pituitary gonadotrophs FSH and LH secreon

Lepn

Ovarian follicle

Insulin Ghrelin

Theca cells

Granulosa cells

Androgens Energy balance (adiposity)

Estradiol

41

FIGURE 3.5 Organized operation of the hypothalamic
pituitaryovarian-endometrial axis. Hypothalamic neurons secrete GnRH that travels though the portal vessels to the gonadotrophs in the anterior pituitary to regulate episodic release of FSH and LH. LH controls theca cell functions including androgen synthesis; androgens diffuse to the granulosa cell compartment, where under FSH influence are aromatized to estradiol. Estradiol exerts a potent negative feedback at hypothalamic and pituitary levels to decrease FSH secretion; by mid-cycle, there is a switch to a positive feedback resulting in the LH surge. The LH surge stimulates resumption of egg meiosis, ovulation, and luteinization, with formation of the corpus luteum. Progesterone is secreted in large amounts by the corpus luteum, and upon estrogen priming induces endometrial decidualization needed for establishment of a window of receptivity to the blastocyst resulting in implantation. Hypothalamic kisspeptin neurons are also regulated by changes in energy balance via circulating metabolic hormones such as leptin, insulin and ghrelin. Kisspeptin neurons may also regulate energy expenditure and adipose tissue levels although the mechanisms underlying these observations are unknown.

Corpus Luteum Progesterone

Other systemic cues

Endometrium Window of implantaon

LH), and the gonadal feedback mechanisms of control of the hypothalamic
pituitary activities, first in a negative fashion and then in a positive way that result in the preovulatory LH surge, ovulation, and corpus luteum formation.

GnRH neuronal system The GnRH neurosecretory system is mainly located in the hypothalamus with other few neurons present in neighboring brain areas. GnRH-immunoreactive cell bodies are more prevalent in the preoptic area, the periventricular (third ventricle) zone from the anterior hypothalamus to the premammillary nucleus, encompassing the median eminence and arcuate nucleus (ARC), and other areas such as the supraoptic nucleus, other septal nuclei, the nervus terminalis, and the amygdala. Moreover, GnRH-immunoreactive axons innervate the portal vessels in the median eminence, the organum vasculosum of the lamina terminalis, the medial mammillary nucleus, and the amygdala [1]. Although diffuse, the GnRH neuronal system is composed of a relatively low number of neurons, ranging from 1,200 to 1,600 in the rat and approximately 2000 in the monkey. In mice and humans, it has been established that GnRH neurons differentiate from the olfactory

placode and migrate through the nasal septum to the forebrain. The clinical relevance of this is seen in Kallmann’s syndrome in which failure of this migratory system is associated with anosmia/hyposmia (absent or reduced olfactory sensitivity) and hypogonadotropism. Multiple factors play a critical role in the guidance of the GnRH neuron migration process. Among them, there are adhesion molecules, such as anosmin (the product of KAL1 gene), polysialic acid form of neural adhesion molecule (PSA-NCAM) and cell surface glycoconjugate [2]. In 1937, a medical student at Cambridge, Geoffrey Harris, was the first one to provide evidence that the pituitary
gonadal axis is under control of the central nervous system [3]. The isolation of a hypothalamic factor that, through the hypophysial portal circulation, regulates the synthesis and secretion of gonadotropins was characterized in a simultaneous fashion by two different groups, of Andrew Schally and Roger Guillemin, from porcine and ovine brain, respectively [4,5]. The active molecule was characterized as a decapeptide, now referred to as GnRH. In the following years, the group of Knobil et al. provided evidence for an episodic manner of luteinizing hormone (LH) secretion, and this was elegantly corroborated in ovariectomized ewes by Clarke and Cummins

42

PART | I Background

[6
8]. In parallel, Gary Hodgen and colleagues provided unequivocal data to support the concepts of follicular recruitment, selection and dominance during the follicular phase of the nonhuman primate cycle, and the regulatory mechanisms leading to timely ovulation and corpus luteum formation [9,10].

The GnRH pulse generator GnRH is the key regulator of the reproductive axis. Its pulsatile secretion determines the pattern of secretion of FSH and LH, which in turn regulate both the endocrine function and gamete maturation of the female gonad. In its unmodulated state the hypothalamic the GnRH pulse generator has a frequency of approximately one discharge per hour, which results in the release of a bolus (pulse) of GnRH to the portal circulation. In simultaneous monitoring recordings of neuronal activity and portal blood secretion, multi-unit neuronal activity of GnRH neurons is synchronized with these secretory pulses. In addition, there is synchronization of the GnRH neuron’s discharge in order to make it a physiologically optimal pulse [6,7]. The pituitary gonadotrophs in turn respond with a pulse of FSH and LH into the peripheral circulation [1]. During the follicular phase, the pulse generator operates in a circhoral fashion, and as the follicle grows and secretes estrogen, this steroid exerts a negative feedback effect on the hypothalamus and pituitary to decrease FSH levels. When in the course of late folliculogenesis estrogen levels exceed a threshold (around 250 pg/mL for 36 hours in the primate) the negative feedback is reversed and a positive feedback results in the release of LH to trigger ovulation. An increase in the frequency of the pulse generator or amplitude are not necessary for the LH surge. Under the influence of LH, the follicle luteinizes and ruptures releasing the egg, and the rapidly developing corpus luteum secretes progesterone. Although progesterone reduces the frequency of the pulse generator the amplitude of the LH pulses increases so that the mean LH levels in the circulation differ little from those found in the follicular phase [1,9]. It is the activity of the GnRH pulse generator that drives the events downstream at the pituitary, ovarian and endometrial levels. A lack of activity of the pulse generator or derangement on the pulse frequency has adverse effects for the reproductive process. In the rhesus monkey it appears that the pulse generator is located within the medio basal hypothalamus, and probably in the ARC. Even with a completely deafferented medio basal hypothalamus, LH pulsatility continued [1]. Using a hypophysiotropic clamp monkey model, Knobil and colleagues showed that changing the mode of GnRH administration from pulsatile to continuous led to refractoriness of the pituitary to GnRH stimulation [7].

After characterization of the GnRH structure (10 amino acid peptide), GnRH analogs were developed. GnRH agonistic analogs cause hypersensitization of pituitary gonadotropin GnRH receptors to first stimulate (internalization of receptor-ligand complexes) and then decrease gonadotropin secretion. On the other hand, GnRH antagonist analogs bind in a competitive fashion to the receptors and have an immediate inhibitory effect on gonadotropin secretion [11]. The development of these molecules, together with newly produced recombinant and highly purified gonadotropins (FSH and LH), are still the basis of controlled ovarian stimulation protocols used in IVF. Additional evidence in support of the view that the GnRH pulse generator is an intrinsic property of the hypothalamus comes from the demonstration of rhythmic pulsatile release of GnRH from a superfused rat, guinea pig, monkey, and human hypothalamic fragments in vitro. Furthermore, studies with the immortalized GnRH neuronal cell line GT-1 have shown that these cells have an endogenous rhythm and release GnRH in pulses [1]. The decapeptide GnRH travels through the portal system to bind to specific receptors on the membrane of the gonadotrophs, the anterior pituitary cells that secrete both FSH and LH. This leads to cellular responses that include stimulation of the biosynthesis, storage, and secretion of FSH and LH. Secondary messengers include calcium- and phospholipid-dependent processes and cAMP. The secretion of gonadotropins is also under the control of inhibin B, a nonsteroidal ovarian inhibitor present in follicular fluid. It is synthetized by the granulosa cells and exerts a suppressive action on FSH secretion from the pituitary gland [1,9].

Kisspeptin neurons Novel concepts have recently emerged related to the regulation of GnRH secretion with the discovery of the kisspeptin
neurokinin
dynorphin neuronal network in the hypothalamus. This mediates both the negative and positive sex steroid feedback control of GnRH secretion, in conjunction with other neuropeptides and neurotransmitters [12,13]. In a general sense, kisspeptin has recently been identified as a key neuroendocrine gatekeeper of reproduction and appears to be essential for the initiation of human puberty and maintenance of adult reproduction. Kisspeptin neurons respond to changes in numerous internal and external factors including nutrient and fat status, stress and sex steroids, thus providing a link between these factors and reproduction. Data suggest that kisspeptin may provide a link between nutritional/metabolic status and reproduction by sensing energy stores and translating this information into the pulsatile secretion of GnRH [14] (Fig. 3.6).

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FIGURE 3.6 Diagram showing the neuroanatomy of the kisspeptin-GnRH pathway and the relationship between KNDy and GnRH neurons in humans and rodents. Kisspeptin signals directly to the GnRH neurons, which express kisspeptin receptor. The location of kisspeptin neuron populations within the hypothalamus is species specific, residing within the anteroventral periventricular nucleus (AVPV) and the arcuate nucleus in rodents, and within the preoptic area (POA) and the infundibular nucleus in humans. Kisspeptin neurons in the infundibular (humans)/arcuate (rodents) nucleus coexpress neurokinin B and dynorphin (KNDy neurons), which via neurokinin B receptor and kappa opioid peptide receptor autosynaptically regulate pulsatile kisspeptin secretion, with neurokinin B being stimulatory and dynorphin inhibitory. Negative (red) and positive (green) sex steroid feedback is mediated via distinct kisspeptin populations in rodents, via the AVPV and the arcuate nucleus, respectively. In humans KNDy neurons in the infundibular nucleus relay both negative (red) and positive (green) feedback. The role of the POA kisspeptin population in mediating sex steroid feedback in humans is incompletely explored. ME, median eminence; 1 , stimulatory; 2 , inhibitory; ERα, estrogen receptor alpha; PR, progesterone receptor; Kiss1/KiSS1, kisspeptin; NKB, neurokinin B; Dyn, dynorphin. With permission from [12] Skorupskaite K, George JT, Anderson RA. The kisspeptinGnRH pathway in human reproductive health and disease. Hum Reprod Update. 2014 Jul-Aug;20(4):485
500. Fig. 1 With permission.

While the pivotal central role played by GnRH remains undisputed, several functional limitations of the GnRH neuronal network have been identified. For example, in rats, GnRH neurons lack estrogen receptor (ER)alpha [15], the principal ER, suggesting the need for an intermediary signaling pathway mediating gonadal feedback. The related discovery of a reproductive role for neurokinin B has stimulated further interest in the field. The same functional neuronal network secretes kisspeptin and neurokinin B; now called kisspeptin-neurokinin Bdynorphin (KNDy) neurons as they also cosecrete dynorphin, a well-established opioid inhibitor [16]. The neuropeptide kisspeptin is produced by two major populations of neurons located in the hypothalamus, the rostral periventricular region of the third ventricle (RP3V) and ARC. These neurons project to and activate GnRH

neurons acting via the kisspeptin receptor (Kiss1R) in the hypothalamus and stimulate the secretion of GnRH. Gonadal sex steroids stimulate kisspeptin neurons in the RP3V, but inhibit kisspeptin neurons in the ARC, which is the underlying mechanism for positive and negative feedback, respectively, and it is now commonly accepted that the ARC kisspeptin neurons act as the GnRH pulse generator [12,13,17]. It is now accepted that the negative feedback regulation by gonadal sex steroids enables the ARC kisspeptin neurons to act as the long sought-after GnRH pulse generator. Previous immunohistochemical data detailed the neural machinery, involving the coexpression of the neuropeptides neurokinin B (NKB) and dynorphin (Dyn) within ARC kisspeptin neurons and autosynaptic control of these “KNDy” neurons [16]. The model stipulates that

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PART | I Background

NKB acts on KNDy neurons to drive pulsatility and Dyn acts as the “brake” halting pulses and kisspeptin is the final output to GnRH neurons. Communication within and between KNDy neurons exists because they express NKB and dynorphin receptors in addition to an ARC network of reciprocal projections and possible cell-to-cell gap junction synchronization [13,17]. Similar data showed electrophysiological multiple-unit activity recordings directed at KNDy neurons depicting “volleys” of activity consistent with GnRH pulse generator activity and appropriate responses to kisspeptin and NKB receptor agonists. Notwithstanding current limitations, new data show optogenetic control of ARC kisspeptin neurons and the generation of GnRH/LH pulses providing the strongest evidence for ARC kisspeptin neurons as the hypothalamic GnRH pulse generator [17]. It appears that in humans KNDy neurons mediate negative sex steroid feedback in the infundibular nucleus by suppressing the secretion of kisspeptin and neurokinin B and stimulating the secretion of dynorphin, which act synergistically to reduce the activity of the GnRH neuronal system, and thus gonadotrophin secretion. Estrogen feedback switches from negative to positive in the late follicular phase to induce the GnRH/LH surge at the time of ovulation. However, the neuroendocrine mechanisms involved in this critical physiological event in humans are still unclear. Emerging data suggest that although the negative feedback of sex steroids is mediated by KNDy neurons in the infundibular/arcuate nucleus, the positive feedback of sex steroids is more site and species specific. There are no studies looking at the anatomical region of kisspeptin expression that mediates estrogen positive feedback in humans and evidence comes from other species, which, like humans, have no homologous area to the AVPV nucleus [12,13,18].

The ovarian cycle Development of ovarian follicles: gonadotropinindependent growth phase During embryogenesis, the germ cells go through migration and homing into the developing gonad. The primordial germ cells soon undergo intraovarian growth and differentiation and establish contact and interrelationships with somatic cells. The ovarian follicle, consisting of an oocyte (germ cell) surrounded first by granulosa and then theca cells (somatic cells) represents the basic functional unit of the ovary. Folliculogenesis can be classified into three phases according to their developmental stage and gonadotropin dependence: (1) follicular growth through primordial, primary, and secondary stages (gonadotropinindependent phases); (2) transition from preantral to early antral stage (gonadotropin-responsive phase). The growth

of these follicles is controlled primarily by intraovarian mechanisms and, although unaffected by the absence of gonadotropins, is stimulated by the presence of FSH. And (3) final growth beyond the early antral stage to antral and Graafian stages (gonadotropin-dependent phase), which includes during any given cycle follicle recruitment, selection, and ovulation. In mammals, a single or small number of germ cell(s) will ovulate during an ovarian cycle, whereas most follicles undergo atresia by follicle cell apoptosis, a selection process that ensures the release of only the healthiest and most viable oocytes [19]. A plethora of growth factors, many belonging to the transforming growth factor-beta (TGF-beta) superfamily, are expressed by ovarian somatic cells and oocytes in a developmental, stage-related manner and function as intraovarian regulators of folliculogenesis [20]. Two such factors, bone morphogenetic proteins, BMP-4 and BMP7, are expressed by ovarian stromal cells and/or theca cells and have recently been implicated as positive regulators of the primordial-to-primary follicle transition. In contrast, evidence indicates a negative role for antiMullerian hormone (AMH) in this key event and subsequent progression to the antral stage. AMH, also known as Mullerian-inhibiting substance, is of pre-granulosa/ granulosa cell origin. Two other TGF-beta superfamily members, growth and differentiation factor-9 (GDF-9) and BMP-15 (also known as GDF-9B), are expressed in an oocyte-specific manner from a very early stage and play key roles in promoting follicle growth beyond the primary stage. AMH is a dimeric glycoprotein and a member of the transforming growth factor β (TGF-β) family of growth and differentiation factors [20,21]. AMH is specifically expressed in granulosa cells of small growing follicles. In rodents, expression is initiated as soon as primordial follicles are recruited to grow, and highest expression is observed in preantral and small antral follicles. AMH is no longer expressed by mural granulosa cells during the FSH-dependent stages of follicular growth, nor is it expressed in atretic follicles [22]. In the human ovary AMH shows a very similar expression pattern [23,24]. Several lines of evidence suggest that AMH acts as gatekeeper of follicular estrogen production. AMH may act as a follicular gatekeeper and ensure that each small antral follicle produces little estradiol prior to selection (i.e., up to a follicular diameter of B8 mm) allowing a direct ovarian/pituitary dialog, regulating the development of the selected follicle that will undergo ovulation, and can be used as a measure of the intrinsic so-called “acyclic” ovarian activity [24]. AMH also reduces follicle sensitivity to FSH in vivo, and in vitro AMH inhibited FSHinduced pre-antral follicle growth [25]. Thus, there is clear evidence that AMH is involved in the regulation of follicle growth initiation and the threshold for FSH

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sensitivity. The rapid decline in AMH expression corresponds with the selection of follicles for dominance, which is characterized by a transition from a low estrogen-producing state to one of rapidly increasing estrogen production. Estradiol is instrumental in this decline through the estrogen receptor β, which interacts with the AMH promoter region [26]. The measurement of circulating AMH has been applied to a wide array of clinical applications, mainly based on its ability to reflect the number of antral and pre-antral follicles present in the ovaries (the so-called ovarian reserve). There is a linear relationship between AMH and oocyte yield after controlled ovarian stimulation, which is of value in predicting ovarian hyperstimulation. Low levels of AMH can also identify “poor responders”; contrarily, women with PCOS (“high responders”) show markedly raised AMH levels, due to both the increased number of small antral follicles and intrinsic characteristics of those granulosa cells, and this may contribute to anovulation [21]. Serum AMH levels were more robustly correlated with the number of early antral follicles than inhibin B, estradiol, FSH, and LH on cycle day 3. This suggests that AMH may reflect ovarian follicular status better than the usual hormone markers such as FSH and inhibin B [27].

Development of ovarian follicles: gonadotropindependent follicular growth Once follicles reach the antral pool, development is controlled by pituitary FSH and LH which maintain follicle growth up to the preovulatory stage. Within these follicles, three types of somatic cells are present. The granulosa cells that surround and communicate with the oocyte differentiate into the cumulus cells (and form the cumulus oophorus complex); the granulosa cells that line the antral cavity and follicular wall become the mural cells (that secrete estrogen and other regulatory molecules); and the theca cells which are located outside the follicular basement membrane where the mural granulosa cells lie (involved in steroidogenesis and receiving critical nutrients from the blood supply). FSH stimulates the production of high levels of estrogen and the appearance of LH receptors, predominantly on mural granulosa cells [28]. The mural cells then respond to the ovulatory LH surge by ceasing proliferation and undergoing luteinization, a terminal differentiation whereby they become highly steroidogenic, synthesizing elevated progesterone that acts on the uterus to prepare it for implantation and subsequent pregnancy maintenance. The cumulus cells undergo cumulus expansion as a secondary response to EGF-like ligands released by granulosa cells in response to LH [29]. The unique extracellular matrix that forms envelops the cumulus

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oocyte complex and is important for successful oocyte maturation and ovulation [30]. At the same time, the oocyte resumes meiosis and undergoes maturation to become competent for fertilization and early embryogenesis [1].

Follicular recruitment, selection, and dominance Whereas, many follicles begin their developmental course at each cycle, typically only a single follicle sustains its inherent gametogenic potential; all others succumb to atresia finally having forfeited their latency [31]. Women are born with around a million eggs present at birth, enclosed in small follicles and arrested in meiosis. Thus, most eggs/follicles are destined to undergo atresia, possibly through apoptosis. Importantly, the provision of exogenous gonadotropins during stimulated cycles such as in IVF violates the normal mono-ovulatory quota. On a controlled basis this is a desirable effect to recover multiple mature eggs. The time required for early growing follicles to attain the preantral stage is unknown, but it is probably in the order of months [32]. Normally, during the early days of the follicular phase a group of follicles begins to develop at the antral stage (around 4
9 mm as observed by ultrasound). This process is known as “recruitment” [33]. Thus, a cohort of growing follicles (of unknown size yet, probably in the order of 5
20 in women, depending on ovarian reserve and age) begins a well-characterized pattern of growth and development, ultimately providing the species’ characteristic ovulatory quota of eggs. This pattern of growth has been termed the “trajectory of follicle growth,” with gonadotropins providing the “thrust” and ovarian factors the “guidance” along the trajectory [31]. There appears to be a threshold level of FSH necessary for the development of a single follicle [34]. By follicular phase cycle day 6
7 only one follicle will be capable of proceeding to timely ovulation, the socalled “dominant follicle.” After selection of the single dominant follicle, all others become destined to atresia. Induction of FSH receptors on the granulosa cells is of paramount importance in the mechanism whereby primary committed follicles become responsive to FSH [35]. LH receptors are present in the theca cells; these cells under LH guidance secrete androgens from cholesterol, which then diffuse to the granulosa cells where they are converted to estrogen via aromatization. Finally, FSH induces LH receptors in granulosa cells, prompting the dominant follicle for ovulation. During the last couple of days before the midcycle LH surge, the incremental rate of blood estradiol parallels that of progesterone and 17hydroxyprogesterone. This concomitant rise of estrogen and progesterone may reflect the acquisition of LH receptors on the granulosa cells [1].

