Clinics in Reproductive Medicine and Assisted Reproductive Technology [First Edition] 9789354669385

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Clinics in Reproductive Medicine and

Assisted Reproductive Technology Volume 4

Clinics in Reproductive Medicine and

Assisted Reproductive Technology Volume 4 BN Chakravarty MO (Cal), FRCOG (Lond), DSc (hon)

Director Institute of Reproductive Medicine Kolkata, WB India

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Disclaimer Science and technology are constantly changing fields. New research and experience broaden the scope of information and knowledge. The authors have tried their best in giving information available to them while preparing the material for this book. Although, all efforts have been made to ensure optimum accuracy of the material, yet it is quite possible some errors might have been left uncorrected. The publisher, the printer and the authors will not be held responsible for any inadvertent errors, omissions or inaccuracies. eISBN: xxxx Copyright © Authors and Publisher

First eBook Edition: 2022 All rights reserved. No part of this eBook 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 authors and the publisher. Published by Satish Kumar Jain and produced by Varun Jain for CBS Publishers & Distributors Pvt. Ltd. Corporate Office: 204 FIE, Industrial Area, Patparganj, New Delhi-110092 Ph: +91-11-49344934; Fax: +91-11-49344935; Website: www.cbspd.com; www.eduport-global.com; E-mail: [email protected]; [email protected] Head Office: CBS PLAZA, 4819/XI Prahlad Street, 24 Ansari Road, Daryaganj, New Delhi-110002, India. Ph: +91-11-23289259, 23266861, 23266867; Fax: 011-23243014; Website: www.cbspd.com; E-mail: [email protected]; [email protected].

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Representatives Hyderabad Pune Nagpur Manipal Vijayawada Patna

to

Dr Subhas Mukherjee Pioneered delivery of first IVF baby in India and second in world and

Dr MN Parikh Founder President of ISAR Who by initiating a solid and stable official organization brought India to a respectable position in the world map of IVF

The author appreciates the services and active support received from his following students during the course of the preparation of the manuscript of this volume. Tushar Kanti Das Sakti Rupa Chakraborty Ratna Chattopadhyay Sanghamitra Ghosh Sunita Sharma Shovandeb Kalapahar Sabnam Parvin

Preface to Volume 4

T

he fourth volume of the book Clinics in Reproductive Medicine and Assisted Reproductive Technology is now complete and ready for publication. I am delighted that almost 50% of the chapters are co-authored by those who, once upon a time, were my students and now are considered to be the experts in their respective disciplines. I am also thankful to the guest authors, Prof Hiralal Konar MBBS, MD, DNB, MNAMS, FACS, FRCOG, for Thyroid and Reproduction: Basic and Clinical Aspects; Prof Subhankar Chowdhury DTM&H, MD, DM, MRCP; and Dr Partha Pratim Chakraborty MD, DM, DNB, FACE, for Thyroid and Reproduction: Endocrinological and Clinical Aspects; Prof Sanjay Ghosh MBBS, MD (DERM & VEN) and Dr Saurav Kundu MD (DVL) . Their contributions enormously upgraded the prestige of this book. This volume contains many chapters interlinking the disorders related to reproduction with endocrinopathy, metabolic problems, dermatology, developmental errors, oncology, etc. Additionally, obesity and cryopreservation of gamete and gonadal tissue included in this volume are two important topics that hold future areas of research to address many unresolved challenges in reproductive medicine. Besides, the chapters like hirsutism and the associated underlying disorders have remained the topic of combined disciplines like endocrinology, dermatology, gynecology, and oncology. Though complicated, an attempt has been made to simplify the problems of hirsutism in three different chapters: basic (more physiological), clinical aspects and more importantly the cosmetic part of the problem. With the advent of the new technology of diagnostic modalities and therapeutic approaches, recent workers have taken a renewed interest in the embryogenesis and classification of urogenital anomalies. This topic has been of interest since my younger days, and has been discussed with a new clinical classification based on my own experience. Customarily it was known that the onset of puberty varies with the geographical location of an individual’s habitation. For example, Eskimos have relatively delayed puberty compared to those living near the equator, like South Asians and Africans. With the alteration of nutrition and lifestyle globally, this phenomenon is also now changing. The general onset of puberty has advanced more or less universally. Two chapters; one on precocious puberty and the other on puberty will help clarify some of the mystiques and miracles of this transitional period of a girl between the immaturity of childhood and gradually approaching maturity of adulthood through semi-maturity of adolescence. Like other disciplines, imaging technologies play an invaluable role in different areas of reproductive medicine including ART, both in diagnostic and therapeutic areas. The related topics have been adequately elaborated in the chapters, namely “Ultrasound Imaging in Female Intertility Management” and “Assessment of Endometrial Receptivety by Ultrasound” by Dr Sunita Sharma MD, FNB, Dr Sanghamitra Ghosh and Dr Kamal Oswal MD. In near future, cryobiology will be a central pillar in many areas of research and development of the science of reproductive medicine. Dr Shovandeb Kalapahar MS, DNB, FNB one of our students and now a junior colleague has assisted in contributing to the basic chapters “Cryopreservation and vitrification: Laboratory aspects” and “Embryo transfer”. Dr Ratna Chattopadhyay MBBS, PhD, has highlighted the importance of In vitro Maturation (IVM) in ART.