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PART | I Background

It is accepted that the dominant follicle is the primary source of the rising tide of estradiol seen in blood by the second half of the follicular phase. This serves several purposes: [1] prepares the endometrium for implantation; [2] probably participates in follicle selection; and [3] while initially suppresses the hypothalamic
pituitary axis (negative feedback, reassuring a mono follicular growth), it will initiate the ovulatory FSH
LH surge (positive feedback). The luteinized, ruptured follicle (ovulation) converts into the corpus luteum and secretes progesterone. A week before next menses, there is a decline in serum levels of estradiol, progesterone, and inhibin due to demise of the corpus luteum, associated with a reciprocal elevation of FSH (the so-called intercycle FSH rise), that will guide new recruitment of follicles in the next cycle. The dominant follicle and its successor the corpus luteum make up the “dominant structures” of the ovarian cycle, having remarkable authority over both intraovarian and systemic events regulating folliculogenesis [31].

Ovulation: the LH surge and its consequences The mid-cycle LH surge (normally accompanied by a smaller FSH rise) results in three fundamental follicular effects: (1) resumption of oocyte meiosis resulting in the release of a mature fertilizable oocyte; (2) follicular rupture with release of the cumulus oophorus complex (ovulation); and (3) luteinization, with transformation of an estrogen-producing follicle to a progesterone secreting corpus luteum. Compelling evidence indicates that activation of the G-protein coupled LH-receptor (LH-R) on mural granulosa cells of mature preovulatory follicles in response to the LH surge stimulates adenylate cyclase, leads to an increase of intracellular cAMP, thereby activating protein kinase A and inducing expression of progesterone receptors (PGR). The LH surge via PKA also activates MAPK [36], which appears to also be essential for the induction of PGR [37], as well as protein kinase C that causes a synergistic induction of PGR [38]. It is now agreed that in addition to its well-known endometrial effects, progesterone also plays a critical role in the ovary. Its effects are mediated by PGRs, which are members of the nuclear receptor superfamily of transcription factors (NR3C3). PGR is a key specific regulator of ovulation across species. Several granulosa cell PGRregulated genes have been identified via their misexpression in progesterone receptor knockout mice or in response to anti-progestins, and a subset appear to be involved in follicular rupture. The currently identified genes that appear to be regulated by PGR include proteases, growth factors, signal transduction components, and transcription factors. For most of these genes, their

specific function in ovulation remains to be completely elucidated, and roles in luteinization (with differentiation into a luteal phenotype manifested by expression of the luteal marker P450 side-chain cleavage enzyme) and/or luteal cell survival cannot be discounted [39]. Most studied genes are proteases (ADAMTS1, cathepsin L and ADAM8) probably involved in follicular rupture and ovulation, growth factors (such as EGF-like ligands related to cumulus expansion and meiotic resumption), COX-2 (cyclooxygenase-2), an enzyme that catalyzes prostaglandin production and is essential for ovulation [39], and transduction factors (endothelin and PPARγ -peroxisome proliferator-activated receptor), involved in the ovulatory cascade. In nonhuman primates and humans, species with long luteal cycles, PGR regulates steroidogenesis in the early corpus luteum [40]. Based on expression patterns, it is likely that PGR-B regulates luteinization, while PGRA controls ovulation. However, whether these are direct transcriptional targets of PGR or indirectly via downstream effects has yet to be clarified. Interestingly, the genes that have been analyzed lack PREs (progesterone receptor elements) in their promoter regions, but rather require constitutively bound Sp1/Sp3 transcription factors. Thus, in granulosa cells, PGR transcriptional activity does not appear to depend on direct DNA binding to proximal promoter elements, rather PGR may be acting as a “coregulator” of Sp1/Sp3 activity to coordinately induce a discrete cohort of essential ovulatory genes. Although PGR expression has been demonstrated in several species, there are very few reports of PGR protein localization in the human ovary. Consistent with all other species examined, PGR is expressed in the dominant follicle at the time of the LH surge and similar to other primates, PGR expression is maintained in the active corpus luteum, but not in its late phases [39,41]. PGR is also consistently observed in the theca of some follicles and the stroma and interestingly, was detected in a small proportion of primordial follicles and preantral follicles [41]. More work is clearly needed to clarify the expression patterns of PGR mRNA and protein isoforms in the human ovary. In addition to the regulation of follicular rupture and egg release (ovulation) and luteinization, the LH surge has another critical function, that is, the stimulation of the resumption of meiosis (egg maturation). At this time, the oocyte resumes nuclear maturation as evidenced by germinal vesicle breakdown, transition from prophase I to metaphase I, and extrusion of the first polar body (metaphase II stage). Nuclear maturation occurs, probably in synchrony with cytoplasmic and zona pellucida maturation [42]. During preantral follicle growth, oocytes are not competent to reenter meiosis and they are arrested at the prophase I stage. With the formation of the antrum, the NPPC/NPR2 paracrine regulation (natriuretic peptide

Female reproductive system Chapter | 3

precursor type C and its receptor, natriuretic peptide receptor 2), becomes active and maintains oocytes arrested in meiotic prophase via regulation of intrafollicular (granulosa cells) and oocyte cGMP. With the LH surge, a switch in paracrine regulation takes place with inactivation of the NPPC/NPR2 module and activation of EGF-like growth factors and the PGE2 paracrine regulations [43,44]. The LH-triggered cellular cascades/pathways act in parallel or sequentially and are necessary to propagate the LH signal from the periphery of the follicle to the center where the oocyte resides (spatial and temporal dependency). There is unequivocal evidence that meiotic arrest of the oocyte is dependent on high concentrations of the second messenger cyclic AMP (cAMP) in the egg [43]. Recent data indicate that the entire molecular machinery required to produce cAMP is expressed in mammalian oocytes and that the activity of these endogenous components is enough to maintain cAMP at levels that prevent maturation. G-protein coupled receptors (GPR) and the transduction machinery are functional in oocytes (including adenyl cyclase and phosphodiesterase PDE3A), and these orphan receptors (GPR3) are constitutively active and are required to maintain high cAMP levels in the oocyte and meiotic arrest. High levels of cAMP in the oocyte suppress the activation of MPF (maturation promoting factor) via the action of cAMP-dependent protein kinase A. The activity of MPF, a complex of Cdc2 and cyclin B, is negatively regulated by the phosphorylation of two highly conserved residues of Cdc2, Thr14 and Tyr15. These inhibitory phosphorylations are catalyzed by the Wee1 kinases; whereas, dephosphorylation of these residues is dependent on the Cdc25 phosphatases. Therefore, meiotic arrest is maintained by cAMP-mediated activation of protein kinase A via the direct regulation of the kinase and phosphatase that regulate MPF activity. Granulosa cells express NPPC, which is the precursor for CNP. Although direct evidence of secretion by granulosa cells is lacking, it is likely that CNP accumulates in the follicular extracellular space and activates NPR2. Activated NPR2 causes accumulation of cGMP in the granulosa cell compartment (mural and cumulus, connected via connexin 43, and cumulus and oocyte connected via connexin 37). cGMP diffuses to the oocyte and maintains PDE3A in an inactive state, resulting in high oocyte cAMP which sustains arrest. Since little cAMP accumulates in the follicle prior to LH stimulation, it is likely that cGMP, rather than cAMP, may be the diffusing molecule critical for maintaining the meiotic arrest. The LH surge results in a decrease of cGMP in granulosa cells, and consequently the PDE3A in oocytes is active decreasing cAMP levels, which releases the inhibition of MFP and determines the resumption of meiosis [44].

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The corpus luteum Upon the completion of the follicular phase and ovulation, the dominant follicle undergoes dramatic morphological and functional changes to become the corpus luteum. This is a robust endocrine organ that biosynthesizes and secretes large amounts of progesterone, the key hormone that prepares the endometrium for embryo implantation. Three critical endocrine events support progesterone secretion in primate ovarian physiology: (1) a timely and adequate LH surge (in amplitude and duration) as the LH surge is the signal for follicular rupture and for luteinization of theca and granulosa cells; (2) LH pulses during the luteal phase are critical to the development and function of the corpus luteum; and (3) hCG secretion by the embryo’s trophoblast sustains the CL function in early pregnancy. LH and human chorionic gonadotropin (hCG) are heterodimeric glycoprotein hormones acting on the same receptor, the LH-human chorionic gonadotropin receptor (LHhCGR), which is found as dimer/oligomer at the cell membrane [45]. LH and hCG differ in their half-life (60
120 minutes for LH, several hours for hCG and in some structural features, such as the presence of a carboxyl terminal peptide and the type and amount of glycosylation [46]. The functional span of the corpus luteum is characterized by early development, acme with highest production of progesterone, followed by demise (luteolysis), unless it is rescued by hCG from an established pregnancy [47]. The rate-limiting step in corpus luteum steroidogenesis is the transport of cholesterol to the site of steroid production. Steroidogenic acute regulatory protein (StAR) is a key player in this process and is positively correlated with progesterone concentrations throughout the early and mid-luteal phase. Progesterone biosynthesis requires only two enzymatic steps: the conversion of cholesterol to pregnenolone, catalyzed by cholesterol side-chain cleavage enzyme P450ssc located on the inner mitochondrial membrane, and its subsequent conversion to progesterone, catalyzed by 3β-HSD present in the smooth endoplasmic reticulum. Before the LH surge, StAR is virtually absent from the human granulosa cells, which are unable to synthesize progesterone from cholesterol precursors. Conversely, StAR is found in high levels in the periovulatory human theca cells that can synthesize androgens from cholesterol. Thus, the rapid increase in progesterone at the time of LH surge suggests that luteinizing theca cells are the possible source of progesterone. Additionally, the limited vascular network of the human periovulatory granulosa cells would limit the ability of these cells to obtain cholesterol (low-density lipoprotein) via vasculature. The establishment of an inadequate vascular supply to the CL is postulated to have significant ramifications on steroid secretion later in the luteal phase [48].

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PART | I Background

During the transformation of the ovulatory follicle to a fully functional corpus luteum, local vascular endothelial cells undergo an intense period of proliferation, followed by the establishment of a rich capillary network. Approximately 30
40% of the cells in a mature corpus luteum are endothelial cells. Factors that regulate luteal vasculature play a major role in the regulation of luteal function. Vascular endothelial growth factor (VEGF) mRNA and protein have been localized in the granulosalutein cells of the corpus luteum. Inhibition of VEGF in vivo during the luteal phase in nonhuman primates prevents luteal angiogenesis and suppresses progesterone secretion. Recently, a novel angiogenic factor endocrine gland
endothelial growth factor (EG-VEGF), with a degree of specificity to the ovary, was detected in human granulosa-lutein cells. In contrast to VEGF mRNA, EGVEGF mRNA increases in mid and late corpus luteum. It is thought that EG-VEGF enables the corpus luteum to respond to hCG in early pregnancy. Immune cells, macrophages, and T lymphocytes are present within the luteal tissue. Macrophages and endothelial cells establish close contact with other luteal cells, which facilitates luteal cell regulation by paracrine mechanisms. The ability of macrophages to secrete IL-1β and TNF-α is significant because both cytokines can modulate luteal steroidogenesis. Under physiologic conditions, it is plausible that these cytokines play a role in functional and structural luteolysis, making possible the beginning of a new cycle. However, the unscheduled activation of these mechanisms may result in corpus luteum dysfunction. Structural regression of the corpus luteum is brought about by apoptosis and autophagy, and hCG can change the apoptotic program of the late luteal phase corpus luteum. A decrease in the proapoptotic protein, Bax, has been reported in the corpus luteum of pregnancy and hCGstimulated late corpus luteum. During a cycle of conception, trophoblastic production of human hCG prevents the regression of the corpus luteum. In monkeys with surgically induced hypothalamic lesions, the maintenance of LH levels during the luteal phase through GnRH administration at a pulse frequency like that of the early luteal phase does not prevent luteolysis. Blocking LH release by the administration of GnRH antagonist results in an abrupt decline in serum progesterone concentrations within 24 hours. This suggests that LH is essential to the development and maintenance of the primate corpus luteum, but luteal regression is not, due to changes in LH pulse frequency and amplitude. It has been demonstrated that luteal regression in the primate’s menstrual cycle is caused by a large reduction in the responsiveness of the aging corpus luteum to LH, which can be overcome in the fertile cycle by elevated concentrations of hCG [47,48].

The endometrial cycle: proliferative and secretory phases and establishment of embryo receptivity (window of implantation) Human pregnancy starts at the time of implantation of an embryo (at the blastocyst stage of development) into the endometrium, initiating a complex and reciprocal series of interactions between the mother and the conceptus. This maternal-fetal dialog involves intimate relationships between the specialized trophectodermal cells of the embryo and the receptive uterine lining. The ovarian steroid hormones estrogen and progesterone play a pivotal role in directing uterine events leading to and during early pregnancy [49]. The human endometrium undergoes extensive growth in a cyclic manner, and it undergoes regular cycles of menstruation, menstrual repair and regeneration, proliferation, and secretory differentiation, which are controlled by a sequential, carefully timed interplay of the female sex hormones during the menstrual cycle. Local levels of autocrine and paracrine molecules have profound roles in endometrial function. The endometrial cycle consists of two dominant phases: the proliferative phase, which follows menstruation and precedes ovulation, and the secretory phase, which occurs after ovulation. During the secretory phase, the endometrium transforms into a temporally receptive tissue that is suitable for embryo implantation [50]. The sequential actions of estrogen and progesterone after ovulation regulate the formation of a differentiated endometrial stromal tissue, known as the “decidua,” which supports embryo growth and maintains early pregnancy. Decidualization occurs during the ovulary cycles, independently of the presence of an embryo in the uterine cavity (as opposed to other animal species). Decidualization of the human endometrium involves a dramatic morphological and functional differentiation of human endometrial stromal cells. Once the luminal epithelium is breached, the embryo is rapidly embedded in the decidual stroma. The decidual reaction plays a central role in the establishment of a pregnancy and continues throughout the pregnancy. Decidualization is the differentiation of elongated, fibroblast-like mesenchymal cells in the endometrial stroma to rounded, epithelioid-like cells. This morphological change is initiated during the mid-secretory phase of the menstrual cycle as a result of elevated progesterone levels and begins with stromal cells surrounding the spiral arteries in the upper two-thirds of the endometrium. Human decidualization begins approximately 6 days after ovulation, at the onset of the putative window of implantation [51]. The process is accompanied by secretory transformation of the uterine glands, an influx of specialized

Female reproductive system Chapter | 3

uterine natural killer (uNK) cells, and vascular remodeling to support the maternal blood supply to the growing conceptus [52]. Progesterone is an essential regulator of decidualization and a prerequisite for successful blastocyst implantation. Decidualization is also controlled by complex interactions of transcription factors, cytokines, and signaling pathways. A critical network for the decidualization of endometrial stromal cells is comprised by progesterone and its downstream molecules, including FOXO1 and HAND2. These decidualized cells contribute to the microenvironment in the human endometrium and have direct and indirect influences on endometrial remodeling, local immune response regulation, antioxidant responses, and angiogenesis [50]. There is evidence of paracrine interaction between the blastocyst and the endometrium before blastocyst attachment. The pre-implantation embryo signals its presence to the mother by both endocrine mediators, such as hCG, and paracrine growth factors, which act locally on the endometrium to facilitate attachment. Following attachment, the blastocyst penetrates the luminal epithelium, breaches the basement membrane and invades into the underlying stromal cells [53]. This is a finely regulated process of limited invasion, and if deranged may lead to severe pathologies. The endometrium differentiates into a receptive state when blastocysts are capable of an effective two-way communication to initiate the process of implantation. This state is termed uterine receptivity for implantation and lasts for a limited period. The coordinated actions of estrogen and progesterone regulating proliferation and/or differentiation of uterine cells in a spatiotemporal manner establish the window of implantation. These coordinated effects of progesterone and estrogen result in the cessation of uterine epithelial cell proliferation, initiating differentiation [54]. When analyzed in isolation, competent embryos may be unsuccessful when placed on a nonreceptive endometrium or vice versa, contributing to the “black box” of implantation failure. It is when the two are assessed together that dissynchrony becomes evident, due to premature progesterone stimulus on the endometrium, physiologic displacement of the window of implantation, or a late blastulation of the embryo, or all combined. From the embryonic component, detailed assessment of the timing of blastulation is essential [53]. Multiple hormones, molecules, and regulators are involved in embryo-uterine interactions during implantation including growth factors, cytokines, homeotic genes, transcription factors, and lipid mediators. Despite the evidence for nongenomic steroid hormone actions, differential uterine cell-specific expression of nuclear ERα and ERβ and the progesterone receptor (PR) during the periimplantation period in mice suggests that the coordinated effects of estrogen and progesterone in uterine events for

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implantation are primarily mediated via these nuclear receptors. Other signaling occurs via multiple pathways that involve adhesion molecules leading to cell
cell interactions (integrins and others), vasoactive factors (prostaglandins and others), cytokines (such as leukemia inhibitory factor and interleukins), homeobox genes and many other transcriptional factors. Further research is needed to determine if these pathways function independently, in parallel, or converge to a common signaling pathway to establish the network of crosstalk between the embryo and uterus that is necessary for implantation [54]. Endometrial receptivity represents the crucial status of the human endometrium. A receptive endometrium regulates the adhesion of the embryo, allowing pregnancy to initiate.

Conclusion When carefully studying the entire scope of the structure, function, and reproductive cycle in the female, one is extremely impressed with the complexity of this finetuned system. The number of complicated, carefully timed, interacting, and on-off switching is staggering. The reader must understand that what is described in this chapter is an extremely compressed summary of several textbooks, some of which have multiple volumes. What has supposedly developed as a functioning human female reproductive system over eons of time under the operation of chance is beyond imagination, what one might consider as miraculous. This chapter has described, in an abbreviated way, a small part of the human existence.

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[8] Clarke IJ, Cummins JT. The temporal relationship between gonadotropin releasing hormone (GnRH) and luteinizing hormone (LH) secretion in ovariectomized ewes. Endocrinology 1982;111(5): 1737
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(25) Durlinger AL, Gruijters MJ, Kramer P, Karels B, Ingraham HA, Nachtigal MW, et al. Anti-Mu¨llerian hormone inhibits initiation of primordial follicle growth in the mouse ovary. Endocrinology 2002;143(3):1076
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32. [29] Park JY, Su YQ, Ariga M, Law E, Jin SL, Conti M. EGF-like growth factors as mediators of LH action in the ovulatory follicle. Science 2004;303(5658):682
4. [30] Russell DL, Robker RL. Molecular mechanisms of ovulation: coordination through the cumulus complex. Hum Reprod Update 2007;13(3):289
312. [31] Goodman AL, Hodgen GD. The ovarian triad of the primate menstrual cycle. Recent Prog Horm Res 1983;39:1
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49 Winter. [34] Zeleznik AJ, Kubik CJ. Ovarian responses in macaques to pulsatile infusion of follicle-stimulating hormone (FSH) and luteinizing hormone: increased sensitivity of the maturing follicle to FSH. Endocrinology 1986;119(5):2025
32. [35] Zeleznik AJ, Schuler HM, Reichert Jr. LE. Gonadotropin-binding sites in the rhesus monkey ovary: role of the vasculature in the selective distribution of human chorionic gonadotropin to the preovulatory follicle. Endocrinology 1981;109(2):356
62. [36] Salvador LM, Maizels E, Hales DB, Miyamoto E, Yamamoto H, Hunzicker-Dunn M. Acute signaling by the LH receptor is independent of protein kinase C activation. Endocrinology 2002;143:2986
94. [37] Fan HY, Liu Z, Shimada M, Sterneck E, Johnson PF, Hedrick SM, et al. MAPK3/1 (ERK1/2) in ovarian granulosa cells are essential for female fertility. Science. 2009;324:938
41. [38] Sriraman V, Sharma SC, Richards JS. Transactivation of the progesterone receptor gene in granulosa cells: evidence that Sp1/Sp3 binding sites in the proximal promoter play a key role in luteinizing hormone inducibility. Mol Endocrinol 2003;17:436
49. [39] Robker RL, Akison LK, Russell DL. Control of oocyte release by progesterone receptor-regulated gene expression. Nucl Recept Signal 2009;7:e012 Dec 31. [40] Stouffer RL, Xu F, Duffy DM. Molecular control of ovulation and luteinization in the primate follicle. Front Biosci 2007;12:297
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74. Casarini L, Lispi M, Longobardi S, Milosa F, La Marca A, Tagliasacchi D, et al. LH and hCG action on the same receptor results in quantitatively and qualitatively different intracellular signalling. PLoS One 2012;7(10):e46682. Devoto L, Vega M, Kohen P, Castro A, Castro O, Christenson LK, et al. Endocrine and paracrine-autocrine regulation of the human corpus luteum during the mid-luteal phase. J Reprod Fertil Suppl 2000;55:13
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Chapter 4

Conception and pregnancy R. James Swanson and Bo Liu Anatomical Sciences, Liberty University College of Osteopathic Medicine, Lynchburg, VA, United States

Introduction Except for the reproductive system, all the individual systems of the body, made up of various organs and tissues, are complete within each person. Each organ within each system has specific roles to play in keeping the person alive and in good health. Some organs share responsibility of functions across multiple physiological systems, all but one are individually complete and competent for each person. The liver is an excellent example. Although the liver develops from the embryological gastrointestinal system, it is not only involved in metabolism and storage of ingested carbohydrates and detoxification of ingested poisons/pollutants from the gut, but it is also involved in non-GI system functions. This includes filtration and storage of blood, bile formation, storage of vitamins and iron, and formation of proteins such as coagulation factors and albumin, to name some important functions. However, when looking at reproduction, this system is totally nonfunctional within any one individual. The reproductive system only works when a male, the source of testicular sperm, can interact with a female, the source of ovarian eggs, to complete the reproductive process. The new human formed will be the product of two functional reproductive systems that required the participation of two separate individuals. Thus unlike all other body systems, the reproductive system is only functional when two people of opposite genomes (XX and XY) come together as a couple, at the proposed exact same evolutionary time period of just a few decades, and in the exact same physical space on this planet, to complete the combined systems’ intricate and perfectly coordinated natural copulation (having sex) event. This process has produced a difficult conundrum for constructing a rational, prospective, research-based explanation for its proposed evolutionarily based origin. Testicular-produced sperm and an ovarian-produced ovum provide the paternal and maternal haploid (23

Fertility, Pregnancy, and Wellness. DOI: https://doi.org/10.1016/B978-0-12-818309-0.00011-3 © 2022 Elsevier Inc. All rights reserved.

unpaired chromosomes, N-1) genomes for the initiation and growth of the human zygote, which would start with 23 paired chromosomes, N-2. To accomplish their biological function, the ejaculated sperm will traverse the female reproductive track, approximately 9
12 cm long, at an average speed of 100 μm/second, which works out to a travel time of about 20 minutes to get to the pelvic exit of the fallopian tube. The ovum, which must be released from the ovarian follicle to be accessible to the single fertilizing spermatozoon, has an available window of 8
10 hours for sperm penetration through its zona pellucida (ZP) shell before this thick surface hardens, blocking all sperm entry. Assuming a healthy normal male and a healthy normal female, intercourse that occurs within a few days prior to ovulation will result in motile sperm being available within the pelvic cavity to interact with a freshly ovulated ovum. This will initiate the fertilization process producing the single-celled human zygote. Both reproductive systems have internal and external structures. The male structures are predominantly external while the female structures are predominantly internal. The anatomy and functional steps leading to successful fertilization and a take-home baby will be expanded in descriptions and figures in the following text.

Short review of male and female anatomy and physiology for understanding conception and pregnancy Male Scrotum and testis The testicles are contained within a sack of skin called the scrotum. When this skin is removed from one side it exposes the oval-shaped testis which is nestled up against the epididymis. The vas deferens will leave the inferior portion of the epididymis to enter the abdominal cavity

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PART | I Background

through the inguinal canal, discussed in the next section. The scrotal sac contains muscle fibers (cremaster muscle) that respond to changes in temperature, allowing the testes to be drawn up close to the body by contracting scrotal skin when scrotal temperature drops significantly below normal body temperature. Conversely, the cremaster muscle allows the testes to drop away from the body by relaxing scrotal skin when scrotal temperature rises significantly above normal body temperature. This testicular movement, closer to or further away from the body, is coupled to a vascular counter-current temperature exchange design. These two parts of the testicular air conditioning system maintain the seminiferous tubules, where sperm develop, at about 2 C (35.6 F) below normal body temperature. Spermatozoa will not mature properly at the core body temperature of 37 C (98.6 F) so this cooling mechanism is very important for maintaining normal male reproduction. Testis and spermatogenesis In an XY fetus, the testis develops from the undifferentiated embryonic gonad and has a thick, strong, creamywhite connective tissue capsule (Fig. 4.1), the tunica albuginea (TAC). This tunic has a rich layer of pain receptors on its surface that comes from a layer of parietal peritoneum lining the abdominal cavity. This pain-nerve-rich layer is picked up by the testis as it descends through the abdominal wall into the scrotum. For this reason, a physical blow to the scrotum that stretches the testicular capsule can cause a significant amount of pain. As an aside, the ovary, developing from the undifferentiated gonad of an XX individual, does not have the same rich pain

FIGURE 4.1 A human pelvis/thigh dissection of an oval-shaped testis covered by the tunica albuginea capsule (TAC) with the epididymal head (EH) at the superior pole (LUCOM model). Copyright by R James Swanson.

receptors on its tunica albuginea capsule, because it remains in the pelvis. However, some women report being able to feel an obvious pain when the follicle ruptures through the capsule to release an ovum into the peritoneal cavity. This pain probably only occurs if the swelling follicle becomes large enough to stimulate sensory fibers that are derived from the same developmental area as the ovary or if the swelling follicle touches and irritates the pain rich lining (parietal peritoneum mentioned above) of the abdomen. Inside the human testis are the seminiferous tubules, a little more than half a meter in length in each testis, that contain the male germinal cells eventually producing mature spermatozoa, the sperm. These cells, called spermatogonia, (Sg) are attached to the inner perimeter of the seminiferous tubes and simultaneously attached to neighboring Sertoli cells (Se). Under the influence of follicle-stimulating hormone (FSH) from the anterior pituitary gland at the base of the brain, some of these cells will be programmed to divide and enter the spermatogenic pathway as spermatocytes (Sc). This pathway allows these germ cells to pass through several mitotic divisions, with 23 paired chromosomes, ending in the two final meiotic divisions producing immature spermatids (Sd), with 23 single chromosomes, without tales. These cells can be seen in a scanning electron micrograph (SEM), depicted in a transverse SEM of a seminiferous tubule showing spermatogonia (Sg), spermatocytes (Sc), spermatids (Sd), Sertoli cells (Se), and spermatozoal tails (Ta) if you go to a medical school library and look for an out-of-print book by RG Kessel and RH Kardon, entitled Tissues and Organs (WH Freeman, San Francisco, 1979, p 263). The end-product is millions of sperm cells within the inner opening, the lumen, of the seminiferous tube. The Sertoli, or sustentacular cells mentioned above, are like cellular nurses that have tight junctions with every spermatogonial cell on its path to becoming a mature spermatozoon. Lying in between the seminiferous tubules are the cells that produce the male steroid hormones, testosterone (T) and dihydrotestosterone (DHT). These Leydig cells, sometimes called interstitial cells, are under the influence of the luteinizing hormone (LH), also coming from the anterior pituitary gland like FSH, and these cells respond to LH by producing the male steroids T and DHT. The final maturation step in progressing from a spermatid to a mature spermatozoon requires both FSH and T. The entire generative process cannot be completed at normal body temperature but requires a 2 C
3 C (35.6 F
37.4 F) reduction from the core body temperature of 37 C (98.6 F). For this reason, the pair of testicles hang outside the pelvic cavity in the bag of skin called the scrotum. Under the skin are several layers, one of which is a thin, sparse sheet of muscle, the cremasteric muscle. These muscle fibers provide half of the cooling system for the testes by relaxing as scrotal temperatures rise, thus

Conception and pregnancy Chapter | 4

FIGURE 4.2 Drawing of the external and internal male reproductive system. VD: vas deferens; TA: testicular artery; PP: pampiniform plexus. From: Netter Atlas of human anatomy, 7th ed, Plate 349-A.

allowing the testes to drop away from the body. When scrotal temperatures fall, these muscle fibers will contract, drawing the testes closer to the body. The second half of the testicular A/C system is based in the anatomical relationship of the testicular blood vessels. The artery supplying each testis is surrounded by a plexus of veins called the pampiniform plexus (PP; Fig. 4.2) [1]. This anatomical relationship allows the heat from the artery, which would be at the body’s core temperature, to leave the artery and warm the blood in the venous plexus as it goes back into the pelvis. This venous blood would be cooler, coming back from the scrotum. This arrangement allows the arterial blood to be cooled as it goes down to the testis and the venous blood to be warmed as it goes back up into the body. This important dual cooling arrangement can be subverted in several ways that will reduce, or completely inhibit, male fertility. Wearing tight jockey-style underwear that compresses the testis against the pelvic floor will not allow the cooling mechanism to work properly. Spending an hour in a hot tub or sauna will greatly increase the testicular temperature. Working in any job that keeps you in a very hot environment for long periods of time will also increase testicular temperature. This could include working in, (1) a kitchen with stacked ovens (like a pizza parlor); (2) a manufacturing job that requires maintenance of a blast furnace mechanism; (3) on a roof in the summer on a hot summer day; and any other situation where you are in a hot environment for long periods of time. The Sertoli cell’s tight junctions, mentioned above, are extremely important in maintaining a barrier between the blood and the spermatic pathway. This blood-testis barrier protects the male from producing antibodies

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against his own sperm, autoimmunity, when he reaches puberty. As the immune system is developing in the first months of life after birth, it analyzes and matches all the antibody-producing cell clones, initially comprised of just a few cells, that can produce antibodies against the body’s own proteins. Any protein that is determined to be self (a protien in your own body), will trigger the immune system to destroy the clone of cells that can produce that antibody thus preventing autoimmunity. Once this process is completed and the baby is progressing into a crawling infant, this “self-protein” analysis comes to an end. Any clone that still exists would then respond to any protein that would encounter the child’s immune system. This would include proteins introduced from outside the body, exogenous proteins, or in the case of a male, proteins from the sperm production process, endogenous proteins, which only begins at puberty. A physical blow to the scrotum after puberty, such as being hit by a baseball or landing on the crossbar of a bicycle, can damage the blood-testis barrier. Proteins unique to the spermatogenic process will not have been processed in the early development of the immune system. Thus the clones that can make antibodies against that person’s sperm proteins would immediately begin making antibodies against self with resultant infertility. Male reproductive tract and sperm pathway during ejaculation Sperm cells develop from the germ cells in the seminiferous tubule of the testes to arrive in the tubular lumen (Fig. 4.3). From there they move through a series of short tubes, rete testis, and vas deferens, to arrive in the epididymis, Figs. 4.1 and 4.4. Along the tubes of the epididymis, spermatozoa will be processed to become motile as they reach the vas deferens. Upon ejaculation the sperm will move from the end, or tale, of the epididymis along with the sperm that are in the vas deferens into the ejaculatory duct. The spermatozoa from the two vas deferens tubes coming from the testes will join with the two tubes coming from the seminal vesicles to form the two ejaculatory ducts as they enter the prostate gland (Fig. 4.5). As the sperm and fluid enter the prostatic urethra (Fig. 4.6A,B) the prostate gland will add the prostatic fluid to this mixture bringing the total volume to average between 2
5 mL for a normal male. This seminal fluid will then move down the remaining length of the urethra to exit from the tip of the glans penis (Fig. 4.2). Just below the prostate gland, the small, paired bulbourethral glands will add 0.1
0.2 mL of fluid to the mixture. Reproductive function in both the male and the female is a biphasic event requiring both sides of the autonomic nervous system for climax and ejaculation in the male and climax in the female. The autonomic nervous system

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PART | I Background

FIGURE 4.3 A human tunica albuginea capsule incised to expose the seminiferous tubules which would contain sperm inside the lumen. Copyright by R. James Swanson.

operates at the subconscious level in the human body and controls things like heart rate, pupil size, bronchiolar dilation, and the reproductive events of excitement with tumescence followed by ejaculation. For sperm to be delivered to the upper part of the vagina, the male penis must become rigid, a characteristic called tumescence. This is achieved by dilation of two arteries (Fig. 4.7), each delivering blood into the center of two of the three tube-like bodies that traverse the penile length. These two structures are called corpora cavernosa, which is Latin for cavernous body. The third body is called the corpora spongiosum, Latin for spongy body. This spongy structure carries the urethral tube along its center and does not become tumescent. Thus during ejaculation, the semen can easily progress along the urethra to be deposited at the superior portion of the vagina. During micturition, the 50-cent word for urinating, fluid moves from the urinary bladder to exit the penis at the glans penis tip. Before ejaculation begins, the external urethral sphincter at the base of the bladder constricts so that urine will not enter the urethra from the bladder during ejaculation and seminal fluid will not proceed up into the bladder. If the urethral sphincter does not constrict sufficiently, semen can go into the bladder, a condition called retro ejaculation. Men that have this problem are candidates for assisted reproductive treatment which would involve immediately urinating, after ejaculation, into a cup and then having the fluid centrifuged to recover the semen for intrauterine insemination. Thus we can see that the process of producing and delivering viable sperm (the male gamete that will fertilize the female ovum) into the superior part of the female vagina, a process called ejaculation, is a long and extremely complicated procedure. We will now outline the multiple steps in the female reproductive system that results in a viable female gamete, the ovum, being released from the ovarian follicle and thus ready to receive a sperm cell.

Female

FIGURE 4.4 Human scrotal dissection of the testis covered by the tunica albuginea capsule with the epididymal head and its appendix at the superior pole. Copyright by R. James Swanson.

Overview of the female gamete: steps to ovulation and fertilization To be fertilized, a carefully prepared ovum must be released from one ovarian follicle. Several events must be carefully orchestrated by the two gonadotropins, discussed in the next paragraph, in a sequence that will properly prepare both the ovum and the follicle: (1) The ovum must be stimulated to complete the first meiotic division. This stimulated ovum will continue into the second meiotic division and stop at the metaphase stage of meiosis two (MII-stage ovum). The MII ovum remains arrested at this stage until ovulation takes place and a sperm is available to fertilize it. (2) The follicular wall must thin out and we can do to enzyme activity for the stigma

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FIGURE 4.5 Plastinated mid-sagittal crosssection of a male cadaver’s external and internal male reproductive system (LUCOM model). Copyright by R. James Swanson.

(pimple-like structure) to form and rupture. This rupturing allows the ovum to be released. At the same time, the follicular volume will increase slowly to pressure the thinning follicular wall to rupture, releasing the ovum. (3) If sperm are present, one will pierce the ZP and touch the oolemma. Immediately upon this sperm-egg fusion, the fertilized ovum, now an embryonic zygote, will undergo a reaction within its membrane, blocking entrance of a second sperm. This new zygote will complete the second meiotic division due to the stimulation by the sperm’s entrance into the ovum and begin organizing its new male and female DNA complements to undergo division to the two-cell stage. Follicle-stimulating hormone, luteinizing hormone, and Estradiol-17β The hormonal control of ovulation begins with release of GnRH (gonadotropin releasing hormone) from the

hypothalamus. This hormone travels the short distance from the brain down to the anterior portion of the pituitary gland to stimulate cells called gonadotrops. GnRH will cause secretion of FSH and LH. As its name implies, FSH stimulates a crop of primordial or primary follicles to begin developing into mature, Graafian follicles. The follicular antral fluid contains a host of compounds that is an ultrafiltrate of the capillaries outside the basement membrane, a connective tissue meshwork, which surrounds the granulosa cells (GCs), ovum, and antrum. No blood vessels or neurons pass through the basement membrane into the GC layer. These cells and the ovum must be fed by diffusion. The antral contents include (1) products of the GCs, and (2) other substances which diffuse through both the basement membrane and the GCs. As a cohort of several follicles responds to FSH stimulation and begins to grow through the secondary, tertiary, and eventually mature or Graafian stages, one follicle will begin to insert more FSH receptors into the GC plasma

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FIGURE 4.6 Plastinated model from a superior and lateral (A and B) view of an isolated male reproductive system including the ureters entering the bladder. Pros, prostate; Sv, seminal vesicle; Ur, ureter; Vas, vas deferens). Copyright by R James Swanson.

Deep penile A

Corpus cavernosum

Deep penile A

Corpus spongiosum

Skin Dorsal penile V

FIGURE 4.7 Horizontal cross-section of a male cadaver’s penis showing the two bilateral erectile bodies, corpora cavernosa. The median corpus spongiosum which contains the urethra is not part of the erectile system. Copyright by R James Swanson.

Conception and pregnancy Chapter | 4

membranes. This more aggressive follicle normally outstrips the other members of the cohort, becomes the dominant follicle, and results in the release of only one egg per menstrual cycle. The remaining follicular cohort becomes atretic and forms a microscopic scar on the surface of the ovary called a cicatrix. Occasionally, multiple follicles can grow at the same rate, simultaneously, resulting in multiple dominant follicles. When this happens, multiple embryos can implant into the uterine wall and develop to produce multiple neonates (twins, triplets or rarely even more). With the development of in vitro fertilization (IVF), multiple dominant follicles are induced to develop via hormonal treatment (called superovulation), so that many eggs can be collected for the assisted reproductive technology (ART). Superovulation, with its copious egg production, illustrates that the dominant follicle development in a natural (nonsuperovulated) cycle is closely regulated by the woman’s endogenous hormones resulting in only one egg normally being released with a single child being born. The one follicle outstripping the others to become the dominant follicle in a normal menstrual cycle contains the GCs that produce steroids. The main steroid at this stage (follicular phase) is estradiol-17β (E2). Because E2 has a negative feedback on the production of FSH and LH, the level of the driving hormone for follicular development, FSH, decreases as the follicles grow and produce an ever-increasing amount of E2 (arrow 2 in Fig. 4.8) [2]. Only the follicle that rapidly responds to the

FIGURE 4.8 Female reproductive cycle hormone graph. 1. As P4 and E2 fall after d24, FSH and LH begin to rise d1. 2. Rising levels of FSH stimulate follicular growth allowing follicles to produce E2. 3. E2 initially feeds back negatively on FSH and LH. 4. E2 levels .600 pg/mL feeds back positively on LH. 5. Increased LH levels causes P4 release initiating mRNA, forming enzymes to weaken follicular wall, degrade OMI, and increase colloid osmotic follicular pressure. 6. Rising P4 and E2 negatively feeds back on LH and FSH. 7. Falling P4 and E2 positively feeds back on LH and FSH. From: Guyton and Hall Textbook of Medical Physiology, JE Hall, 13th edition, Fig 82-4, p. 1039. Elsevier

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decreasing FSH levels by producing and inserting many FSH receptors on the GC membranes survives and continues to grow. This upregulation of receptors keeps the FSH level within those GCs at a sufficient level to maintain continued growth and division while the remaining cohort follicles become atretic. As the dominant follicle continues to grow, E2 blood levels continue to increase which negatively feeds back on the two gonadotropins (arrows 3 in Fig. 4.8). At some point the E2 reaches a value (approaching 600 picomole/ mL) that, by some as-yet-unknown mechanism, triggers a positive feedback release of both FSH and LH (arrow 4 in Fig. 4.8). Prior to this peak level of E2, few if any LH receptors are present on the GC membranes. But at the same time, as LH is stimulated to be released from the anterior pituitary, the GCs synthesize and insert the necessary LH receptors into their plasma membranes. This LH rise triggers two events in the GCs: (1) the GCs change their steroid production from producing primarily E2 to producing progesterone (P4, arrow 5 in Fig. 4.8), and (2) the GCs begin undergoing a radical change in both structure and function to become corpora luteal cells. This morphological change produces the CL, or “body of yellow,” and the CL becomes a steroid factory eventually synthesizing large amounts of P4 and E2. The initial change from E2 to predominantly P4, causes antral levels to be very high (2
4 nanomol/mL, again arrow 5 in Fig. 4.8), profoundly affects the GCs. All steroids function by passing through the cell membrane and bind to an intracellular receptor protein. This complex then couples to a very specific receptor on the DNA in the nucleus. This binding process allows P4 to initiate transcription of a number of mRNA strands that are transported through nuclear pores to bind to ribosomes in the cytoplasm of the GCs. The proteins being translated from those mRNAs are enzymes that: (1) degrade the extracellular matrix, made up of collagen and several proteoglycans like chondroitin sulfate and polysaccharides like hyaluronate; (2) degrade the ovum maturation inhibitor (OMI) protein that keeps the egg arrested in the dictyate stage of development and allows it to develop to the MII stage; and (3) increase the colloid osmotic pressure in the follicular antrum by inserting proteins into this cavity. Fig. 4.8 [2] summarizes all these hormonally interacting events with a graph. Progesterone (P4) and ovulation of a mature ovum Progesterone has long been known to be important for (1) creating a secretory endometrium that accepts the fertilized ovum, and (2) maintaining pregnancy. The first use of progesterone as a contraceptive was reported by C. J. Andrews from Norfolk, Virginia, in 1936. However, the importance of progesterone in ovulation has not been

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conclusively confirmed in the literature, although the role of P4 in ovulation has been implied over the past several decades. As seen in Fig. 4.8, arrow 5, P4 levels rise prior to ovulation due to the LH spike. One of the main roles of steroids is to increase mRNA transcription from the DNA. The various mRNAs will then be translated into the appropriate proteins for which they code. This P4 production seen in the graph at #5, while not initially large in the serum samples from which this graph was produced, is coming from the GCs that surround the fluid-filled antrum with the ovum inside. This early P4 production is high enough to cause the GCs to produce the enzyme milieu to bring about the events necessary for ovulation to take place. Over the space of 5
10 hours these enzymes will (1) produce the necessary thinning of the follicular wall, (2) degrade the OMI to allow for egg maturation, (3) increase the antral colloid osmotic pressure, and (4) begin the transition of GCs into luteal cells of the CL [2].

chromatin in the sperm head was condensed six times tighter than normal chromosomes that would be involved in mitosis. This sixfold packing is necessary for the sperm heads to be small enough to swim efficiently through the female reproductive tract in order to arrive in the peritoneal cavity to meet the egg as it ovulates out of the ovarian follicle. Decondensation of the single sperm that has been brought into the egg cytoplasm is stimulated by another set of oolemmal enzymes. These unpaired male chromosomes are now called a pronucleus. The female pronucleus, which will have formed in response to the sperm head binding to the egg’s membrane, will migrate towards the male pronucleus, pairing its 23 single chromosomes (1N) with the 23 single male chromosomes (1N). This forms the 23 paired chromosomes (2N) of the new single-celled zygote with all the DNA information necessary to direct the formation of a new human being.