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For writing the sequential volumes of this book, I got the initial incentive from my junior colleague Dr Gita Ganguli Mukherjee MBBS, DGO, MD, FICOG, FRCOG; who keeps on inspiring me for the addition of chapters especially containing my own experiences. Further in this journey, I have received constant encouragement from my colleagues and juniors like Prof BB Hore, DA, MS; Prof BB Sarkar MO; Dr Subhas Halder MD; Dr Biman Kumar Ghosh FRCOG, and many other coworkers to continue my work till the last day of my life. I offer my s p e c i a l t h a n k s t o Prof Syed N Kabir PhD ; Dr Pratip Chakroborty PhD and Dr Sanghamitra Ghosh MBBS; for their help and assistance in providing me with some constructive criticisms during the compilation of different chapters of this volume. My wife, Dr Manjusree Chakravarty, as usual, inspires and enjoys all my activities. I thank and appreciate all the assistance from Late Ashis Shit (no more with us), M r Sushanta Chakraborty, Mr Arup Ranjan Sarkar, Dr Tushar Kanti Das PhD and Dr Sakti Rupa Chakraborty MBBG, DGO, MD, FICOG, for their tireless efforts in compiling by rewriting the manuscript and, thereafter, revising each chapter thoroughly. I will be happy, if the contents of this volume are of benefit to the readers.

BN Chakravarty

Preface

ix

Preface

A

round 1950s, soon after the Second World War, there were numerous innovations in the vast field of medical science. As a consequence, several superspecialties were identified for the purpose of specialised training and treatment. Most of them have already been recognised for postdoctoral training and specialisation. Examples include cardiology, ophthalmology, otolaryngology, paediatrics, radiology and oncology. On the other hand, recognition of reproductive medicine, a newborn superspecialty in medical curriculum, was relatively late. This discipline, as a superspecialty, was well-established following the advent of assisted reproductive technology (ART). Though science of embryology was documented as early as 1875 by Oscar Hertwig in Germany, it was a difficult subject to study in humans. At that time most knowledge of human reproduction was achieved through animal experiments. Scope of observation and research directly in humans has only been possible following the introduction of assisted reproductive technology in the treatment of infertility. The process of gametogenesis, their maturation, sperm egg interaction, fertilisation and implantation are a few of the major information which have been derived through clinical application of assisted reproduction. Endocrine background of human reproduction was realised much earlier from late 1920s. Fertility enhancing drugs like gonadotropin, clomiphene and bromocriptine, in addition to fertility preventing drugs like oral contraceptives were discovered and introduced for clinical use around the early 1960s. Knowledge and expertise for clinical use of these hormones expanded further with introduction of gadgets and technologies like ultrasound, laparoscope, RIA and EIA. These technologies have opened up avenues for better understanding of physiology at molecular level of folliculogenesis and ovulation, pathophysiology of PCOS, precocious puberty, premature menopause and many others. ART has brought about an unprecedented revolution in the treatment of human infertility. The procedure has not only provided help in relieving the distress of childless couples but also has opened up avenues of potential research. One of them is stem cell research which is heading towards another medical discovery through tissue engineering and has already established the foundation stone of a new superspecialty—regenerative medicine. Apart from stem cell research, ART has also provided novel protocols for fertility preservation during and preceding oncotherapy in young cancer victims, both boys and girls. In 1990s, there has been a breakthrough in the treatment of male infertility through introduction of intracytoplasmic sperm injection (ICSI) procedure. However, till now we do not know the exact treatment of male infertility because in ICSI gametes are treated, but not the individual. Andrology and spermatology are expanding fast and it is expected that many obscure areas in male reproduction will be explored in the near future. Expansion of genetic and immunological knowledge has widened our views on amenorrhoea, recurrent miscarriage and sexual ambiguity. In spite of all these advances in science and technology, ART still has practical limitations, primarily in four areas which have become a source of concern for the clinician.