Implantation and pregnancy Fertilization

Maternal changes in pregnancy

The egg becomes a single-celled embryo, the zygote, when a sperm penetrates the ZP shell and enters the egg cytoplasm to fuse the father’s chromatin with the mother’s chromatin. To do this, hundreds of sperm surround the ovulated ovum, releasing the enzyme (acrosin) that will disperse all the GCs surrounding the ovum. Releasing this enzyme means that the sperm arriving early at the egg will have spent their acrosin and be unable to fertilize the egg. However, once the path is clear to the ZP, a spermatozoon that arrives a bit later will be able to release its acrosin as it touches the ZP shell, allowing it to arrive at the oolemma (outer membrane of the egg). At the moment that this first sperm touches the oolemma, the ovum undergoes a microsecond-long cortical reaction on the inside of this egg’s membrane. This cortical reaction involves thousands of small vesicles (cortical granules) releasing on the inside of the oolemma, an enzyme that changes the egg membrane surface in a way that blocks any other sperm from binding to the membrane. In this way, the ovum is protected from polyspermia (more than one sperm inside the egg), a lethal condition that cannot produce a live baby. This single sperm that has fused with the oolemma will be drawn into the egg’s cytoplasm, but the egg has enzymes that will cut the attachment of the tail away from the sperm head. Since the ATP energy for the sperm to swim is produced by the mitochondria that reside in the tail, male mitochondria do not normally enter into the egg’s cytoplasm and even if a few do get into the cytoplasm, the egg will “spit them out” by exocytosis. The sperm head will now go through a process called decondensation. This step is necessary because the

Maternal, placental, and fetal conditions are the primary concerns for normal fetal development. Therefore routine maternal evaluations for blood pressure, group B streptococcus colonization, glucose tolerance test, as well as cardiac, pulmonary, and thyroid functions are regularly conducted at most women health centers. The fetus is regularly monitored by a nonstress test (NST) to catch a fetus early on with undetermined NST or a mother with a disease status such as gestational diabetes mellitus or preeclampsia (high blood pressure). A fetus is regularly monitored by a biophysical profile (BPP) which includes NST, AFI plus fetal movement, fetal tone, and breath. Of course, the fetus is not breathing air in the uterus, but the diaphragm is contracting, which can be monitored, and a small amount of amniotic fluid will be moving in and out of the pulmonary system. A fetus should begin fetal movement roughly around 20 weeks and $ 3 movements per hour is considered normal. However, frequent or decreased fetal movements indicate intrauterine fetal distress. Nonreassuring BPP warrants prompt delivery. As the fetus is growing, maternal weight is measured each week with 0.5 kg/week growth considered normal. Fundus height is also measured at each visit to determine the growth velocity. Normally, fundus height reaches the level of the umbilicus at roughly 20 weeks, whereas one finger below the xyphoid process (inferior tip of the sternum, breastbone) corresponds to 38
39 weeks. Estimated fetal weight (EFW) is used to assess fetal growth, often by ultrasound. The EFW ,5
10 percentile of gestational age, or ,2500 g at term, is considered intrauterine growth restriction (IUGR). Symmetric IUGR is caused by the fetus due to various things, that is, infection or

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aneuploidy, whereas an asymmetric IUGR would come from maternal causes, that is, placental insufficiency or malnutrition. EFW over 90
95 percentile of gestational age or .4000 g at term is called macrosomia, which is often due to gestational diabetes or twin-twin transfusion syndrome. Maternal changes during pregnancy begin during placental invasion and hormonal (steroids) secretion from the placenta. However, after 12 weeks of pregnancy several maternal changes are more evident and related to the correct fetal development and maternal physiologic adaptation. By the early second trimester, the circulating blood volume increases 40%
50% which results from increases of both red blood cell (RBC) mass and plasma volume. The increase in plasma volume is higher than the RBC mass, which in turn decreases the mean level of hemoglobin from 12 to 10.5 g/dL. This phenomenon is referred to as the physiologic anemia of pregnancy even though there is an increase in RBC. The patient will not be considered as having pathological anemia until the hemoglobin is reduced to less than 10.5 g/dL. A patient is usually prescribed iron tablets when the complete blood count lab result is close to that value. Due to the increase of circulating blood volume, the cardiac output increases 50% to accommodate this change. Some patients may physically present with II/VI systolic ejection murmur during the physical exam. A patient who suffers heart disease is particularly at risk of developing pulmonary edema, due to an increased cardiac output demand and decreased pulmonary volume as described below. Additionally, peripheral vascular resistance is reduced by nearly 45% that in turn decreases blood pressure 20% in the first trimester. The decrease in blood pressure is compensated by an increased heart rate by 20% to meet the metabolic needs. In the third trimester, the large uterus, when supine or in the right decubitus position (lying flat on the right side) will further reduce blood pressure by compressing the inferior vena cava, which reduces venous return to the right atrium. Pregnant women are recommended to lie in the left decubitus position whenever sleeping or lying to avoid this compression. At the same time, due to the enlarging uterus, the diaphragm is gradually displaced superiorly. The displacement subsequently reduces the functional residual volume of the lung which is the term describing the leftover volume after forced expiration and is the measure of the lung capacity. Especially in the third trimester, the increased demand for oxygen and production of carbon dioxide drives an increase in the tidal volume (spontaneous natural breath volume) rather than the respiratory rate. The minute ventilation, which is tidal volume multiplied by time, the critical quantitative measurement of respiration, is also increased. Some pregnant women may experience shortness of breath when lying supine (on the back) since carbon dioxide diffuses faster

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than oxygen and is a weak acidic gas; this decrease in pH is a major stimulator of respiratory rate. Increased minute ventilation can put pregnant women in a more chronic respiratory alkalosis status, because the increased respiratory rate blows off more of the carbon dioxide thus making the pH increase. The increase in circulating blood volume also increases both renal blood flow and the glomerular filtration rate (GFR) by about 40%. This increase results in a decrease of blood urea nitrogen (BUN) and creatinine, which are both quantitative measurements for evaluating renal function. At the same time, increases in GFR accompanied by increased basement membrane permeability also increases urine protein and sodium output. As the uterus enlarges, lumbar lordosis, where the lumbar section of the vertebral column excessively protrudes anteriorly, will cause pain in the lumbar, and hip areas. Sometimes the pubic symphysis can separate up to 1 cm to accommodate the passage of the infant. Reinforcement of lumbar muscles instead of bedrest is highly recommended for pregnant women. Pregnant women are in relative hypercoagulative status due to a 50% increase in coagulative factors (fibrinogen), decrease in anticoagulation factors (protein C and S) and decrease in platelet numbers (physiologic thrombocytopenia). Regular exercise, 5 days a week, doing 30-minute aerobic exercise is recommended to reduce chances of deep vein thrombosis and gestational diabetes. The hPL, is the major driving force of insulin resistance and gestational diabetes. hPL, estrogens, and progestin concentrations are increased throughout pregnancy. Weight increase in the pregnant woman is expected, as mentioned above, at rate of 0.5 lb/ week and normally should not exceed a limit of 30
35 lb for the entire pregnancy.

Embryo development Embryo development during the first 7 weeks applies to both the XY (male) and XX (female) undifferentiated time of growth. The tissue that will eventually become a testis or an ovary is undifferentiated (i.e., it cannot be distinguished in the baby as either male or female during these first 7 weeks). These undifferentiated gonads are exactly the same histologically (under the microscope) and are also functionally quiescent at this time. Once the undifferentiated gonad begins to become a testis or ovary, the subsequent male or female secondary structures (e.g., prostate gland or uterus) will start to grow. The following description is extremely complicated and full of many technical scientific terms which we have defined parenthetically. We decided to take you through this fascinating, multistep, convoluted process to give you an idea of how miraculous it is that anyone of us makes it to parturition (birth) without multiple defects, or even being born at all. Every step along the way requires various DNA

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FIGURE 4.9 (A)
(H) Mouse embryo micrographs from 2-cell to hatched blastula from Dr. Swanson research laboratory. Copyright by R. James Swanson.

codes to be turned on, read, transcribed into mRNA, and translated into proteins. When moving from one earlier step to the next step in line, the DNA codes for the first step must be turned off and a new set of codes for the next step must be turned on. Sometimes a miscommunication occurs in this switching process and the embryo will either not develop properly or will not develop at all. For example, it would be a disaster if hair began to grow in the embryos pictured in Fig. 4.9, before the embryo hatched, or if fingers began to grow from the spinal cord. The potential miscommunications in development are essentially infinite but normally take place in a wellordered, design-implied fashion, as described below. The embryo’s first week is referred to as the preimplantation stage and afterwards is referred to as an embryo up until the end of week 11 or essentially the end of the first trimester. The early embryo developmental stage is defined by a period that starts from the beginning of week 2 (bilaminar embryo formation) to the end of week 8 (completion of major organogenesis). It is a highly orchestrated and strictly genetically coded process in which advanced genetic expressions manifested in a spatial, sequential, and quantitative manner, brings drastic qualitative anatomical and physiological changes. Since it is a qualitative transformation period, the development is heavily influenced by environmental hazards, TORCH infections (acquired in the utero or during the birth process the acronym comes from five infections: toxoplasmosis; other-STDs like syphilis; rubella; cytomegalovirus; and herpes simplex virus) and drugs with teratogenic potential. The hazards can come from many different

sources; chemicals (for example: organic mercury, lead, potassium iodide, radiation); sickness (for example: HIV, rubella, cytomegalovirus, toxoplasmosis, herpes, syphilis); or drugs and addiction (for example: heroin alcohol, nicotine, phenytoin; or warfarin). Many pregnant women are unaware of pregnancy when they make contact with these substances, biohazards, or organisms until the qualitative change of the embryo has already been completed, which is roughly before 11 weeks (embryonic week 9) after their LMP last menstrual peroid (LMP). Thus avoidance of these kinds of items, if possible, should be followed 3 to 6 months ahead of a planned pregnancy. Additional prenatal nutrient supplementation, systemic baseline preexisting disease control and trimester embryo-fetal monitoring are essential for early embryo developmental stage. Zygote to hatching blastocyst, sometimes called the preimplantation embryo 1. Zygote: After approximately 16
18 hours postspermoocyte fusion (fertilization), the two pronuclei in the zygote (Fig. 4.10A) are the central feature of the newly fertilized egg. The presence of two round pronuclei with a thin perivitelline space (the space between the embryo and the ZP shell) and two small polar bodies observed under the light microscope is perceived as normal fertilization. One of the remaining polar bodies can still be seen in a healthy 2-cell embryo (Figs. 4.9A and 4.10B), and following embryonic micrographs, from research in Dr. Swanson’s lab (Fig. 4.9). For normal zygote development, the

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FIGURE 4.10 (A)
(F) Human embryo micrographs from 2-pronuclear stage to late blastula from Dr. Lisiane Knob, Insemine Human Reproduction Center, Brazil. Copyright by R. James Swanson.

a: 400x, 2 pronuclei

d: 400x, 8-cell

b: 400x, 2-cell

e: 400x, morula

c: 400x, 4-cell

f: 400x, late blastula

chromosomes from each pronucleus are aligned in pairs on the newly formed spindle apparatus. The aligned chromosomes are thus prepared for the first mitotic division without the formation of a nuclear envelope. The transition from 1-cell to 2-cell is the transition from using maternal gene expression for driving the embryo’s function, to embryonic gene expression. Maternal materials from the oocyte cytoplasm provides essential support for meiosis, fertilization and the first mitotic division (Figs. 4.9A and 4.10B) and are degraded during 2-cell stage as the embryo begins to take on the responsibility of its own development. Cleavage stages: In the following cleavage stages, the zygote normally divides into a 4-cell embryo on day 2 and an 8-cell embryo on day 3 (Figs. 4.1B and C and 4.2D and D). Each individual embryonic cell is a blastomere. They are equal in size without fragmentation and packed tightly in the ZP so that each blastomere will not have a round shape. The perivitelline space is squeezed to the point where it is almost nonexistent. Each blastomere, before merging into the morula stage (Figs. 4.1D and 4.2E), is omnipotent, that means it has the potential to develop into a whole mouse or human being if separated from the rest of the blastomeres. Fewer numbers of cells, widening perivitelline space, blastomere asymmetry, polarity, cytoplasmic inclusions, and blastomere multinucleation are all considered abnormal embryo development and are used as morphological scoring criteria to determine the competence of an embryo in the IVF procedure. The plasma membranes of blastomeres in the 8-cell stage begin to be connected with each other by gap junctions, which allows exchange of nutrients and signaling information. 2. Morula, blastocyst, and hatching stage: The 8-cell embryo begins to fuse together from the loosely associated group of cells on day 3.5 and divide into approximately 32 cells inside the ZP. The tight

association of cells forms the compacted morula (Figs. 4.9D and 4.10E). The name morula originated from the Latin name for the mulberry fruit. The process of the fusion is called compaction. The failure of compaction is a genetic failure and indicates a loss of viability. This type of embryo will be unable to develop further. On day 4, the morula begins to differentiate to form a round fluid-filled chamber called a blastocoel, which reaches 50
150 cells during the stage. The hollow cavity, or blastocoel, develops from a small chamber to approximately 120 μM (mouse Figs. 4.9D and 4.10E). The embryo in this expanded space is called an expanded blastocyst (human Fig. 4.10F). The outer layer of the blastocyst is called the trophectoderm (TE); the inner layer is the inner cell mass (ICM). ICM cells are often considered as pluripotent stem cells. The ICM can develop into any cell type (ectoderm, mesoderm, or endoderm) and will later differentiate to cells forming various tissues, organs, or extraembryonic tissues such as amnion and yolk sac. The TE, on day 6, which further differentiates into cytotrophoblast and syncytiotrophoblast, is the precursor components of the entire epithelial layer of the placenta. Vitrification (plunging the embryo into liquid nitrogen) is a common procedure in embryo cryopreservation and results in contraction of the blastocyst. On day 6
7, the expanded embryo hatches (mouse Fig. 4.9G and H) out of the ZP (blastocyst hatching or embryo hatching). The embryoderived enzyme, plasminogen activator, seems to be a proteolytic enzyme participating in the hatching process by degrading the ZP. The hatched embryo is irregular in its border and ready to implant in the uterus (Fig. 4.9H). Implantation After 31/2 days of propulsion by the cilia of the uterine tube to arrive in the uterus, the blastocyst embryo will

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hatch after another 31/2 days in the uterine chamber and begin the process of embryo implantation, at the end of week 1 (day 7). The implantation is a complicated process which involves multiple events and signaling pathways. The events include but are not limit to a complicated series of DNA-guided events called endometrial decidualization, receptive state transformation, trophoblast invasion, embryo-endometrial interaction, and immune modulation. We will not describe all these events, but there are many resources on the Internet that you may look up if you are interested. Leukemia inhibitory factor (LIF) secreted by blastomeres plays a central role prior to and during the crosstalk between the blastocyst’s trophoblastic cells and the endometrium [3]. The implanting embryo, which bears a 50% chromosome difference from the maternal genetic material, miraculously gains total immunity exemption from the maternal immune system. Current research has not yet discovered the necessary steps to explain how this protection occurs, but without this protection, the mother’s immune system would reject and destroy what would be considered a tumor of nonself tissue. In addition, in order to have a successful embryo implantation, four major criteria have to be met simultaneously: (1) the late-stage blastocyst embryo must be hatched from the ZP; (2) an adequate maternal serum progesterone level must be maintained; (3) the secretory phase of uterine endometrium must be maintained; and (4) completion of the differentiation of cytotrophoblast

and syncytiotrophoblast cells in the embryo. The ideal implantation location for the embryo is at the posterior wall of the uterine chamber when the uterus is at its normal anatomical position. However, due to the combination of various factors, such as pelvic position of the uterus, previous uterine trauma, or surgical manipulation (for example, a uterine scar by cesarean section, elective abortions or leiomyoma tumor removal), intrauterine infections, congenital uterine anomalies (presence of a uterine septum and bicornuate uterus), implantation of the embryo can occur either at unusual intrauterine locations or locations outside the uterus, predominantly in one of the fallopian tubes. Formation of bilaminar embryo, placenta, amnion, chorion and yolk sac The following paragraph description can be followed in the illustration (Fig. 4.11) [3]. After the successful beginning of implantation on embryonic day 7.5, the syncytiotrophoblast cells contact with the endometrium of the uterus which will then be named the decidua basalis. The endometrium will grow over the embryo as it is buried (implanted) into the uterus and will be termed the decidua capsularis. Simultaneously, the ICM, sometimes called embryonic stem cells, develop into two cell layers, the top epiblast layer and bottom hypoblast layer. This developing FIGURE 4.11 The first and second weeks. Cell division and the elaboration of structures that will be outside the embryo (extraembryonic) characterize the first 2 weeks. The morula, a ball of cells, becomes hollow to form a blastocyst that develops into a placenta and membranes that will surround the future embryo. The embryo is first identifiable as a mass of cells within the blastocyst late in the first week. By the end of week 2, the embryo will be a disk, two cell layers thick. The conceptus (all of the intraembryonic and extraembryonic products of fertilization) takes most of week 1 to travel down the uterine tubes to the uterine cavity. In week 2, the blastocyst sinks within the endometrial wall of the uterus (implantation). From: Netter’s Atlas of human embryology, p. 2, figure 1.1.

Conception and pregnancy Chapter | 4

embryo is now transformed from a blastocyst into the bilaminar embryo. Soon after the formation of the bilaminar embryo, a thin layer of cells will cover the inner surface of the cytotrophoblast layer. This space will be divided into two separate spaces on either side of the bilaminar embryo called the amniotic cavity and the primitive yolk sac (Fig. 4.3). On day 12, some small fluid-filled spaces begin to form inside the extraembryonic mesoderm and soon coalesce into a big space, the extraembryonic celom. The extraembryonic mesoderm connecting between the cytotrophoblast and the embryo is called the connecting stalk which later develops into the umbilical cord. The maternal blood vessels, syncytiotrophoblast, cytotrophoblast, and connective tissue are knitted together to form the placenta, which serves as the fetal endocrine organ for nutrition and growth as well as developing into a physical and immunological barrier. Gastrulation or trilaminar embryo formation Gastrulation, a term that describes the process of development from the bilaminar embryo to the trilaminar embryo (three-layer embryo), takes place during week 3. In the beginning, at the caudal (tail) end of the epiblast layer, the embryo forms the primitive streak, a cell proliferation center for the subsequent events. The primitive streak cells proliferate and completely replace the hypoblast cells and insert an additional layer of cells between the original two epiblast and hypoblast layers except for the area of the future mouth and anus. At the end of gastrulation, the epiblast layer is re-termed as the ectoderm. The replaced hypoblast layer is called endoderm and the insertional layer is named mesoderm. All three layers are referred to as germ layers, because all future development of human structures (germ layer derivatives) can be traced back to these three layers. At the same time, there is another midline structure developed from the primitive streak called the notochord, which induces the formation of the vertebral column and central nervous system (CNS) (signals necessary for the development of axial, i.e., midline structures) and the notochord defines the primordial longitudinal axis. Within the ectoderm, the layer further divides into surface ectoderm and neuroectoderm. The surface ectoderm develops into epidermis (top layer of the skin) and epidermal appendages (hair, nails, sweat and sebaceous glands) whereas the neuroectoderm develops into the CNS (brain, spinal cord) and neural crest. The neural crest cells develops into the peripheral nervous system [all the paired nerves coming from the brain (12) and spinal cord (31)] plus a temporary group of cells which will develop into various cell lineages including melanocytes (cells that produce melanin to darken the skin), adrenal medulla (the central part of the adrenal gland that produces epinephrine

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and norepinephrine), and the aorticopulmonary septum (the developmental structure that eventually divides aorta from pulmonary trunk). The neuroectoderm, under the guidance of the notochord, soon folds into a tubular structure with closure at the caudal and cranial (head) ends on day 24 and 27, respectively. Folate (vitamin B9), which is abundant in green leafy vegetables, is a critical nutrient for this process. Pregnant women require at least 400 mg/ day to fully supplement the ongoing DNA synthesis. For a pregnant woman who is unable to wean off valproate (seizure control drug), a dose of folate may need to be given up to 4 g/day to prevent neural tube defects. A neural tube defect refers to a neural tube closure failure which results in anencephaly (failure to close at cranial end), spina bifida (failure to close at caudal end) and polyhydramnios (excessive amniotic fluid as the result of a neural tube defect). Acetylcholinesterase concentration in maternal serum and amniotic fluid (collected through amniocentesis) can often be used as a biomarker to determine the likelihood of a neural tube defect. Within the mesoderm, the middle germ layer, development proceeds into three clusters of cells: paraxial (next to notochord), intermediate, and lateral plate mesoderm. Paraxial mesoderm develops into most of the head structures and 42
44 pair of somites (the parallel, segmented tissue clumps on each side of the midline notochord). The somites further develop into the axial (midline) skeleton, skeletal muscles and dermis (the bottom layer of skin). Intermediate mesoderm, however, develops into the future urinary system and gonads (ovary, uterus or testes, prostate). Lateral plate mesoderm divides into a somatic (of the body linings) layer and splanchnic (of the organs of the body) layer. The somatic layer forms the linings of the organs (pleura, pericardium, peritoneum) and the splanchnic layer develops into solid organs such as liver and cardiovascular system. Mesoderm is also responsible for forming hematological and immunological components such as RBC, white blood cells especially lymphocytes, macrophages, and neutrophils. Finally, endoderm forms the epithelial linings of the GI tract, respiratory tract, biliary apparatus, urinary tract, inferior 2/3 of vagina, auditory tube, middle ear cavity, and the lung. The development of the lung requires a sufficient amount of amniotic fluid. The fetus with a hypoplastic kidney (small and/or low functioning) and renal dysfunction will result in hypoplastic or agenesis of the lung. Embryonic folding After gastrulation, embryo development enters into the fourth week. Embryonic folding and the beginning of organogenesis is the hallmark event of this week of development. Embryonic folding occurs due to developmental

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PART | I Background

increase in the two ends of the embryo and different layers of the embryo. The cranial end and caudal end of the embryo develop faster than the body, and the ectoderm develops faster than the endoderm. Therefore the embryo experiences both cranio-caudal and lateral foldings that all meet together at the umbilicus (belly button). Endoderm is folded into a tube-like structure which is the prototype of the gastrointestinal (GI) system. At the end of this tubular structure, there is a branching blind tube called the allantois. The allantois and connecting stalk of extraembryonic mesoderm form the future umbilical cord with two umbilical arteries carrying deoxygenated blood from the fetus to the mother and one umbilical vein carrying oxygenated blood from the mother to the fetus. The heart prototype that once was at the cranial end of the future mouth folds caudally into the chest area. The yolk sac diminishes in size but still connects to the GI tube via vitelline duct, a patent opening at the umbilical area which is critical for GI development. Organogenesis Organogenesis that occurs simultaneously with the embryonic folding is the process forming the prototype of major system organs. The majority of the organ system will be completed by the end of week 8 except for the CNS, the ear, and auditory apparatus, and the male/female reproductive systems. Heart development and fetal circulation To understand the heart and fetal circulation we must first briefly describe the anatomy and physiology of the adult heart. The adult heart has two completely separate, walled off pumps, each with two chambers, an upper atrium and a lower ventricle with this completely formed septum between the left and right sides. The right atrium receives its blood from the body (called the systemic circulation) and sends the blood down into the right ventricle through the tricuspid (right atrioventricular) valve which then pumps the blood to the lungs. The left atrium receives the blood back from the lungs (called the pulmonary circulation) and sends this blood down into the left ventricle through the bicuspid (left atrioventricular or mitral) valve which then pumps this blood to the body, excluding the lungs. The heart formation begins with two endocardial tubes merging to form the primitive heart tube which then folds and twists to be walled off into four separate chambers, a left and right atrium and a left and right ventricle. Two separate walls (called septa) form individually between the left and right atria. The two atrial septa do not close completely at a hole called the foramen ovale. This foramen allows blood to pass from the right atrium into the left atrium for the purpose of proper fetal circulation. The lungs, which are supplied by the right side of the heart,

receive only enough blood to maintain the life of the pulmonary cells because oxygen and carbon dioxide exchange occurs through the placenta and not the lungs while the baby is still in the uterus without access to air. Once the baby is born and takes the first breath, the microscopic balloons, called alveoli, expand with the surrounding capillaries which greatly reduces the pressure in the lungs. This change in pressure triggers the two atrial septa to immediately grow and close so there is no longer a right-to-left shunt of blood away from the lungs and to the body. During the next week there are many complex changes taking place in heart development that we will leave to your imagination or studying from a pulmonary textbook. After completion of the prototype heart development, at the end of week 8, fetal circulation is established. Placental blood oxygenation begins for the embryo when the uterine spiral arteries penetrate and provide blood to the intervillous space where oxygen will diffuse through the maternal-fetal-placental barrier via simple diffusion to the fetal umbilical vein. The vein then penetrates and passes over the liver by a venous duct. The blood flows into inferior vena cava via the hepatic vein. The mixture of blood from inferior vena cava (deoxygenated blood) and umbilical vein causes a decrease in fetal oxygen saturation. This relative hypoxemic status puts the fetus in a more oxygen deprived vulnerable situation and is more susceptible to events of maternal and fetal hypoxemic diseases, such as maternal smoking, heart and lung diseases, and placental insufficiency. The fetus physiologically tends to have more RBC mass and hemoglobin (Hbg) concentration to compensate for this relative hypoxemic status. This higher oxygenated blood circulates into the right atrium and through the foramen ovale and moves into the left atrium and then left ventricle. The left ventricle pumps the blood into the aortic arch where the majority of the blood runs to the fetal head. However, the lessoxygenated blood from the superior vena cava drains into the right ventricle via the right atrium and then is pumped into the pulmonary trunk. The majority of the less oxygenated blood bypasses the lung (due to the high resistance of the unexpanded lung) by taking another right to left shunt from the pulmonary trunk to the aortic arch through an open (patent) vessel called the ductus arteriosus where it will then circulate to the rest of body. Thus the head always gets the first priority to receive the oxygenated blood, whereas the less oxygenated blood in the rest of body will flow back to the mother via two umbilical arteries. After birth, the expanding lung dramatically decreases the pulmonary circulation resistance, so the high-pressure systemic circulation allows the closure of the foramen ovale (now called fossa ovalis) and patent ductus arteriosus (now called ligamentum arteriosum) which redirects the blood through the pulmonary circulation for the

Conception and pregnancy Chapter | 4

independent life-sustaining oxygenation. However, a cyanotic congenital heart defect in the infant (e.g., Tetrad of Fallot) usually keeps or medically sustains the patent ductus arteriosus open to let the brain receive oxygenated blood. Digestive system development and lung development After embryonic folding, the prototype of the GI tract divides into three portions: foregut, midgut, and hindgut. Foregut further develops into the esophagus, the stomach, and the proximal duodenum and additionally the liver, pancreas, and biliary apparatus. The foregut also gives rise to the trachea and lung buds. The midgut provides the prototype for the gut from the distal duodenum to the transverse colon. The hindgut is the section from the descending colon to the anus. The most miraculous changes to the GI tract are from the protrusion of the midgut through the umbilicus outside the body (physiological herniation), then rotating 270 degrees counterclockwise and returning back to the abdominal cavity. This externalization, twisting, and then returning into the abdominal cavity results in the beginning of the colon or large intestine (the cecum with the attached wormlike appendix) to be located in the right lower quadrant of the abdomen and the rest of the colon will reside in front of the small intestine. Errors happening in this process results in multiple congenital defects, such as volvulus (malrotation), gastroschisis, and omphalocele (unable to return internally and/ or abdominal closure failure) and umbilical hernia (incomplete closure of the abdominal wall at the belly button). Interestingly, lung development arises from the endoderm of the foregut to form the lung bud and trachea. The lung bud branches into lobes, segments, bronchioles, terminal bronchioles, respiratory bronchioles, alveolar sacs, and alveoli. The entire lung maturation process can continue to the adult age of 25. One major survival component, the surfactant, significantly decreases the tension and increases the compliance of the alveoli. This surfactant begins to be secreted at week 16, but the amount of the surfactant is not sufficient to sustain life until the gestational age of 35 weeks. With increasing advancement of medical management, synthetic surfactant can significantly improve the respiratory function of preterm infants, bringing the viable age to as early as 21 weeks. Betamethasone, a glucocorticoid (steroid), is often clinically administered to the preterm labor patient (,37 weeks) 12 hours ahead of delivery to maximally decrease the incidence of neonatal respiratory distress syndrome. Muscular skeletal development Somites of paraxial mesoderm, mentioned above, are the primary source of the musculoskeletal system. The somites further differentiate into sclerotomes, myotomes, and dermatomes which represent future axial skeletal bones, skeletal muscles and

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dermis. However, the appendicular skeleton is derived from lateral plate mesoderm. The notochord induces the midline axial vertebral column formation and itself degenerates into the nucleus pulposus, the central part of intervertebral disks. Beginning at the end of week 4, limb buds begin to form. At week 6, digital rays and notches between rays develop and digital webs link the digits together and at the same time, chondrification (cartilage development) of the future limb bones starts to form. The long bones are formed by sequential stages from mesenchyme (undifferentiated mesodermal cells) replaced by cartilage and then replaced by bone (ossification). The process of long bone formation is different from flat bone (such as in the skull bones) formation which starts from mesenchyme between two membranes and then replaced by fibrous tissue and bone without any involvement of cartilage formation. The characteristic developmental event in the limb formation takes a different direction in limb rotation between upper and lower extremities. The upper extremity rotates 90 degrees laterally which results in the lateral placement of the thumb whereas the lower extremity rotates 90 degrees medially which results in the medial placement of the big toe. At the end of week 8, all digital webs disappear, and the prototype of the appendicular skeleton formation is complete. Central nervous system development Neural tube formation from neuroectoderm is induced under the guidance of the notochord which process is referred to as neurulation. The CNS development includes both brain and spinal cord development. During the 4th week, the cranial end of the neural tube develops into forebrain (prosencephalon), midbrain (mesencephalon) and hindbrain (rhombencephalon). Then during the 5th week, the forebrain splits into telencephalon and diencephalon. The hindbrain differentiates into metencephalon and myelencephalon. The midbrain remains the same. These five brain portions develop into all the parts of brain and related spaces inside of the brain (ventricles). The telencephalon differentiates into the cerebral hemispheres and lateral ventricle; the diencephalon becomes the thalamus, hypothalamus, and third ventricle; the last three sections become the brain stem (midbrain, pons, medulla oblongata, and 4th ventricle). For the spinal cord development, the caudal neural tube divides into ventral basal plate and dorsal alar plate which later develops into motor and sensory centers. The remaining white matter of the spinal cord forms the ascending and descending tracts with multiple synapses (connections between nerves) along the way. The placenta as an endocrine organ The placenta secretes human chorionic gonadotropin (hCG), human placental lactogen (hPL), and steroid

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PART | I Background

hormones. The hCG, secreted by the syncytiotrophoblast, functions similar to LH from the corpus luteum (CL) of the ovary. This hCG will rescue the CL so that both progesterone and estradiol will continue to be produced by the CL, maintaining the placenta and allowing for continued development of the embryo and eventual fetus. The hCG can be detected as early as 7
10 days in the maternal serum and 11
14 days in the urine after fertilization. Women who miss a period can easily ascertain pregnancy by a random sensitive urine hCG strip test. The hCG is also present and increases in choriocarcinoma (a trophoblast cell cancer), hydatidiform mole (abnormal proliferation of trophoblast cells through abnormal fertilization), germ cell tumor and twin pregnancies. Some patients may even experience symptoms of hyperthyroidism (sweating, weight loss, heat intolerance, and tremor) due to a shared α unit of hCG with TSH. These two hormones, along with LH and FSH, have two subunits, α and β, and all four are thus referred to as dimers. The hCG can also be used in detecting chromosomal disorders primarily in trisomy 13, 18, and 21 which provides critical screening information for further fetal evaluation, such as chorionic villous sampling (CVS) or amniocentesis. The placenta also secretes hPL steadily throughout pregnancy. Due to the structural similarity to growth hormone (GH), hPL increases maternal lipolysis (breaking down body fat), amino acid placental transfer into the embryo, and increased glucose availability to fulfill fetal energetic substrate requirements. In addition, the placenta produces large amount of progesterone and estrogen which are needed to maintain the pregnancy. The placenta provides the fetus with the oxygen and nutrients while removing metabolites, carbon dioxide, urea as urine and any items from the fetus that need to be placed in the maternal feces. The exchange ranges from just simple diffusion (water, O2, CO2) to complicated pinocytosis (active transport) of items that the fetus needs to acquire or to remove. Thus the placenta serves as maternal-fetal blood barrier which restricts the embryonic environment so that infectious and teratogenic (birth defect-causing) hazards do not reach the fetus. Fetal development Most organogenesis processes are complete at the end of week 8 except for the female reproductive and renal systems which begins at week 11. Embryo development transitions into what will now be called fetal development at either week 9 for males and week 11 for females which marks the presence of all the major body systems at an immature level. Until the baby is delivered, the development of the fetus will then mainly follow the route of quantitative change and organ maturation rather than developing new structures. Ultrasound dating in the first

trimester until early second trimester is often used when LMP is uncertain due to irregular menstruation or pregnancy under oral contraceptive (OCP) use. Later ultrasound dating is not very accurate due to many variables such as speed of fetal development, placental function, or maternal status (e.g., gestational diabetes mellitus). The reproductive and urinary system development in the male reproductive system does not start to develop until week 7. The availability of the Y chromosome guides the sexual differentiation of the male. There are only two biological sexes, male (XY) and female (XX). Intermediate mesoderm differentiates into left and right urogenital ridges and then further develops into a nephrogenic ridge (urinary parts) and a genital ridge (productive parts). Intermediate mesoderm also develops two tubes for future reproductive tracts: mesonephric (male Wolffian) and paramesonephric (female Mu¨llerian) tubules and ducts. Under the influence of the sexdetermining region of the Y-chromosome (Sry) gene, the genital ridge forms the testis. The Mu¨llerian tubules and ducts degenerate under the influence of the anti-Mu¨llerian hormone secreted from the Sertoli cell of the testis. The Wolffian ducts form the epididymis, vas deferens, seminal vesicle, and ejaculatory duct under the influence of testosterone from the testicular Leydig cells. Male external genitalia including the prostate is formed under the influence of DHT which is transformed from testosterone. However, if there is no Y-chromosome or Sry gene available at the 12th week, the gonads will, by default, develop into ovaries. The inferior third of Mu¨llerian ducts merge to form the uterus and upper third of the vagina. The upper two thirds of the Mu¨llerian ducts remain separate to form the two fallopian tubes which open into the peritoneal cavity. The Wolffian ducts will degenerate due to the absence of testosterone stimulation. Female external genitalia including the lower two thirds of the vagina is formed under the influence of estrogen from the ovary. All prototypes of the reproductive organs are complete by about week 16. The ovaries contain 5
10 million oocytes at the 5th in utero month, at which time mitosis of the female germ cells ceases. At parturition, the ovaries will contain 0.5
2 million oocytes and these eggs will remain in their primordial or primary follicles until they ovulate or become atretic (a fancy word for dying). At puberty there will be approximately 50,000 ova remaining. A normal female reproductively competent would last approximately 40 years, having approximately 13 menstrual periods per year from puberty to menopause. This results in approximately 520 ovulations which numbers could be reduced by approximately 10 menstrual cycles/ovulations per pregnancy or more ovulation reduction if the mother chooses to breast-feed her baby. Menstrual cycles during breast-feeding very widely depending on the mother’s ethnicity and also whether the infant is sustained

Conception and pregnancy Chapter | 4

exclusively with mother’s milk or is supplemented with other forms of nutrition. The urinary system, as stated above, develops from the nephrogenic ridge. These ridges undergo serial progression from pronephroi to mesonephroi to final metanephroi. Ureteric buds from metanephroi induce the growth and connection with surrounding mesenchyme which in turn generates the permanent kidney (renal cortex and medulla). As the formation and development of kidneys progress, the kidneys extend from the pelvic region to the retroperitoneal (posterior to the pelvis) region. Failure to do so will result in a horseshoe shaped kidney. Urine produced by the kidneys is the primary source of the amniotic fluid. The volume at term is roughly 800 mL and ranges from (400
1200 mL). The amniotic fluid index (AFI) is a result of this fluid volume and if ,5 cm it is termed oligohydramnios, or $ 24 cm it is termed polyhydramnios. Both volumes would be considered abnormal. Adequate amniotic fluid is necessary for normal lung, limbs, and craniofacial development. Amniotic fluid also serves the purposes of shock absorption, thermoregulation, and labor augmentation.

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uterine fundus (the most superior part) and progressing to the lower portion of the uterus. The power of the contraction is measured by Montevideo units with one unit equaling 1 mmHg force within 10 minutes. Two hundred Montevideo units are considered an adequate contraction power. A sample calculation, for example, could be four contractions in 10 minutes producing 50 mmHg of power for each contraction. The Montevideo power is calculated by 4 3 50 mmHg 5 200 U. Oxytocin, a hormone secreted from the posterior pituitary, is the primary driving force for uterine contraction. In women who experience prolongation of stage I labor due to hypotonic contraction, oxytocin can be used as a labor augmentation to reach the adequate contraction power. Not all uterine contractions are considered as labor. The uterine contraction power varies throughout late third trimester. However, as time gets close to term, the contractions become more frequent, intense, and longer lasting. The intensity usually begins at around 30 mmHg for a 30 second duration every 10 minutes. The sign of the irreversible active labor is widely accepted as 5
1
1 rule: 5 minutes. Intervals, lasting 1 minute per contraction and continuing for 1 hour. The entire labor process is a positive feedback process until the fetus and placenta are delivered.

Parturition Term pregnancy Term pregnancy is defined as being between week 37 and 42. If calculated by the start of the LMP, the expected delivery is 40 weeks after LMP which an obstetrician roughly calculates by LMP plus 9 months and 6 days. Normal parturition ranges from 3 to 20 hours. The elements of labor are (1) the birth canal, (2) the object of labor (passenger), and (3) the power of uterine contraction. The birth canal consists of bony and soft tissues. The bony portion is made by the fusion of the hip bone, sacrum, and coccyx. The obstetrician must measure both the bony birth canal by measuring instruments, and the head of fetus (largest portion of the fetus) by ultrasound to determine the feasibility of vaginal delivery. The soft tissue canal is made by the uterine cervix, vagina, and vulva and to a larger extent, it also includes the surrounding structures (bladder, rectum, perineal and levator ani muscles). For the passenger, the ideal fetal position at the time of delivery is left occiput (back of head) anterior (LOA) position which means the occiput of the fetus is facing the left side of the pubic symphysis. Any other fetal positions, such as breach, transverse or other head presenting positions (e.g., right occiput posterior), are obstetric-challenging situations. Finally, the main power of parturition is primarily the uterine contraction, though other forces such as abdominal muscle contraction may take part in increasing the force. The smooth muscle of the uterus contracts and generates power starting from the

Phases of labor Labor is divided into first, second, and third stages (Fig. 4.12) [4] from the onset of regular uterine contraction to the delivery of the placenta. The mechanism causing labor onset is not yet fully understood. However, most women will experience an increase of vaginal discharge several days before labor when a thick mucus plug is expelled from the cervix. Also, a patient may experience “lightening” once the head of the baby engages in the pelvis, with an increased ease of breathing and increased frequency of avoiding due to the pressure on the bladder. As stated above, regular contraction with adequate intensity marks the onset of labor. First stage The first stage is defined from the onset of labor to the complete dilation of the cervix (10 cm). The first stage is further divided into latent and acceleration (active contractions) stages. The latent stage represents the dilation of the cervix from 0 to 6 cm. The hallmark of this stage is cervical effacement (thinning) where the uterine cervix (normally 3
4 cm thick before labor) gradually will efface to the thickness of several pieces of paper. It should take primipara (first childbirth) less than 20 hours (16
18 hours average) or multipara (subsequent childbirths) less than 14 hours (2
10 hours average) to complete. The most common cause of prolongation of the latent stage is due to analgesia (pain reducers). Rest and

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PART | I Background

FIGURE 4.12 Composite of the average dilatation curve for labor in nulliparous women. The curve is based on analysis of data derived from a large, nearly consecutive series of women. The first stage is divided into a relatively flat latent phase and a rapidly progressive active phase. In the active phase, there are three identifiable component parts: an acceleration phase, a linear phase of maximum slope, and a deceleration phase. From Cunningham FG et al., chap 21, Physiology of Labor, Williams Obstetrics, 24 ed., p. 409.

sedation are the primary ways to manage the latent stage as long as the fetal monitoring is normal. The acceleration stage of cervical dilation begins from 6 cm to full dilation, usually 10 cm with a speed of .1.5 cm/hour in primipara or .1.2 cm/hour in multipara. A slower speed is considered as prolongation and no cervical change over 4 hours is considered as active phase arrest. The causes can be the passenger (big fetal size or an abnormal presenting part) or pelvis (cephalopelvic disproportion, i.e., to narrow) or power (inadequate or excessive contraction power). Some situations can be solved by medications, such as oxytocin for inadequate contraction power or morphine sedation for hypertonic contraction. However, some situations will end up with an emergency cesarean section (active phase arrest). Second stage The second stage starts from full cervical dilation to delivery of the infant. Normal duration of this stage is much shorter, less than 2 hours for primipara and 1 hour for multipara. Add one hour epidural anesthesia. Women are instructed to push by simultaneously contracting abdominal muscles to augment the power of the uterine contraction. Contractions reach their maximum intensity to nearly unbearable.