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These are (a) cost, (b) complexities of treatment, (c) complications and (d) results, at least the prediction of outcome. Clinicians and researchers alike have joined hands to overcome these problems. Enormous volumes of publications have accumulated over the years discussing ways and procedures for solving these persistent deficiencies of ART through development of good quality embryos, generating effective endometrial receptivity and finding ways for performing smooth and atraumatic embryo replacement. Though to some extent some of the objectives have been achieved, yet many more remained elusive. Recent publications emphasise more on optimising stimulation protocol, redirecting approaches for mild ovarian stimulation, a move towards single embryo transfer, reducing embryo stress by introducing metabolomics in culture system, time-lapse embryoscope, and attempting to predict endometrial receptivity through non-invasive markers like uterine fluid, follicular fluid components and many others. From the academic point of view, it is apparent that comprehensive knowledge in reproductive medicine demands a sound background of different branches of basic science and their intelligent application in clinical medicine. Unfortunately, in our medical postgraduate teaching and examinations, these two aspects of the same superspecialty have been segregated. For example, in MD (Obs and Gynae) examination, the emphasis is more on clinical aspect, whereas the PhD course syllabus and training has been oriented more on the basic aspect rather than its clinical application. There is an urgent need for bridging the gap between the two. However, as far as I am concerned, during my professional career which covers nearly a period of five decades, I had the privilege of being continuously associated with medical teaching, both undergraduate and postgraduate. This uninterrupted commitment has helped me immensely in keeping myself updated with contemporary advances, both basic and clinical aspects of my respective discipline. I have been teaching reproductive medicine in fellowship and PhD course for the last 20 years. Over the years I have updated and upgraded my teaching slides with contemporary information and novel experiences that I had gathered during my clinical practice. This has provided some opportunities for me to unify both basic and clinical aspects together which I learnt during the last decade of my teaching career. The incentive for writing a book came from my students whom I taught and from my colleagues with whom I have worked from the beginning of my career. Nevertheless, one of my ambitions was not fulfilled owing to my busy schedule during my working years. I could hardly get some time to concentrate and write a book, which I felt was a difficult task for me. The chapters of this book have been written in the way in which I teach my students—meaning thereby that each topic has been discussed from different angles. For example, the topic of PCOS has been covered in 5 different lectures: a. Adolescent PCOS—current management strategy b. Pubertal metabolic and endocrine changes—their relevance to adolescent PCOS c. Overview and management of PCOS d. Optimising ovarian stimulation of PCOS patients e. Changing concept in PCOS. Each of these lecture notes has been converted into an individual chapter. To accommodate all these lecture notes, it was not possible to include them in a single publication. Therefore, it has been decided to publish the book in three or four different volumes. My primary interest in infertility and subsequently in the discipline of reproductve medicine was created by my renowned colleague late Prof Subhas Mukherjee MBBS, DGO, PhD (Edin)

Preface

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who had pioneered the delivery of first ‘test-tube baby’ in India in 1978 and the second in the world. My initial experience in ART was gathered through a small team of doctors (all my students) organised by me following the tragic death of Dr Mukherjee in 1981. The team primarily consisted of Dr Sudarshan Ghosh Dastidar, Dr Siddhartha Chatterjee, Dr Arup Kumar Majhi, Dr Sourendra Kanta Goswami, Dr Bani Kumar Mitra, Dr Sanghamitra Ghosh and Dr Ratna Chattopadhyay. For writing this book I got continuous encouragement from Prof Dr Gita Ganguly Mukherjee MD, FICOG, FRCOG; Prof BB Hore MBBS DA, MS (Cal); Prof Subir Kumar Dutta MBBS, DCP, MD (Cal); Prof BB Sarkar MBBS, DGO, MO (Cal); Dr Biman Kumar Ghosh MBBS, DGO (Cal), DRCOG, EPA, FRCOG; Prof Hiralal Konar MD, DNB, FRCOG, FICOG; Dr Arup Kumar Majhi MBBS; DGO, MD (Cal); and all my students, present and past. I express my deep appreciation to Dr Ratna Chattopadhyay, Dr Sourendra Kanta Goswami, Dr Sanghamitra Ghosh, Dr Radhika Kandula, Dr Rita Modi, Dr Geetha Rani BS, and Dr Anwesha Ghosh for their meticulous care and painstaking effort and for their assistance in repeated corrections, criticism and revision of my manuscript. I acknowledge and sincerely thank Dr Ratna Chattopadhyay and Dr Pratip Chakraborty for their individual contribution of two chapters on ‘Nutraceutical in Male Infertility’ and ‘Insulin Resistance’ respectively in this publication. I also thank and appreciate the efforts and devotion of my computer assistants Mr Ashis Shit, Mr Arup Ranjan Sarker and Ms Ria Chakraborty who have worked continuously and retyped the corrected manuscripts time and again. Lastly, I have no words to appreciate the silent help offered by my wife Dr Manjusree Chakravarty, who has spared me from my domestic commitments and has continuously encouraged me to express my ideas through writing a book which might help the future generation. I will be only too happy if the contents of the current and future volumes of this book are of benefit, both for information and for practice of the students and practitioners in reproductive medicine, for whom the book has been compiled.