The fetus passes through the birth canal by experiencing the following processes: (1) Engagement—fetal vertex (in most favorable position, the occiput) enters the pelvis with the sagittal suture of the calvarium aligning with the transverse pelvic diameter (10 cm). The measurement of the engaging diameter of the fetus is usually the anteroposterior diameter (9.5 cm) and is critical for the feasibility of vaginal delivery. (2) Descent—fetal position is usually measured by the relative position with the maternal ischial spines. When the vertex is at the level of the ischial spine, it is referred to as position 0 (zero). A “ 1 ” or “
” placed before a number, for example, 13 position, means the centimeters below or above the ischial spine. The obstetrician uses these numbers to assess the process of fetal descent. Full engagement is defined when the fetal head is at the “0” position which allows obstetric forceps or vacuum to usually be used if the second stage is prolonged and the fetal head is at 12 position. If the fetal head is at
3 position over a normal time duration, then cephalopelvic disproportion is diagnosed and an emergency cesarean section follows. (3) Flexion and internal rotation—the pelvic inlet, with which the fetal head initially engages, has its widest

Conception and pregnancy Chapter | 4

diameter at the transverse plane. However, as the fetal head descends to the level of pelvic outlet, the widest portion is the sagittal diameter, between the tip of the coccyx and the inferior border of the pubic symphysis. Therefore flexion and internal rotation allows the fetal head to rotate 90 degrees to accommodate the directional change of the widest diameter. (4) Extension and expulsion of the head—as the fetal head is passing over the pubic symphysis, the extension of the fetal head causes the body to be expelled through the vagina. (5) External rotation—the fetal head then rotates back to its engaging position and is expelled out by the final push. The shape of the head at the time of delivery is often very flexible by having the skull bones overlap each other to accommodate the rigid birth canal. In extreme circumstances, the clavicle can be broken to relieve the pressure caused by an oversized fetal body. However, a fetal weight over 4500 g estimated by ultrasound, is used to prescribe a cesarean section to avoid unnecessary risks of obstetric complications. Third stage The third stage of labor starts with fetal expulsion and ends with delivery of the placenta. Delivery of the placenta usually takes less than 30 minutes. The placenta is normally delivered on its own by uterine contractions. If the delivery is prolonged more than 30 minutes, the obstetrician is forbidden to manually pull the umbilical cord because it may cause uterine inversion and massive hemorrhaging (over 500 mL). A physiological hemorrhage up to 500 mL is expected during the delivery of the placenta. Uterine massage or oxytocin augmentation is recommended when placental delivery is delayed. Placenta accrete/increta/percreta (terms for when the placenta grows into myometrium or through the entire thickness of uterus) is often the preexisting condition for placental delivery difficulties. After delivery, the placenta will be visually inspected for its integrity. A placenta that has ruptured into pieces and retained in the uterus often causes massive uterine bleeding and subsequent intrauterine infections. Manual extraction from the uterus is often needed together with oxytocin for complete placental removal. Hysterectomy is recommended when all above attempts have failed. Some experts also include an

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additional stage of parturient recovery after delivery of the placenta. The parturient recovery refers to the uterus returning to the status prior to the pregnancy which is usually 6 months.

Conclusion Conception and pregnancy, much like gamete production and intercourse, require an exceptionally large number of fine-tuned, exquisitely timed, and skillfully orchestrated events to take place for the successful delivery of a normal take-home baby for the parents. In 1978, the first IVF baby in the world was born in Oldham and District General Hospital, Manchester, England, producing the first IVF baby using a natural cycle. The first American IVF baby was born in 1981, in Norfolk, Virginia, United States, being the first baby produced by a hormonally super ovulated cycle. As IVF technology soon mushroomed around the world, IVF programs began to realize that normal conception in a fertile couple was inefficient in producing the desired take-home baby. In an unpublished conversation that Dr Jim Swanson had with Dr Howard Jones in the mid-1990s, Howard felt that the data indicated a 1-in-5, or at best a 1-in-4 successful term pregnancy for every initially fertilized ovum in a normal reproductively competent couple. This would help confirm the multiple number of critical instructions, or steps, necessary to be rigorously followed within the DNA blueprint information residing in the 23 pairs of chromosomes found in the cells that make up all the various tissues and organs that result in successful reproduction. Our chapter is a very constricted description of a beautifully choreographed dance that would take a number of volumes to fully describe what is known, at this time, about this process. Many volumes are yet to be filled with the rest of the story that we do not understand at this time.

References [1] Netter Atlas of human anatomy, 7th ed, Plate 349-A [2] Guyton and Hall Textbook of medical physiology, 13th ed, JE Hall, p. 1039, Figs. 82
4. Elsevier. [3] Netter’s Atlas of human embryology, p. 2, Figure 1.1. [4] Williams Chap 21, Physiology of labor. In: Obstetrics, 24th ed, FG Cunningham et al., p. 409.

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Chapter 5

Genetics and epigenetics of healthy gametes, conception, and pregnancy establishment: embryo, mtDNA, and disease Ciro Dresch Martinhago1 and Cristiana Libardi Miranda Furtado2 1

Chromosome—GeneOne/DASA, Sa˜o Paulo, Brazil, 2Drug Research and Development Center, Postgraduate Program in Medical and Surgical Sciences, Federal University of Ceara´, Fortaleza, Brazil

Gametogenesis and genetic healthy gametes Gametogenesis (for review, see [1
3]) is defined as the development of mature haploid gametes from precursor cells [primordial germ cells (PGCs)] that undergo a series of cell divisions (mitotic and meiotic) followed by cell differentiation to become gametes [1
3]. Gametogenesis takes place within the gonads (testes or ovaries in males or females, respectively); in males this process is called spermatogenesis and produces spermatozoa, while in females this is oogenesis and results in eggs or oocytes [4]. During mitosis a cell makes a complete copy of its genetic material, and after cell division both daughter cells receive a complete and exact copy of the parent cell’s genetic information (DNA). Meiosis is a two-step cell division process. The first step involves DNA replication [meiosis I (MI)]; during MI, homologous chromosomes pair and exchange genetic information (chromosomal crossover). During meiosis II (MII) the sister chromatids separate, resulting in haploid cells that become sperm or eggs [5
7]. Meiotic recombination is essential to produce gametes, and is key to genetic variation in the population. To ensure that MI and MII occur correctly, the spindle assembly checkpoint regulates chromosome segregation. If a failure occurs at this checkpoint for any reason, aneuploid germ cells could result; in most cases these cells cannot sustain embryo development, resulting in lower chances of pregnancy or higher rates of early pregnancy loss. Some autosomal trisomies and some sex

chromosome aneuploidies are compatible with birth in humans, however. Approximately 1% of chromosomal aneuploidies result in a live birth, while 6% of stillbirths show chromosomal abnormalities [8]. While in principle the process of gametogenesis is identical in males and females, oocytes and sperm cells differ in terms of chromosomal events leading to meiotic division [5,6,8]. In general, most human trisomies are derived from errors during female gametogenesis, especially during the MI stage. Nearly 30% of fertilized oocytes are aneuploid (for review, see [9,10]). Some features of meiosis during female gametogenesis, such as the prolonged resting phase in MI (dictyate) may predispose the oocyte to chromosome missegregation and the transmission of aneuploidies to the offspring. The rate of embryo aneuploidy increases with advanced maternal age. On the other hand, failure to form a crossover during oocyte development contributes to aneuploidy, regardless of maternal age [5,6]. Although aneuploidy affects roughly 1
2% of sperm cells and is less frequent than in oocytes (Fig. 5.1), some polymorphic variants on chromosomes seem to affect meiotic segregation during spermatogenesis (for review, see [11,12]). The variants most frequently associated with infertility are related to chromosomes 1, 9, and Y; polymorphic alterations in chromosome 9 are correlated with higher rates of sperm aneuploidy, poor semen quality, and infertility in men, while Y chromosome polymorphisms are more frequent in azoospermic and oligozoospermic men than in normospermic men [13]. Although aging is slower in the testes than the ovaries, age can diminish

Fertility, Pregnancy, and Wellness. DOI: https://doi.org/10.1016/B978-0-12-818309-0.00016-2 © 2022 Elsevier Inc. All rights reserved.

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PART | I Background

FIGURE 5.1 Gametogenesis. Numerical chromosome disorders include duplication or loss of entire chromosomes, as well as changes in the number of complete sets of chromosomes. They are caused by nondisjunction, which occurs when pairs of homologous chromosomes or sister chromatids fail to separate during meiosis. (A) Spermatogenesis. a. Normal spermatogenesis. a0 . Aneuploidy in spermatogenesis. (B) Oogenesis. b. Normal oogenesis. b0 . Aneuploidy in oogenesis.

proper function of the male reproductive organs [8]. Advanced age in men has been correlated with a higher rate of XY sperm, which increases 3.5% per year. This abnormality results in a XXY karyotype, corresponding to Klinefelter syndrome [14]. However, there is no consensus on the potential effect of paternal age in Klinefelter syndrome. For other autosomal aneuploidies, such as trisomy 21 (Down syndrome), 22, 13, 14, and 15, the father’s contribution has been estimated at 5% (trisomy 21), 11% (trisomy 22), 12% (trisomy 13 and 15), and 17% (trisomy 14) [15]. Psychiatric and developmental disorders, such as autism spectrum disorder, schizophrenia, epilepsy, and intellectual disability, are also related to paternal age. These disorders are often associated with de novo mutations, mostly due to de novo single-nucleotide variants (dnSNVs), but the risk of such mutations in spermatozoa is unknown [16]. Risk factors that increase the

de novo mutation rate include advanced paternal age, local genomic architecture containing segmental duplications, and genetic variation; the combined de novo mutation frequencies equal the risk of Down syndrome [17] Since age and lifestyle are strongly correlated, it can be difficult to distinguish maternal and paternal effects. Environmental factors are found worldwide, so some studies have shown the effects of lifestyle on fertility. Adverse environmental factors such as aging, oxidative stress, obesity, smoking, alcohol, stress, poor diet, and sedentary lifestyle may affect human reproduction. The rate of sperm aneuploidy may be higher in men who consume large quantities of alcohol and caffeine or pesticide factory workers. Sperm DNA can also be affected by nonionizing radiofrequency radiation, while smoking may cause oocyte spindle abnormalities [5,18]. Endocrine-disrupting compounds may interfere with hormone-regulated cell

Genetics and epigenetics of healthy gametes, conception, and pregnancy establishment Chapter | 5

signaling pathways and affect the expression of genes that regulate reproductive functions, and oocyte aneuploidy may be caused by bisphenol A [5,19].

Gametic mitochondrial DNA and embryo quality Assisted reproductive technologies (ARTs) have made considerable progress over the years but outcomes for in vitro fertilization (IVF) remain relatively low [20,21]. Embryo viability is mainly assessed by morphology and morphometric scores, which are imprecise and subjective. Several invasive and noninvasive selection methods can directly or indirectly determine an embryo’s implantation potential, but even despite this selection a significant proportion of euploid embryos will not result in live births. Various hypotheses have been suggested to explain implantation failure, such as endometrium receptivity, increased progesterone levels, and more recently, energy production by the mitochondria, which is essential for embryo development [22
26]. Mitochondria (for review, see [27,28]) are cytoplasmic organelles, and vary in number according to cell type and energy demand; in other words, the more energy required, the greater the number of mitochondria [27,28]. Among eukaryotic cells, oocytes have the most mitochondria. Mature human oocytes (MII) contain approximately 100,000 mitochondria; their levels increase considerably during oogenesis, but do not change after fertilization until the implantation stages are complete. The energy produced by the mitochondria [adenosine tri-phosphate (ATP)] is needed for fertilization and early embryo development [21
25,29]. For this reason, assessing oocyte competence may be useful to improve embryo selection for IVF [20]. Some researchers have shown that the number of oocyte mitochondria may change due to aging, body mass index, cigarette smoking, and embryonic stress prior to implantation [25]. For this reason, mitochondrial efficiency may have a significant impact on embryo viability in terms of implantation potential [26]. According to St. John et al., oocytes with reduced mitochondrial DNA (mtDNA) copy number frequently fail to fertilize or arrest during early embryonic development [27]. Additionally, embryos under conditions that deviate from the steady state (such as aneuploidy, advanced maternal age, or chemically induced stress) tend to be associated with higher mtDNA content; in other words, these embryos are of poor quality and have less chance of developing until birth [28,30]. According to Fragouli et al. and Diez-Juan et al., one explanation for these findings could be that stressed embryos have fewer energy sources, and consequently compensate by resuming mitochondrial activation and replication to obtain the

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energy they need in the blastocyst stage [25]. On the other hand, embryos with “quiet metabolism” (the “quiet embryo” hypothesis; for review, see Leese, 2016) and more likely to implant would show lower mtDNA content [31]. Two recent studies have investigated mtDNA content for determining embryo quality [25,32]. Both noted that during the blastocyst stage, a greater number of mtDNA copies were associated with poorer embryo quality (for review, see [33
35]). Another study by Scott et al. found that mtDNA content in human euploid blastocysts is not predictive of reproductive outcomes [36]. Previous published studies indicating mtDNA as a good biomarker for embryo quality considered the relative rather than absolute quantification of mtDNA in trophectoderm (TE) cells [34,40]. Relative quantification permits comparison of the amount of mtDNA in different samples, but the exact copy number of each sample cannot be determined. Scott et al. [39] corroborates earlier publications showing that healthy babies were born from embryos with high mtDNA copy numbers [30,37]. Regarding earlier stages of embryo development, some studies were not able to use mtDNA as a parameter for embryo viability, since they analyzed a small sample of embryos [24,33]. In 2019, Bayram et al. assessed cleavage-stage embryos and ploidy status using mtDNA content; these authors found that mtDNA can be used as a biomarker of human embryo quality, and that early blastulation was correlated with lower mtDNA content and higher euploidy [22]. mtDNA can also be assessed using cumulus cells (CCs), an alternative and noninvasive method for evaluating oocytes and predicting embryo quality [20,38
40]. A number of discrepancies related to the use of mtDNA as a biomarker of embryo viability which can be seen in some studies result from experimental design, technical challenges of mtDNA quantification, patient-specific variables, and embryonic variables that can impair data analysis [21]. According to Cozzolino et al., mtDNA appears to be a better biomarker of poor embryo quality, rather than a means of selecting the best embryo to transfer [41]. The lack of consensus makes it difficult to draw definitive conclusions, and, consequently, the use of mtDNA as a biomarker of embryo quality in clinical practice remains unresolved and merits further investigation as a robust predictor.

The genetics of fertilization Fertilization is a highly complex sexual reproductive event that requires recognition and fusion between one sperm and one egg. Fertilization enables haploid gametes to recreate a genetically distinct individual and restore the normal number of chromosomes. This process is regulated by several signaling molecules, and basic genetic

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principles are well conserved through eukaryotic organisms. These mechanisms require a sequence of coordinated molecular events between the sperm and egg to guarantee that male and female gametes form a single diploid cell, the zygote. Gamete health is crucial for successful fertilization and early embryo development, and as a result alterations in gametogenesis may lead to poor fertility outcomes and major impacts on couple fertility [19,42]. An oocyte is ready for fertilization when it reaches the metaphase stage of MII. Meanwhile, only sperm cells that pass through the epididymis are mature, motile, and capable of fusing with the oocyte. There are three main stages in fertilization: sperm capacitation and acrosome reaction, sperm-egg binding and fusion, and cortical reaction and oocyte activation. Briefly, sperm capacitation occurs in the female genital tract and prepares sperm cells for the acrosome reaction. This process includes flagellar hyperactivation (removal of inhibitory factors such as glycoproteins, seminal plasma proteins attached to the surface of the sperm cells, and depletion of membrane cholesterol) without morphological changes. For the acrosome reaction to occur, spermatozoa must recognize specific glycoproteins in the zona pellucida, called ZP3, which are specific to each species [43]. The contents of the acrosome (peptidases) are then released, digesting the ZP and allowing the sperm membrane to fuse to the oocyte membrane. Sperm-egg binding and fusion triggered by sperm binding to ZP3 involve several molecules such as fertilins (α and β), a disintegrin and metalloproteinases (ADAMs) 1 to 3, Izumo (sperm cells), and Juno (oocytes). After sperm-egg fusion, the sperm nucleus is incorporated into the oocyte cytoplasm. The oocyte membrane undergoes depolarization (through an influx of sodium ions) and cortical granules promote ZP hardening; both events are essential to create a permanent barrier to sperm entry and impede polyspermy. The last phase of oocyte activation is resumption of MII, polar body (PB) extrusion, and zygote cell division (for review, [42,44
46]). As we have seen, mammalian fertilization is a robust process, but the molecular apparatus intrinsic to each step is not yet fully understood. Failures during any component of the gametogenesis or fertilization process may consequently lead to abnormal fertilization and impaired embryo development. Some of the factors that can affect fertilization may stem from failures in the molecular bases of cell division during gametogenesis, while lifestyle, endocrine, immunological, oncological, and congenital factors may also compromise gametogenesis and, in turn, fertilization events [44,46,47]. Errors in the division of germ cells lead to chromosome abnormalities and can cause health problems in offspring, as we have seen. Chromosome aberrations are detected in more than half of all human spontaneous

abortions; no autosomal monosomy and only a few autosomal trisomies (13, 18, and 21) can survive to term. Approximately 84% of all embryonic trisomies are found to derive from female meiosis. Embryos with sex chromosomal aneuploidies, such as 45,X, and 47,XXY do not survive to term in approximately 95% and 45% of cases, respectively, while most 47,XYY embryos seem to survive in all pregnacies. Over 50% of all sex chromosome aneuploidies are thought to result from nondisjunction in male meiosis. On the other hand, structural chromosome rearrangements are 6% less frequent than numerical abnormalities in cases of spontaneous abortion [5,6,8,14,15].