BN Chakravarty

Contents Preface to Volume 4

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Preface

ix

1 Puberty

1

2 Precocious Puberty

12

3 Polycystic Ovary Syndrome and Infertility

30

4 Azoospermia

42

5 Unexplained Spontaneous Miscarriage

60

6 Obesity: Definition and Etiology

70

7 Obesity and Reproductive Health: Molecular and Clinical Aspect

76

8 Hirsutism: Basic Aspects

90

9 Hirsutism: Clinical Aspects

96

10 Hirsutism: Cosmetic Aspects

119

11 Thyroid and Reproduction: Endocrinological and Clinical Aspects

125

12 Mullerian Anomalies: A New Clinical Classification and Overview

144

13 Ultrasound Imaging in Female Infertility Management

158

14 Assessment of Endometrial Receptivity by Ultrasound

173

15 In vitro Maturation: Overview

182

16 Embryo Transfer

188

17 Endometrial Preparation in Frozen Embryo Transfer Cycle

201

18 Mitochondrial Transfer: Current Status

212

19 Cryopreservation and Vitrification: Overview

226

20 Cryopreservation and Vitrification: Laboratory Aspects

237

21 Oncofertility: Science of Collaboration Between Reproductive Medicine and Oncology

256

22 Thyroid and Reproduction: Basic and Clinical Aspects

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Index

277

1

Puberty BN Chakravarty

INTRODUCTION

GnRH in a pulsatile fashion which stimulate gonadotroph cells in pituitary. These cells, in turn start secreting follicle stimulating hormone (FSH) and luteinizing hormone (LH), also in a pulsatile rhythm. Pituitary gonadotropin stimulates maturation of ovarian follicles. Granulosa and theca cells of the ovary, under influence of FSH and LH, also start secreting in a similar pulsatile manner. To stimulate ovarian steroids (estrogen and progesterone) in a similar pulsatile manner. Consequently, the pattern of pituitary gonadotropin and gonadal steroid secretion during the entire period of life, foetal, infancy, childhood, adolescence and adulthood will reflect changes in the activity of the hypothalamic pulse-generator.

Events Preceding Advent of Puberty Puberty is a transitional period between immaturity of childhood to semimaturity of adolescence. Apart from genetic influence, endocrinological, nutritional and environmental factors primarily initiate and regulate the advent and onset of puberty. Endocrinological factors primarily initiate—nutritional and environmental factors subsequently control and regulate advent and onset of sequential events of puberty. Endocrinological modulation through hypothalamic-pituitary-ovarian (HPO) axis is the primary trigger which initiates and coordinates different physiological and physical changes of puberty from intrauterine (foetal) to pubertal development of human life. The initiative starts from hypothalamic pulse generator, located in hypothalamus.

Pattern of gonadotropin and gonadal steroid secretion from intrauterine life till onset of puberty: Hypothalamic neurons which synthesize GnRH originate primarily in the olfactory placode and migrate to the hypothalamus during 6–9 weeks of gestation.1 By 10 weeks, hypothalamus contains significant number of GnRH neurons. Meanwhile, hypothalamicportal venous system develops from 9–10 weeks and the development is completed by 19–20 weeks. GnRH secreted by hypothalamic neurons can now be transported through hypothalamic-pituitary portal venous system to stimulate pituitary gonadotropin release. Foetal serum gonadotropin rises progressively

What is Hypothalamic Pulse-Generator? The term “hypothalamic pulse-generator” was coined by Ernst Knobil. The hypothalamic pulse generators are clusters of neurohormonal secretory cells (specialized) located in t h e arcuate nucleus in the medial basal hypothalamus. The number is approximately 1500 to 2000. These are known as GnRH neurons. They have ‘autorhythmicity’. They perform their function as oscillator for the pulsatile release of GnRH. Under the influence of ‘signal’, they start secreting 1

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reaching a peak between 20 and 24 weeks.2 Thereafter, the level starts declining progressively during the last 10 weeks of pregnancy possibly because of negative feedback effect of placental oestrogen and progesterone.3–5 After birth, following a small gap of persisting negative feedback of placental steroid effect, FSH and LH in the newborn start rising again (Fig. 1.1). This is because auto-rhythmicity of neonatal GnRH pulse-generator starts functioning again following removal of negative feedback effect of placental sex steroid hormones. The characteristic pulsatile pattern of GnRH function now emerges.5,6 The serum gonadotropin level (both FSH and LH) rise again. Type of gonadotropin rise has a specific sex difference. FSH rises to a greater extent in females and LH in males. 7,8 The maximum rise reaching to a peak is observed about 3–6 months in boys and 12–18 months in girls. The gonadotropin starts to decline thereafter presumably because normal feedback mechanism becomes fully functional. By

approximately 9–12 months of age in boys and 24–36 months in girls, gonadotropin concentration reaches to a typical pre-pubertal level remaining at a very low concentration till the advent of puberty.8 The suppressing effect is more intense in boys than in girls which probably reflects the influence of testosterone on hypothalamic programming.9 Advent of Puberty Biological expression of interlinked hormones (neurohormone-GnRH), protein hormones (FSH and LH) and sex steroid hormone (estrogen and progesterone) remain suppressed during childhood and early puberty. With advent of puberty, the expression of these hormones become apparent both biochemically and clinically. The controlling mechanism of this dramatic change is not very clear. Previous theory which tried to explain ‘Juvenile pause’ which precedes puberty, speculated, a hypothalamic ‘gonadostat’ was probably controlling the level of ‘sensitivity’

Fig. 1.1: Pattern of GnRH release (GnRH pulse generator); functioning from intrauterine life till onset of puberty

Puberty

of the central negative feedback action of gonadal steroid. The view was based on the fact that changing pattern of gonadotropin secretion depends on changes in the gonadostat setting, i.e. primarily on the gonadal steroid (estrogen) feedback mechanism. But it is becoming more clear that the typical ‘biphasic pattern’ of gonadotropin secretion from infancy to puberty is primarily because of changing level of central inhibition of pulsatile GnRH secretion and to an insignificant extent from a high sensitivity to low level of gonadal steroid feedback. In other words, the control of ‘pulsatile release’ or ‘inhibition’ is more on central factors and not on simple ‘gonadal steroid’ feedback mechanism as was explained by ‘gonadostat’ theory. The central factor has a neuroendocrine switch and this switch controls the GnRH pulse-generator. The switch when ‘on’ will release GnRH, pituitary gonadotropin

3

and ovarian steroids in a pulsatile fashion and when the controlling switch is ‘off’—the entire chain becomes non-functioning. Some of the factors governing the ‘neuroendocrine switch’ for the GnRH pulsegenerator have now been identified. The list of factors which modulate the activity of hypothalamic-pituitary gonadal axis include both ‘inhibitory’ and ‘excitatory’ neurotransmitters and peptides. In summary, it appears that primary controlling factors for ‘initiation’ and ‘inhibition’ of HPO axis for pulsatile release of GnRH are centrally located ‘neurotransmitters’ and ‘peptides’. ‘Negative ovarian steroid feedback mechanism’ has minor role to play. Neuroendocrine Factors Controlling Neurotransmitters and Peptides Some of these factors (neurotransmitters and peptides) (Fig. 1.2) governing the functioning

Fig. 1.2: Control of GnRH pulse-generator activity; central-peripheral-genetic-familial

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of HPO axis have been identified and they are briefly outlined as follows: • Gamma amino butyric acid (GABA): It is an inhibitory neurotransmitter. Experimental evidences have suggested that declining concentration of GABA at the onset of puberty helps to accelerate increasing release of pulsatile GnRH. In other words, central GABA signaling is one of the factors which inhibits GnRH neuronal activity during childhood. • Neuropeptide (NPY): This is also a hypothalamic peptide involved in the control of food intake behaviour and reproductive function in adults. Majority of experimental evidences have suggested that NPY, like GABA is an important component of ‘neurobiologic break’ that inhibits activity of GnRH in the pre-pubertal period.10 However, the observation was not substantiated by other observers who hold an opposite view.11 Additional work will be required to verify the role of NPY in regulation of hypothalamic pulse-generator and the onset of puberty. • Glutamate: This is an excitatory neurotransmitter and stimulates release of pulsatile GnRH both in vivo and in vitro.12 Experimental evidences13,14 suggest that glutamate stimulates resurgence of pulsatile GnRH release at the onset of puberty. • Kisspeptin: Kisspeptin is a neuropeptide (encoded by kiss-1 gene) and like GnRH neurons, are resident in hypothalamus. Evidences suggest that Kisspeptin signaling might play a major role in resurgence of pulsatile GnRH secretion at puberty. 15 Melatonin produced by the pineal gland also regulates the production of kisspeptin, and synthesized mostly in the hypothalamic tissue. Also, decreased melatonin blood levels lead to increased kisspeptin. These are two different views of controlling the level of kisspeptin for the developmental of puberty. The results of subsequent studies in non-human primates and also in human, strongly support this view.