Embryonic genome activation After fertilization, a zygote is formed, with both genetic and epigenetic contributions from the paternal genomes. At the beginning of development an embryo has totipotent cells, which have the potential to generate an entirely new individual. Early embryo development is marked by two main events: active maternal degradation of messenger RNAs (mRNAs) and proteins that were stored in the oocyte, and embryonic genome activation (EGA), namely emancipation from the maternal genome and replacement with the products of the embryo. Prior to EGA, embryo development is orchestrated by maternal-effect genes, in the absence of RNA transcription. EGA is species specific, but is not specific to vertebrates. The maternalzygotic transition has been studied for decades in various organism models [48
51]. The transition from silence to widespread gene expression requires precise regulation. Several EGA promoters and transcription factors contribute to maternal mRNA clearance by activating factors that mediate mRNA degradation. Maternal transcript degradation is driven by RNAbinding proteins (RBPs). The first RBP identified was Smaug (SMG), in Drosophila. In zebrafish, the involvement of some microRNAs (short regulatory RNAs, often 6
8 nucleotides) has been identified in the maternal pathway of transcript degradation. Further studies in Xenopus laevis, Caenorhabditis elegans, and mice found another type of RBP related to maternal RNA clearance called adenylate-uridylate-rich elements (AU-rich elements, or ARE) and a molecule known as the bicoid stability factor (BSF). There are various conjectures as to why maternal products degrade; some researchers believe that clearing the maternal transcript allows its embryo counterpart to have a broader spatial expression profile. Others describe an instructive function, with maternal RNAs degraded to restrict their function or remove transcripts that are no longer needed, preventing abnormal mRNA dosages in the embryo. An alternative hypothesis is that maternal products must degrade to delete the old and highly

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differentiated program which is replaced by the embryonic program. In any case, the molecular mechanisms of maternal transcript degradation remain to be determined [50,51]. For embryo development, transcription factors must access the DNA to start gene expression. After fertilization and during EGA, the onset of histone exchange allows a permissive and loosely packed chromatin structure in the embryo. Histone acetylation is associated with chromatin accessibility and active transcription, and in many species it increases around EGA (for review, see [52,53]). Some transcription factors such as RNA polymerase II (Pol II) and preinitiation complex (PIC) are located in the oocyte cytoplasm, and transcription and gene expression only begin after their nuclear localization in the early embryo. In Drosophila, zygotic transcription is induced by a transcription factor known as Zelda (Zld); in zebrafish, EGA is controlled by Pou5f3, Nanog, and Sox19b (for review, see [51]). These factors are believed to open the chromatin structure for high transcription. This hypothesis is not entirely accepted, however [48,51,54]. Studies of these transcription factors in human embryonic stem cells (hESCs) have indicated that factors such as ARGFX, CPHX1, CPHX2, LEUTX, and DUX5 activate EGA, but these factors still need to be investigated in human embryos [55,56]. The exact mechanism that regulates each step of gene expression remains to be determined, but failures in these processes are known to potentially result in abnormal cell differentiation into somatic cell types (such as neurons, muscles, hair, or teeth), as manifested in teratomas. Some RBPs are responsible for maintaining the germ cell and inhibiting precocious reprogramming; examples include DND-1 (dead end 1; mouse) and GLD-1 (defective in germ line development 1; Caenorhabditis elegans). However, EGA is a complex event and we are only beginning to understand its mechanisms [48].

Embryo implantation Embryo implantation occurs about 7 days after fertilization when the embryo reaches the blastocyst stage and hatches from the zona pellucida. At this time the luminal endometrial epithelial cells are able to interact with the blastocyst. This process occurs over 4
6 days during the mid-luteal phase of the menstrual cycle, and is known as the implantation window (for review, see [57]). An embryo passes through different stages of blastocyst development before implanting into a receptive endometrium. The TE, which gives rise to the placenta, differentiates into the proliferative cytotrophoblast (CTB) and syncytiotrophoblast (STB). The inner cell mass (ICM), which becomes the embryo itself, differentiates into epiblast and primitive endoderm cells [58
60].

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Implantation is a process that begins with apposition, continues through attachment of STB outgrowth to the underlying stroma (blastocyst invasion), and ends with decidualization (for review, see [57,61]). The molecular mechanisms of implantation are poorly understood because of the limitations of both animal and in vitro models, but some adhesion molecules and extracellular matrix proteins have been suggested to mediate the first steps of embryo implantation. Some adhesion molecules like L-selectin are present in the TE, and may allow the embryo to initially attach to the endometrium. Other molecules such as integrins and transmembrane glycoproteins function as links between the extracellular matrix and the cytoskeleton [58]. MicroRNAs are emerging as important factors in human embryo implantation (for review, see [61
63]). Human blastocysts are known to release microRNAs that bind to complementary regions of mRNAs, inhibiting translation or destabilizing the gene. The human endometrium also expresses a large number of microRNAs, facilitating endometrial receptivity and embryo implantation. Diferencial expression of micro RNAs have been reported in implanted and nonimplanted embryos and in euploid vs aneuploid blastocysts. Because of the complexity of microRNA signaling, studies so far have been inconsistent in terms of reproducibility of results, and additional research is required [61,64]. The success of pregnancy is based on communication between the endometrium and embryo during the implantation window. During this period, it is important to consider embryo quality and the receptiveness of the endometrium. When these factors are altered, cross-talk between embryo and uterus may be impaired and implantation may fail. The most common complication of pregnancy is miscarriage, ending roughly 15% of gestations; despite technological advancements, the reasons for approximately half of miscarriage cases are still unclear [69]. Although not all the elements that impede endometrial receptivity have been recognized (for review, see [65]), they include female factors (impaired endometrial function, anatomic issues, thrombophilia, etc.), male factors (such as sperm chromatin fragmentation), immunological factors, lifestyle factors (e.g., smoking), genetics, and embryo-related issues [60,66
72]. In terms of genetics, chromosomal abnormalities are the main recognized genetic cause for miscarriage and may account for 60% of cases [73]. Chromosome aneuploidy and polyploidy constitute more than 96% of chromosomal abnormalities in spontaneous abortion and X, Y, 13, 16, 18, 21, and 22 are frequently involved [71]. In a retrospective study, Franasiak et al. demonstrated that the prevalence of aneuploidy is lower in patients between the ages of 26 and 30 (20
27%) than in aging patients in the 31
43 age range (85%) [74]. These same authors also

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determined that the complexity of aneuploid errors also increased with age, and found higher rates of monosomies in younger patients and more trisomies in older women. According to Colley et al., it is important to identify numerical chromosome errors (monosomy, trisomy, or polyploidy) since these can lead to miscarriage [69]. Yet it has been suggested that 86% of chromosomal abnormalities are numerical, 6% are due to structural alterations, and 8% result from other genetic alterations such as mosaicism [75]. More recently, a study of 1,106 embryo samples found that 59.4% had classic cytogenetic abnormalities [73]. These authors stated that aneuploidy accounted for 85.4% of these abnormalities, triploidy for 10.3%, and structural errors or tetraploidy for the remaining 4.2%. One year later, Jia et al. showed that trisomy 16 was most common among all the aneuploidies and polyploidies analyzed by this group (39.03%), followed by trisomy 22 and X monosomy [71]. In 2018, a retrospective analysis by Pylyp et al. found that approximately 50% of first-trimester miscarriages exhibited chromosomal abnormalities; of this group, 59% were trisomies, 22% were polyploidies, 7.5% were monosomies, 7% were unbalanced structural errors, and 3.8% resulted from multiple aneuploidies [72]. These same authors also noted that maternal age increased chromosomally abnormal miscarriages [75]. Apparently, normal euploid embryos may also fail to develop until birth; other genetic abnormalities may be involved in these cases of pregnancy loss, such as singlenucleotide variants (SNVs) that affect individual genes or clusters of genes (either as gains or losses) that are deleted, duplicated, or disrupted. This is a significant genetic alteration, since it may follow a recessive or X-linked mutation. It is important to note that little is known about the genes and pathways involved in pregnancy loss, and for this reason the clinical significance of many copy number variants are uncertain. Many genetic mechanisms may also be influenced by environmental factors such as diet, medication, pollutants, and lifestyle, which could lead to a cumulative effect culminating in pregnancy loss [69].

Mitochondrial diseases The central role of the mitochondria in eukaryotic cells is to produce energy (ATP synthesis). However, this organelle is also essential for other cell functions such as regulating apoptosis, calcium handling, biogenesis of iron-sulfur clusters, fatty acid degradation, innate immunity, and phospholipid synthesis. Because mitochondria participate in various functions, they are more prone to dysfunction in tissues with greater energy demand (namely the brain, heart, muscle, liver, kidney, and central nervous system). Mitochondrial dysfunction that causes

severe disease may affect 1 in 4300 people worldwide [76
78]. Mitochondria have their own DNA, which is believed to have come from bacteria that underwent an endosymbiotic event with an early eukaryote. The mammalian mtDNA genome has 13 protein-encoding genes that are pivotal for oxidative phosphorylation (OXPHOS; for review, see [77]), which is responsible for cell energy production. To translate these protein-encoding genes, mtDNA also has two ribosomal RNAs (rRNA) and 22 transfer RNAs (tRNA) [76,79]. mtDNA is often exposed to a powerful source of oxygen free radicals, which together with histone deficiency makes it vulnerable to mutations. Mutated mtDNA causes a blended population of normal and altered mtDNA (heteroplasmy) [80]. Since the entire mitochondrial genome is involved in OXPHOS, increased mtDNA mutation leads to altered ATP production. To guarantee optimal function in energy generation, mitochondria exhibit dynamic properties such as fusion, fission, and degradation (autophagy). The interplay of fusion and fission (for review, see [76,81]) is beneficial to the mitochondria, since it promotes homogenization of the organelle population and helps ameliorate the adverse effects of heteroplasmic mtDNA mutations (coexisting normal and mutated mtDNA). For this reason, when fusion and fission are inhibited serious organelle dysfunction can result. In some cell types, a severe decrease of mitochondrial fusion leads to abnormal OXPHOS activity. On the other hand, the degradation of mitochondria is important to control its quality (for review, see [82]). Once mitochondria demonstrate altered function, they are directed to the lysosome for degradation [76]. Mitochondria are inherited from the maternal line, but their pattern is poorly understood. Moreover, mtDNA variations between the mother and offspring and even between siblings are known in higher quantities than expected from one generation to another. This means that a female with heteroplasmy can give birth to a homoplasmic child carrying only the mutant mtDNA. These markedly variable levels of mutated mtDNA between offspring in the same generation are described in what is known as the mitochondrial bottleneck hypothesis, which explains the sudden changes in allele frequency seen during transmission from one generation to the next (for review, see [83]; Fig. 5.2) However, the mechanisms that govern the mitochondrial bottleneck are not fully understood [79]. Several pathologies are caused by mitochondrial dysfunction. Mutated mtDNA is not frequently found in populations worldwide but may increase with aging and is associated with a broad range of incurable congenital disorders. Nondividing cells such as neurons and skeletal and cardiac muscle tend to accumulate mtDNA mutations [80]. To alter cell function and lead to

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FIGURE 5.2 The mitochondrial bottleneck hypothesis. During oogenesis, a selected number of mitochondrial DNA (mtDNA) molecules are transferred into each oocyte. During oocyte maturation this mtDNA population replicates quickly, which can lead to a blended population of normal and altered mtDNA (heteroplasmy). Possible repair mechanisms are fission, fusion, and degradation; fission and fusion promote homogenization of the mitochondrial population. Degradation of mitochondria by lysosomes is important to control the quality of these organelles.

disease, the percentage of mutated heteroplasmic mtDNA must exceed 60%
80% or reach homoplasmy (a single mtDNA variant that may be mutant or wild-type) [77,84]. Disease severity depends on the proportion of inherited abnormal mtDNA molecules and the affected tissue; symptoms may vary from mild to fatal and commonly affect the nervous system, where tissue has high energy demands. Unfortunately, palliative treatment rather than cure remains the standard of care. The best treatment for so-called heteroplasmic disease at this time is to avoid mutated mtDNA through generations [77,84]. When oocytes come from a patient with known high levels of mutated mtDNA, mitochondrial replacement therapy (MRT) so far is the only strategy to deliver a healthy baby (for review, see [79]). This technique is based on nuclear transfer from the patient affected by the mtDNA mutation to an enucleated recipient oocyte that has

nonaffected (“healthy”) mitochondria. This same procedure is feasible for zygotes at the pronuclear stage [pronuclear transfer (PNT)]. Both techniques have shown adverse results, with 15% of cases reverting back to the homoplasmic donor mtDNA [77
79,82,84]. Moreover, because this procedure involves DNA transfer it is considered genetic manipulation, raising a variety of ethical, social, theological, and safety concerns (“three-parent children”; for review, see [85
87]). Oocyte donation is a strategy to address all maternally transmitted mtDNA diseases, since it reduces the risk of transmission to the prevalent population. Another possibility for reducing transmission of mitochondrial diseases is the use of preimplantation genetic testing (PGT), which has been used to evaluate mtDNA mutation during early embryo development. To assess the mutant load of embryos produced in vitro, 1
2 or 5 cells (cleavage-stage

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embryos or blastocysts, respectively) can be removed by biopsy. However, this technique is feasible only for embryos with low or no levels of mtDNA mutations [77,82]. The feasibility of PGT for those embryos is partly due to the threshold effect of some mtDNA mutation, which means that there are no symptoms unless the mutant load exceeds a certain level; this cut-off point varies between tissue types as well as different mutations. Other concerns related to PGT in mtDNA disorders concern the possibility that the mutation load could increase with time, variable mutant load among embryonic cells (mosaicism), and the strong impact of the bottleneck effect [77,88
90]. For this reason, it is not clear whether the biopsied blastomeres or the TE can precisely represent the entire embryo, or whether PGT can guarantee prevention of mtDNA diseases in embryos with low mutation loads [91,92].

Epigenetic modifications Since they were first described by Conrad Waddington (1942), the epigenetic mechanisms that control gene expression have been extensively investigated, due to the important role these mechanisms play in cell growth and differentiation, providing cellular identity during mammalian development. During differentiation, the epigenetic landscape modifies the genetic information from the totipotent zygote to a differentiated multicellular organism [93
95]. Once differentiated, in the somatic cell the epigenetic memory is maintained stable during mitotic cell division [96]. Disruption of epigenetic programing (epimutation) is related to the pathophysiology of many human diseases, including growth and developmental disorders, the process of carcinogenesis, type 1 diabetes, and neurological and cardiovascular diseases [97]. Epigenetic marks such as DNA methylation, histone modification, and noncoding RNA (ncRNA) regulation promote chromatin remodeling, leading to differential gene expression without changes in the DNA sequence and resulting in transcriptional activation and/or silencing [93,94]. DNA methylation is a well-understood epigenetic mark in the human genome; the main modification occurs with the addition of a methyl group (
CH3) at cytosine (C) residues in a cytosine-phosphate-guanine (CpG) dinucleotide, resulting in 5-methylcytosine (5-mC) [98]. Usually, 5mC is a repressive epigenetic mark, and DNA cytosine-5 methyltransferases (DNMTs) are a conserved family of enzymes responsible for this modification [99]. Five DNMTs are encoded in the human genome: DNMT1, DNMT3A, and DNMT3B are a group of canonical methyltransferases with catalytic activity. The methylation pattern is maintained by DNMT1 during replication, while DNMT3A and DNMT3B are responsible for the de novo establishment of DNA methylation in

hemimethylated and/or unmethylated CpG sites. The noncanonical methyltransferases, DNMT2 and DNMT3L, demonstrate sequence conservation but do not have catalytic DNMT activity [100]. The global mammalian genome is CpG-depleted and hypermethylated, while the promoter regions and regulatory sequences are rich in CpG dinucleotides termed CpG islands (CGI), which are usually hypomethylated. Loss of DNMT function leads to global hypomethylation and genomic instability, as well as the activation or repression of important genes [101,102]. DNA methylation is essential for gametogenesis and embryo development, and directly affects pregnancy rates [103,104]. The paternal and maternal epigenome have distinct DNA methylation patterns; the male genome is mostly methylated and covers 90% of the spermatic genome, while 40% of the mature oocyte genome is methylated and most germline CGIs are differentially methylated regions (DMRs) [105]. Before activation of the embryonic genome, the oocyte form of DNMT is required to maintain DNA methylation in preimplantation embryos in specific genomic regions in early stages [99]. DNA methylation is a dynamic process throughout development [106]. Passive DNA demethylation occurs in the absence of DNMTs during replication, but active demethylation is catalyzed by the ten
eleven translocation (TET) protein family, in which 5-mC is oxidized to 5-hydroxymethylcytosine (5-hmC) [107]. Even though hydroxymethylation is not abundant in the human genome (B10% of 5-mC) and seems to be a transient epigenetic mark, it plays an important role in tissue-specific gene regulation during differentiation [107]. Methylation in adenine, N6-methyladenine (N6-mA) DNA modification, is also reported in the mammalian genome and seems to be a repressive epigenetic mark responsible for silencing long interspersed nuclear element (LINE) transposons in mouse embryonic stem cells (ESC) [108]. The role of N6mA in gametogenesis and embryo development is still poorly understood. Histone modifications are a posttranslational epigenetic mark also responsible for chromatin remodeling and control of gene expression. Nuclear chromatin is a complex structure of DNA wrapped around two copies each of histones H2A, H2B, H3, and H4, an octameric nucleosome core linked by the histone H1. The histone proteins package the DNA in the transcriptionally repressive heterochromatin, and can be modified by the addition of chemical groups to the tails of these nucleosome-proteins [109,110]. Many chemical modifications have been reported, such as ubiquitination, phosphorylation, deamination, and sumoylation, but acetylation and methylation are the most studied and are closely linked to enhancers and promoter regions. Histone modifications change the chromatin to a transcriptionally active or inactive state,

Genetics and epigenetics of healthy gametes, conception, and pregnancy establishment Chapter | 5

directly affecting gene expression. Acetylation is usually linked to functionally active chromatin, and methylation is related to both active and inactive genes [111,112]. Aberrant posttranslational modifications (PTMs) in histone acetylation and/or methylation during spermatogenesis alters protamination, an essential process for sperm maturation with implications for male infertility and embryo development [113]. In the maternal epigenome, these modifications are essential to maternal-to-zygote transition in the EGA [114]. Epigenetic modification in the histone proteins and chromatin organization is a dynamic and well-controlled process that takes place across the entire genome, and is directly related to early development, implantation, and pregnancy success [115]. However, histone PTMs in preimplantation embryos are still poorly understood due to technical issues (such as small amounts of molecular material). One epigenetic mechanism which has been studied the most is the regulation of gene expression by ncRNAs. The human genome has a wide variety of ncRNAs, since 80% of these molecules are transcribed but not translated into functional proteins; they can be divided into micro(miRNAs) and long ncRNAs (lncRNAs, .200 nt), which regulate posttranscriptional gene expression in binding to RNA or DNA and PTM in association with proteins (ribonucleoproteins) [116
118]. The regulatory network of miRNA is complex, targeting many human genes or transcripts, which in turn regulates a variety of biological processes including cell growth, proliferation, and differentiation [116,119]. The lncRNAs interact with DNAshaping chromosome conformations, such as the XIST (X inactive specific transcript) transcript expressed in the inactive X-chromosome [120]. Besides X-chromosome inactivation (XCI), lncRNAs are also involved in genomic imprinting phenomenon, and as a result aberrant expression of these transcripts is closely linked to growth and developmental disorders [117]. Recently, epigenetic modifications at the RNA level have added even greater complexity to the study of the epigenome. Adenine methylation in the mRNA, N6methyladenosine (m6A), is the most abundant epitranscriptome modification, usually located in regulatory sequences. Modification of m6A regulates splicing control, export, stability, and translation of mRNA, as well as the synthesis and function of microRNAs and lncRNAs [121,122]. Because of the important role it plays in regulating gene expression, m6A methylation is required for embryo development by controlling cell fate transition, and deletion of RNA methyltransferases in mouse ESC is related to lethality [131]. The implications of m6A for human development have been poorly investigated [122]. High-throughput strategies have facilitated the understanding of epigenetic modifications during early life, a complex network of interactions with spatial and temporal

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control of gene expression. The disruption of epigenetic marks has considerable consequences for human biology and long-term health in adulthood. Considering the dynamic and variable nature of the epigenome and epitranscriptome, additional research is required on risk factors and specific epimutations in humans, especially during gametogenesis and embryo development. The limiting factor for use of epigenetic modifiers as a biomarker for embryo development is the yield of genetic material to quantify DNA methylation in TE biopsies for PGT, and consequently few studies have been published in this area [123]. The gold standard technique for DNA methylation analysis is bisulfite modification of CpG dinucleotides via next-generation sequencing (NGS). While unmethylated cytosines are converted to uracil, 5mC remains unaffected, and after PCR amplification the uracil (U) is read as a thymine (T), which is identified by genomic DNA sequencing techniques [124]. Highthroughput NGS is an accurate and sensitive method, but additional sequencing (covering) is required for the epigenome to obtain representative results after TE biopsies. This increases costs, especially for diagnosis or prevention of imprinting disorders in the newborn after ART [123] Most studies regarding whole genome bisulfite sequencing (WGBS) in preimplantation embryos have utilized animal models, but recently, increased global DNA methylation (hyperventilation) was observed in aneuploid human blastocysts after IVF treatment, with higher methylation levels in embryos from older parents [125]. As a biomarker of embryo viability, the epigenome and correlation with implantation and pregnancy rates may not be sensitive enough to improve diagnosis and therapeutic strategies; furthermore, the intrauterine environment and maternal exposures may also affect epigenetic marks in fetuses during pregnancy and disease in adulthood.