Neurons expressing kiss-1 gene are located exclusively in arcuate nucleus16,17 where GnRH neurons also express their neurohormones.18 Hypothalamic kisspeptin secretion is distinctly pulsatile and highly correlated with GnRH.19 The biological expression of kisspeptin regulates pulsatile GnRH secretion.20 It has also been observed that kisspeptin might only amplify and not stimulate GnRH pulsegenerator directly.15 In summary, it is more or less accepted that kisspeptin signaling is the key component of the neurobiological mechanism that triggers the onset of puberty. Kisspeptin neurons provide the ingredients for functioning of the hypothalamic GnRH pulse-generator. These are all about central activity of kisspeptin in initiating pubertal resurgence of hypothalamic pulsatile GnRH release at puberty with activation of HPO axis. There is another mechanism through which kisspeptin controls advent of puberty. It has been suggested that kisspeptin mediates the negative feedback action of both testicular and ovarian hormones. There are evidences which suggest that kisspeptin neuros play an important role in the negative feedback loop that regulates hypothalamic GnRH secretion in the male which also involves the opioid and GABA neuronal input.21 Similar activities of kisspeptin have also been observed in the ovarian steroidal feedback mechanism. Hence, it is obvious that kisspeptin neurons are critical components of neurohormonal mechanism which activate and regulate hypothalamic pulse-generator at puberty. Impact of Peripheral Signaling In addition to ‘central’ and ‘loop feedback’ mechanism, GnRH pulse generator is also controlled by peripheral signaling. Since last few decades, the age of onset of puberty has been declining steadily. This may be also due to increasing prevalence of obesity. It is possible that critical body weight 22 or body composition23 may be important factors in determining the timing and progression of puberty. The negative impact of fasting (anorexia nervosa) is well known. Possibly the impact of

Puberty

malnutrition and lean body weight negatively affects pulsatile release of hypothalamic GnRH secretion.24 Impact of Leptin Adipocyte (fat) cells produce leptin. Leptin may provide metabolic signals and indirectly, have peripheral control on the higher center, regulating the activity of the hypothalamic pulse generator at the onset of puberty. The clinical observations suggests that leptin plays an important, but only permissive role in the onset of puberty. However, available evidences suggest that leptin might have the ability to influence or modulate the activity of the hypothalamic GnRH pulse generator.9 Other Metabolic Signals In addition to leptin, various other metabolic signals have been implicated in the process of nutritional regulation for the normal function of hypothalamic GnRH pulse generator. Some of the metabolites which provide these signals are—insulin, ghrelin (endogenous ligand of growth hormone secretagogue with a small role of energy balance),25 gallamine like peptide (potential neuronal target of leptin and free fatty acid).26 However, the exact mechanism in which these peripheral impulses regulate release of pulsatile GnRH and onset of puberty is not known.

Clinical Landmarks of Puberty Puberty is a period of immense physical, physiological, endocrinological and psychological changes. The physical and physiological changes are regulated primarily by endocrine maturation and partly by environmental and ethnic variation. Physiological changes are totally dependent on a combination of physical, endocrinological and psychological changes. Although the timing of appearance may vary, the sequential events of puberty generally follow a predictable pattern. The sequence of events are briefly summarized in following paragraphs: Adrenarche: The first physical indicator of puberty is adrenarche. This means activation and stimulation of adrenal androgen. Adrenal androgen originates from zona reticulosa of

5

adrenal cortex. It is significant to realize that adrenarche is independent of maturation of hypothalamic-pituitary gonadal axis, but the two (GnRH and ACTH) often are temporarily interrelated.27 The steady increase in adrenal androgen stimulates growth of pubic hair (pubarche) and activates pilosebaceous unit consisting of hair follicles and sebaceous gland. 27 Androgen increases bone density, suggesting that androgen also contributes growth of cortical bone.28 Adrenarche precedes maturation of HPO axis by 2 to 3 years. This observation initially suggested the idea that ‘adrenarche’ stimulates ‘gonadarche’ or in other words, adrenarche has the fundamental role for the ‘onset of puberty’. But other clinical evidences do not support this view. The contradictory evidences are: • Premature ‘adrenarche’ is not generally associated with earlier onset of thelarche or menarche. • Adrenarche occurs in those girls who have hypergonadotropic hypogonadism (gonadal dysgenesis), hypogonadotropic hypogonadism (Kallmann syndrome). • Even without adrenarche like Addison’s disease (hypoadrenalism) gonadarche occurs. • In precocious puberty under the age of six gonadarche precedes adrenarche. If adrenarche occurs earlier than gonadarche, then pubic hair should appear before ‘growth spurt’ and ‘thelarche’. But during normal pubertal development, pubic hair develops after growth spurt and thelarche. Possibly in adrenarche, weak androgen DHEA is secreted from zona reticulosa for which clinical impact (pubic and axillary hair) appears after full development of gonadarche, i.e. after the onset of growth spurt and thelarche. Growth Spurt ‘Growth spurt’ is an important landmark at puberty. Growth includes both in height and body composition. 17–18% of adult height is gained at puberty.29 Pubertal growth occurs usually two years earlier in girls than boys and in girls the peak velocity reaches approximately 6 months before menarche.30

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Accumulation of bone mass is critical during puberty. This is a major determinant of occurrence of osteoporosis in later life. Bone mass accumulates from circulating calcium. About one half of total body calcium is utilized during puberty in girls and one half to two third in boys.31, 32 In girls the maximum accumulation of bone mass occurs 9–12 months after peak height velocity has been reached. Apart from height velocity, pubertal weight changes, reflect deposition of lean body mass and fat. Adolescent girls have more body fat than boys—the maximum deposition occurs in upper arms, thighs and back. The difference in increase in girls and boys continues throughout puberty. The increase in BMI before the age of 16 is primarily due to increase in lean body mass and, thereafter, due to fat mass.33 Apart from genetic factor, pubertal growth spurt is primarily dependent on growth hormone, including IGF-1, nutritional factors and also on sex steroid. These factors are being briefly described below. Growth Hormone Growth hormone (GH) is synthesized and released by ‘somatotrophs’ (like gonadotroph) in the pituitary. It is also released in a pulsatile fashion. Release and rhythmicity are controlled by central (growth hormone releasing hormone) and peripheral negative feedback effect of IGF-1 (insulin-like growth factor 1) which inhibit GH release. The peripheral action of growth hormone on target cell is mediated through IGF-1. Other peripheral factors like nutritional or hormonal (oestrogen, glucocorticoids) also contribute to release and functioning of growth hormone. Fasting34 and ‘high’ protein meal35 stimulate GH release, whereas hyperglycemia and leptin inhibit GH secretion.35 Similarly oestrogen stimulates and excess glucocorticoids inhibit GH release. The peak rise of growth hormone is observed during puberty and thereafter declines with aging. Approximately every 7 years GH acts through stimulation of hepatic synthesis and secretion of IGF-1 which

accelerate bone growth and differentiation. GH is also involved in a number of metabolic factors, namely increase of lipolysis, stimulation of protein synthesis, insulin antagonism, water and sodium retention. Insulin Like Growth Factor 1 (IGF-1) IGF-1 is synthesized and released by liver in response to GH stimulation. IGF-1 circulates in serum in bound form—IGF binding protein (IGFBP). The family of IGFBP includes six proteins having greater affinity for IGF-1. The IGFBP-1 concentration is regulated by insulin, increases during fasting when insulin levels are low and decreases after fasting or administration of insulin.36 IGF-1 augments the effect of FSH and LH in the ovary—the effect of ACTH on adrenal steroidogenesis and also thyroid response to thyroid stimulating hormone (TSH). IGF-1 levels are low at birth; peak values are observed during puberty, thereafter, fall rapidly approximately by 50% by the age of 20 and then decline gradually as age advances.37 Impact of Sex Steroid Hormone Undoubtedly, pubertal growth is primarily dependent on growth hormone and IGF-1. But clinical evidences suggest that sex steroid hormones also play important role. This is evident from the fact that precocious puberty can induce a substantial growth spurt even in the absence of a normal pubertal increase in circulating hormone or IGF-1. Normally, pubertal ‘growth spurt’ requires combined action of sex steroid and growth hormone. It is already well known that sex steroids limit adult height by stimulating epiphyseal fusion. The Age of Onset of Puberty The triggering mechanism of onset of puberty has still remained unknown. Apart from genetic factor, the age of onset of puberty and the mechanisms are influenced by overall health, social environment and environmental exposure. Family history is also closely linked with early or delayed onset of puberty or menarche. Age of onset of puberty and menarche are often well correlated between mother and daughter and between sisters.38 Apart from

Puberty

family linkage, environmental factors play a great role. Girls living close to equator, at lower altitude in urban areas, and those who are mildly obese, generally begin puberty earlier than those who live away from equatorial region, at higher altitude, in rural areas and those who are of normal weight. Environmental pollution, which probably acts as endocrine ‘disrupters’ may also be involved in the timing of sexual development.38 The age of onset of puberty is gradually declining globally over the past few decades irrespective of location and ethnicity. The trend to an earlier onset of puberty and sexual development has been attributed to improved nutrition and stressful living conditions (Fig. 1.2).38 Early onset of puberty is directly associated with increased weight and higher body fat mass.39,40 Early pubertal development has two adverse impacts during adult life: (1) increase of obesity; and (2) decrease in adult height.41,42 The age of pubertal development, thelarche and menarche in India are 10.2 and 12.6 years respectively (1988 to 1991). Landmarks of Onset of Puberty— Sequence of Development There are basically four landmarks of onset of puberty. These are: (1) Growth spurt, (2) thelarche, (3) pubarche, and (4) menarche— onset of menses.