Genomic imprinting and developmental disorders Genomic imprinting is an epigenetic process characteristic of placental mammals, in which only one member of an allele pair (maternal or paternal) is expressed in a parentof-origin manner, and consequently both maternal and paternal genomes are required for normal development [126]. Approximately 107 human genes have been confirmed to be imprinted and many others are predicted or still unconfirmed [137]. In most tissues these genes are usually located within gene clusters with a conserved pattern of expression in humans and other mammals (such as mice and bovines); the placenta is an exception, with its own pattern of expressing some imprinted genes [127
129].

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This non-Mendelian monoallelic gene expression is crucial to normal human development and placentation, and loss of imprinting (LOI) is consequently related to developmental disorders, fetal growth disturbances, and carcinogenesis [130,131]. Regulation of imprinting gene expression is complex and coordinated at the genomic level [129]. The main epigenetic mechanism behind this control is DNA methylation. The imprinted loci or imprinting control regions (ICRs) contain DMR depending on paternal or maternal origin. The DNA methylation pattern of ICRs are determinate during gametogenesis and must be maintained after fertilization and embryo development. Even so, a complex molecular interaction network including posttranslational histone modifications, DNA binding proteins, and noncoding RNAs is part of the epigenetic control of imprinted loci [132,133]. Normal development requires both maternal and paternal contribution of imprinted genes [130]. LOI or biallelic expression of imprinted genes in the human chromosome 11p15.5 region is related to Beckwith-Wiedemann (BWS) and Silver-Russell syndromes, which involve congenital malformations and predisposition to tumors [134]. This domain contains the KvDMR1 (ICR2) and H19DMR (ICR1) regions, responsible for the imprinting regulation of several genes involved in placental and embryonic growth and development [134
136]. KvDMR1 is a telomeric region methylated on the maternal allele (Fig. 5.3). This DMR is located in a promoter region of the paternally expressed lncRNA KCNQ1 overlap transcript 1

(KCNQ1OT1) that overlaps within an intronic region at the maternally expressed potassium voltage-gated channel subfamily Q member1 (KCNQ1). Other important genes are controlled by the KvDMR1 region, such as the maternally expressed Cdkn1c, Slc22a18, Ascl2, Phlda2, and Tssc4 genes in mice [136]. In humans, only CDKN1C, SLC22A18, and PHLDA2 seem to be imprinted with maternal gene expression, while ASCL2 and TSSC4 are biallelic [137]. The H19DMR controls imprinted expression of the lncRNA H19 and insulin-like growth factor 2 (IGF2) (Fig. 5.3). The genomic DMR is located in a centromeric region upstream from the H19 gene, and expression depends on a shared enhancer. The maternal unmethylated allele contains a CTCF binding domain, an enhancer-blocking or insulator element that prevents IGF2 activation, and for this reason H19 is maternally expressed. On the paternal allele, methylation induces changes at the H19 promoter that is silenced, and the insulator activity is blocked, consequently permitting IGF2 activation by the paternally expressed enhancer [137]. The lncRNA H19, despite its tumor suppressor characteristics, may also act as an oncogene during carcinogenesis, and is a key gene in cancer initiation and progression [138]. Prader-Willi and Angelman syndromes are imprinting disorders characterized by neurodevelopmental alterations and epigenetic mutations on imprinted genes at 15q11q13. Several genes are controlled by this region,

FIGURE 5.3 Schematic representation of human imprinting control regions KvDMR1 (ICR2) and H19DMR (ICR1) at human chromosome 11p15.5 and targeting strategy. The KvDMR1 is methylated on the maternal allele. The differentially methylated region (DMR) is located in a promoter region of the KCNQ1 overlap transcript 1 (KCNQ1OT1) that is paternally expressed, while KCNQ1 and CDKN1C are maternally expressed. The H19DMR region is located near the H19 gene, and controls expression of this gene as well as IGF2. Control of gene expression depends on a shared enhancer. The maternal unmethylated allele contains a CTCF binding protein that prevents IGF2 expression, and H19 is maternally expressed. Paternal methylation blocks the insulator activity and H19 is silenced, allowing IGF2 activation by the paternally expressed enhancer. Black circles 5 methylated; White circles 5 unmethylated.

Genetics and epigenetics of healthy gametes, conception, and pregnancy establishment Chapter | 5

such as the paternally expressed SNURF-SNRPN sense and the maternally expressed UBE3A genes [139]. Transient neonatal diabetes mellitus (TNDM) is related to LOI of maternally imprinted PLAGL1/ZAC (6q24), characterized by intrauterine growth retardation (IUGR), hyperglycemia, and congenital abnormalities. Multiple congenital disorders and growth alterations (such as central precocious puberty) have identified epimutations in imprinted genes [131]. Other molecular alterations like uniparental disomy (UPD) inheritance, chromosomal rearrangements, and genomic mutations in imprinted genes are observed in imprinting disorders, and the phenotypic outcome depends on the related epigenetic alteration. Since chromosome rearrangements lead to UPD, with the homologous chromosomes inherited from only one parent, it is important to identify the parental origin of aneuploidy in PGT for aneuploidies (PGT-A) in order to prevent clinically relevant diseases such as imprinting disorders. NGS strategies like single-nucleotide polymorphism (SNP) array or whole genome sequencing may be performed during PGT to detect genetic variants in the embryo [131,140].

Epigenetic reprogramming in the gametes and embryo Since the genetic information is the same, epigenetic marks are responsible for cellular identity that is maintained after differentiation through an inherited epigenetic memory. The epigenetic landscape modifies the genetic information from a totipotent zygote, controlling cell fate transition to a differentiated cell lineage from a multicellular organism. During human development, genome-wide reprogramming of epigenetic marks is responsible for establishing pluripotency and differentiation [94,96,141]. Global epigenetic reprogramming occurs during gametogenesis in the germ cells, and after fertilization in preimplantation embryos (Fig. 5.4). Most research on epigenetic reprogramming in germ cells utilizes mouse models, and this process has not been studied in detail in humans. During embryonic development prior to sexual determination, the epigenetic marks in PGCs are erased, with extensive genome-wide DNA demethylation, chromatin remodeling, and loss of the genomic imprinting pattern. The lowest level of methylation in PGCs (6
7%) is observed around 10
12 weeks of gestational age, and male and female epigenetic marks are established at different times during spermatozoa and oocyte maturation [142]. During spermatogenesis, establishment of global DNA methylation and genomic imprinting marks occurs during the first part of male germ cell differentiation, in the prospermatogonia stage that precedes mitosis/meiosis. Chromatin remodeling via histone modification and

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ncRNAs plays an important role in spermatozoa maturation, especially the replacement of histones with protamine in mature sperm [142]. Protamine proteins are essential for maturation of the spermatozoa nucleus, facilitating chromatin hypercompaction and in turn preventing DNA fragmentation. Aberrant epigenetic reprogramming and protamination during spermatogenesis are related to a number of negative reproductive outcomes such as reduced fertilization capacity, impaired embryo development, and miscarriage [143]. In the female epigenome, DNA methylation is established after birth during folliculogenesis and oocyte growth. However, oogenesis is a complex process that begins in early embryo development and continues during puberty, with maturation only complete after fertilization [144]. In humans, the DNA methylation pattern seems to be established during the germinal vesicle stage for most CpG sites and continues later in development, especially in the CpG-rich domains where methylation may happen in MII oocytes [145,146]. During meiotic oocyte maturation in mice, histone deacetylation and increased gene expression are essential for oocyte competence and embryonic development [142,145]. Since the oocytes are responsible for initiating embryo development, altered DNA methylation patterns (especially in maternally imprinted genes) are related to many diseases in adult offspring, and also may result in embryo lethality [144]. Proper reprogramming of oocytes and spermatozoa is required to establish maternal and paternal genomic imprinting [145]. The dynamics of epigenetic reprogramming in humans have been extensively investigated through single-cell sequencing techniques. The second round of epigenetic reprogramming occurs in preimplantation embryos, where the paternal genome undergoes genome-wide demethylation, chromatin accessibility, and replacement of sperm protamines by acetylated histones after fertilization. These epigenetic changes contribute to the acquisition of totipotency and early embryo transcriptional activation to generate a multicellular and complex organism [96,145]. Both active and passive DNA demethylation is observed in early embryos, and 5-hmC and TET expression seem to be involved in embryonic reprogramming and pluripotent ESCs [107]. Maternal-to-zygotic transition (MZT) requires maternal mRNA and protein degradation and de novo DNA methylation in the embryo. The major transcriptional activity in human embryos is observed during the eight-cell stage in the EGA, which is accompanied by microRNA and lncRNA transcription and activation of endogenous retroviruses and repetitive elements, important regulators of gene expression. Despite global DNA methylation, the imprinting pattern established during gametic reprogramming must be maintained throughout preimplantation development

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FIGURE 5.4 Epigenetic reprogramming in mammalian development. Primordial germ cells (PGCs) are demethylated, including genomic imprinting erasure, early in development. Male remethyalation and imprinting establishment begins early, in the prospermatogonia stage, while female reprogramming occurs in growing oocytes after birth. After fertilization, male and female genome are demethylated depending on DNA replication, and the embryonic methylation pattern is established at implantation, in different manners in embryonic and extraembryonic cells. Preimplantation reprogramming appears to take place with EGA, around the 4
8 cell stage. In this round of reprogramming, the maternal and paternal imprinted pattern must be maintained according to parental origin, and other epigenetic marks are reprogrammed in somatic cells for proper embryonic and fetal development.

[96,142]. Maintenance of the methylation pattern in these regions in preimplantation embryos seems to require the somatic form of DNMT1 from the maternal pronuclei [144]. Disruption of epigenetic programming in early life can influence fetal development and placentation, leading to infertility, developmental disorders, and increased risk of gestational complications and miscarriage [147].

Environmental factors and epigenetic changes Conrad Waddington, in his studies on the canalization of development, was the first to report how genomic

information interacts with the environment to change cellular identity and how these changes are inherited and fixed throughout epigenetic memory during cell division [94]. Unlike the genome, the epigenome has significant plasticity and the specific program of gene expression of a particular cell can be reprogrammed [141,145]. Epigenetic reprogramming can be affected by many factors including genetic mutations and chromosomal alterations, leading to epimutations and disruption of tissue-specific gene expression. Gene mutation or altered expression of the cell machinery responsible for the establishment and maintenance of epigenetic marks such as DNMTs, TET, and histone modifier enzymes, as well as

Genetics and epigenetics of healthy gametes, conception, and pregnancy establishment Chapter | 5

regulatory ncRNAS, is related to aberrant epigenetic reprogramming and has various implications for human health. Environmental factors like age, health status, and lifestyle have a major influence on the epigenome, changing the cellular landscape [148]. Placental and intrauterine exposures to such environmental factors during early life and fetal development are related to aberrant epigenetic reprogramming in the offspring, leading to an increased risk of congenital disorders and long-term health issues in adulthood [147,149]. Recently fetal origins have been proposed to explain adult diseases, in which the epigenetic programming of gene expression is thought to be modified by intrauterine environmental conditions. Disturbance during implantation and placental development leads to pregnancy-related complications such as infertility, preeclampsia, intrauterine growth restriction, and recurrent pregnancy loss [150,151]. Intrauterine disturbances are also related to increased risk of metabolic disorders, type 2 diabetes, obesity, and cardiovascular disease in adulthood [152,153]. Because of the essential role they play in cellular growth and differentiation-related pathways, epimutations are also related to congenital malignancy and tumorigenesis, for example the increased risk of developing Wilms’ tumor in BWS [154,155]. Assistive reproductive techniques such as in vitro maturation of oocytes, controlled ovarian hyperstimulation, IVF, embryo culture, and cryopreservation of gametes and embryos are associated with altered epigenetic reprogramming and birth defects, principally imprinting disorders in newborns. As mentioned, BWS and Silver-Russell syndrome are rare genetic diseases with growth alterations and congenital formations that are more frequent in children conceived via ART [156]. These technologies also influence the development of metabolic and endocrine alterations in infants and adults [142]. Understanding epigenetic modifications during early life and fetal development, as well as the mechanisms underlying cell differentiation and totipotency, adds important information to aid in developing new treatment strategies. Furthermore, the potential use of induced pluripotent stem cells in regenerative medicine through epigenetic erasure of cellular memory, as well as epigenetic reprogramming of cancer cells using epigenetic drugs in treatment, contribute to general knowledge [142,157]. Research on epigenetic reprogramming during gametogenesis and embryogenesis and the associated risk factors for human disease is also important to improve ARTs and reduce epigenetic defects in offspring.

Conclusions Genetic and epigenetic mechanisms are essential for gametogenesis, fertilization, and proper embryo

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development and successful pregnancy as well as health in adulthood. Chromosomal abnormalities are the main recognized genetic causes of failures in embryo development and miscarriage. Most aneuploid human embryos result from errors during gametogenesis, with altered parental chromosomal rearrangements during meiotic cell division, especially in the oocytes. Although aneuploidy affects sperm cells, polymorphic variants and increased DNA fragmentation are the most common genetic alterations in spermatozoa. Euploid embryos may also fail to develop until birth, and some genetic aberrations such as SNVs and single gene mutations may be involved in these cases of pregnancy loss. Disruption of epigenetic reprogramming during gametogenesis and prior to implantation also leads to impaired fertilization and embryo development. Epimutations are related to many human endocrine and metabolic diseases, carcinogenesis, and growth and developmental disorders such as imprinting syndromes. Many factors can affect epigenetic programming of tissue-specific gene expression, including genetic mutations, chromosomal aberrations, and environmental factors like age, health status, and lifestyle. Despite advances in ARTs, the associated technologies may modify the embryonic epigenome, increasing the risk of imprinting disorders. Gametogenesis, fertilization, and early embryo development are well-controlled and complex events in which genetic and epigenetic factors directly impact pregnancy success and offspring health. Lifestyle changes may prevent impaired gametogenesis and errors during early embryo development in naturally conceived children. Advances in the NGS techniques used for PGT can also improve pregnancy and healthy live birth rates.

Acknowledgments We wish to thank the members of the Experimental Oncology Laboratory at the Federal University of Ceara´’s Drug Research and Development Center (NPDM), and Dr Nathan Treff for the review.

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PART II

Fertility - IIA

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Chapter 6

A contemporary view on global fertility, infertility, and assisted reproductive techniques Natalibeth Barrera1,2, Temidayo S Omolaoye3 and Stefan S Du Plessis4,5 1

In vitro Fertilization Laboratory, Montevideo Sterility Center, Montevideo, Uruguay, 2Andrology Research Laboratory, Reprovita (Reproductive

Medicine Division of Fertilab Laboratory), Montevideo, Uruguay, 3College of Medicine, Mohammed Bin Rashid. University of Medicine and Health Sciences, Dubai, United Arab Emirates, 4Basic Medical Sciences, College of Medicine, Mohammed Bin Rashid University of Medicine and Health Sciences, Dubai, United Arab Emirates, 5Medical Physiology, Faculty of Medicine and Health Sciences, Stellenbosch University, Tygerberg, South Africa

Introduction Fecundity is an integral part of human life which makes reproduction and childbirth pivotal. From 1800 to 1900, fertility was not regarded as a global health issue because the fertility rate among women who wanted to have children ranged from 5 to 7 children per woman across different populations and regions of the world (Fig. 6.1A) [1,3]. Despite that a slight decline started to occur between 1900 and 1950, the total global fertility rate was 5.4 children per woman. Still, fertility was not yet regarded as a global health problem. However, by the late 1970s, the decline in fertility was striking as the median number of children was calculated to be 3.5 children per woman (Fig. 6.1B, C, and D) [4]. During the period 2010
15, it was established that fertility reached an all-time low when the worldwide median fertility rate fell to 2.5 children per woman. On a global scale, the average number of births per women declined by 44% over a 40-year period, from 4.5 births per woman in 1970
75 to 2.5 births per woman in 2010
15. At regional level significant differences in fertility rates are observed. These variances could be ascribed to lifestyle, environmental, and socioeconomic factors. During the same period (1970s to 2010
15) across different regions of Asia, the total fertility rate dropped from 5.1 children per woman to 2.2 children per woman (
57%), across South America from 5.0 children per woman to 2.2 (
56%), in Central America from 6.5 children per woman to 2.4 (
60%), across different regions of Oceania from 5.6 children per woman to

3.2 (
43%), in Europe and North America from 2.8 children per woman to 2.1 (
25%) and in Africa from 6.7 children per woman to 4.7 (
30%). Thus Africa, except for South Africa, remained the continent with the highest fertility rate in 2010
15 (4.7 children per woman) with predominantly high levels in sub-Saharan Africa (5.1 births per woman) (Fig. 6.2) [2]. It is generally accepted that replacement-level (average number of children born per woman at which a population exactly replaces itself from one generation to the next) is 2.1 children per woman. In 2015, 46% of the world’s population lived in areas where the fertility rate was less than that; including Europe, Northern, Southern and Central America, Asia, and parts of Australasia. Another 46% lived in countries where the fertility rate ranged between 2.1 and ,5 children per woman; these included parts of Oceania. Only 8% of the world’s population lived in high fertility countries ( . 3.5 children per woman) which were mostly African countries, except for South Africa. A summary of the percentage of global total fertility rates at different time points are provided in Fig. 6.2 [2]. The decline in fertility rates observed during the previous decades and predicted to be further exacerbated in the near future, is experienced in both males and female alike. Extrapolating from the existing data, it is expected that by the year 2100 the fertility rate would further decline to ,1 child per woman. Hence, fertility has become a global health threat as many fear that this trend will ultimately lead to humans becoming extinct [5].

Fertility, Pregnancy, and Wellness. DOI: https://doi.org/10.1016/B978-0-12-818309-0.00009-5 © 2022 Elsevier Inc. All rights reserved.

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A

C No of children per woman

No of children per woman

5.8 5.75 5.7 5.65 5.6 5.55 5.5 1780

1800

1820

1840

1860

1880

1900

2.8 2.75 2.7 2.65 2.6 2.55 2.5 2.45 2.4 1995

1920

D

B 6

2010

2015

2020

8 7

No of children per woman

No of children per woman

2005

Total fertility rate trend (2000 -19)

Total fertility rate trend (1800-99)

5 4 3 2 1 0 1880

2000

6 5 4 3 2 1

1900

1920

1940

1960

1980

2000

2020

Total fertility rate trend (1900-99)

0 1750

1800

1850

1900

1950

2000

2050

Decline in fertility over the years USA

Europe

Africa

Asia

FIGURE 6.1 Summary of fertility rate from 1800 to 2019. (A) Total fertility rate from 1800 to 1899. (B) Total fertility rate from 1900 to 1999. (C) Total fertility rate from 2000 to 2019. (D) Decline in fertility rate over the years. Adapted from Rosling O. Gapminder_data [Internet]. 2017. Available from: https://github.com/Gapminder-Indicators/tfr/blob/master/tfr-by-gapminder.xlsx%0A; United Nations, Department of Economic and Social Affairs PD. World Fertility Report 2015 (ST/ESA/SER.A/415) [Internet]. 2017. Available from: http://www.un.org/en/development/desa/population/publications/fertility/world-fertility-2015.shtml.

Definitions of infertility From the Longman Dictionary of Contemporary English, infertility is defined as the inability to have babies [6]. However, in the scientific community, infertility is yet to have a uniform definition as authors tend to explain it with regards to the features displayed by the population they are studying. The World Health Organization (WHO) and the International Committee for Monitoring Assisted Reproductive Technology (ICMART) defines infertility from the epidemiological approach, as the failure to achieve a clinical pregnancy after 12 or more months of regular unprotected sexual intercourse [7,8]. As mostly used by obstetricians, gynecologists, and reproductive endocrinologists in the United States, infertility is defined as the inability of a woman under or over 35 years of age to conceive after 12 or 6 months of contraceptive free sexual intercourse, respectively [9]. In the United Kingdom, the National Institute for Health and Clinical Excellence (NICE) guideline defines infertility as the failure to conceive after regular unprotected sexual

intercourse for 2 years in the absence of known reproductive pathology [10]. Demographers define infertility as the absence of a live birth after 5 years of a noncontracepting sexually active woman [11].

Prevalence of infertility Due to the various existing definitions of infertility, it is challenging to make a general estimate of global infertility as the outcomes are measured differently, and this allows variation in estimating infertility prevalence [11
13]. The global prevalence rate for 12-month infertility was reported by Boivin et al. to range from 3.5% to 16.7% in more developed countries and from 6.9% to 9.3% in less-developed nations [14] following data extrapolation from Olsen et al. and Karmaus and Juul [15
17]. Boivin et al. further estimated that globally, 72.4 million women are infertile, of which 40.5 million seek medical help [14]. Following an application of current duration approach in measuring the prevalence of infertility in low-to-middle-income countries, another

A contemporary view on global fertility, infertility, and assisted reproductive techniques Chapter | 6

95

70

Percentage (%)

60 50 40 30 20 10 0

≥5

4