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Growth spurt in preadolescent girl is due to combined effect of “adrenarche” followed by ‘gonadarche’. It is due to combined effect of adrenal and gonadal hyperactivity, which includes increase both in height and bulk. Details have already been discussed in earlier part of this chapter. A staging system to describe physiological changes of puberty was first described by Marshall & Tanner in 1969 for girls43 and for boys in 1970.44 The staging system describes secondary sexual characteristics, including breast development in girls, pubic hair growth in both sexes and genital development in boys. There are five Tanner stages of breast and pubic hair development, in girls. This has been discussed in further details in Chapter 2: “Precocious Puberty”. Stage 1 represents the prepubertal state and stage 5 maintaining the adult development. Menarche occurs about 2.6 years after the onset of puberty and after completion of attaining the peak growth.45 The total period from onset to completion of pubertal growth (accelerated growth, thelarche, pubarche and menarche) covers a period of 4.5 years (range 1 to 6 years). After menarche, the growth rate declines and generally does not increase more than 6 cm (2.4 inches). Initially menses are infrequent and anovulatory (Fig. 1.3). Anovulation and infrequent menses continue to persist on

Fig. 1.3: Sequence of chronological development of clinical landmarks of puberty-regulated by combined endocrinological, environmental, genetic and ethnic control

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an average about 12 to 18 months to as long as up to 4 years after menarche.45,46 The criteria for completion of pubertal development and landmarks for maturity of hypothalamic– pituitary ovarian axis is the development of oestrogen function which stimulates the midcycle LH surge and ovulation. Take Home Message • Onset of puberty depends on step-wise development, maturation and regulation of HPO axis. • HPO axis develops and differentiates during early intrauterine life (6–10 weeks) and becomes fully functional before birth. • Activity of HPO axis reaches a peak around 20 week of intrauterine life and thereafter, declines and remains at a low level through later part of intrauterine and earlier part of neonatal life after birth. • The decline is due to negative feedback effect of placental oestrogen which continues up to earlier period of neonatal life. • HPO axis becomes active again in the later neonatal period because of its autorhythmicity and LH and FSH remain elevated for 3–6 months in boys and 12–18 months in girls. • Again there is decline in HPO axis and FSH, LH levels remain very low during the entire period of childhood till onset of puberty due to higher sensitivity of ovarian steroid negative feedback by kisspeptin. • Somatotrophic axis (GHRH) through growth hormone (GH) and insulin-like growth factor-1 (IGF-1) provide partial support (adrenarche) to GnRH-HPO axis. • This support also helps in ‘growth spurt’ of puberty. • The ‘prime mover’ however is HPO axis. • The ‘specialized’ neurohormonal secretory cells within the hypothalamus (n = 1500–2000) aggregate to form a functional organ within the hypothalamus known as ‘hypothalamic pulse generator’. • The cells of pulse generator originate from olfactory placode, and migrate to arcuate nucleus and are known as GnRH neurons. • The pulse generator has an ‘oscillatory’ pattern of function and also has ‘autorhythmicity’. • Hypothalamic pulse generator produces neurohormone ‘GnRH’ in a pulsatile fashion—which stimulate pituitary gonadotroph cells to synthesize and release pituitary hormones—FSH and LH also in a pulsatile fashion.

• Gonadotropins (FSH and LH) induce release of episodic ovarian steroids (oestrogen and progesterone) from granulosa and theca cells of ovary. • This chain of events starts functioning when hypothalamic pulse generator becomes fully active (around 18–20 weeks of intrauterine life) and again become suppressed in later part of pregnancy because of negative impact of placental oestrogen. • The activity of hypothalamic pulse generator and consequently HPO axis is controlled primarily by ‘central’, ‘excitatory’ and ‘inhibitory’ factors and secondarily by oestrogen ‘feedback’ mechanism. • The central control, which is more significant, is a composite combination of several neurotransmitters, which are the products of neuro secretory cells and peptides. • All of them are residents of hypothalamus. Some of them act as inhibitors while others work as excitatory stimulators. • Kisspeptin has been accepted as the most significant ‘excitatory’ neurotransmitter for GnRH pulse generator. • In addition to its excitatory function, kisspeptin also provides a ‘high’ hypothalamic sensitivity to respond to the regulatory mechanism of negative feedback loop of ovarian steroid hormones. • These functions become fully functional before birth. • After birth, beginning in late infancy and continuing through childhood, the HPO axis remains inactive because pulsatile release of hypothalamic GnRH is suppressed to a very low level. • The suppression is due to low level of activity due to central inhibitory mechanism, and to a lesser extent, due to high sensitivity to low ovarian steroidal feedback. • The first endocrine change in prepuberty is ‘adrenarche’. • The stimulus is unknown. ACTH does not increase. Adrenal androgen is liberated from ‘zona reticulosa’ of adrenal cortex which differentiates around 3 years of age. • Weak adrenal androgen DHEA is elevated. It increases slowly and stimulates ‘pubarche’ (growth of axillary and pubic hair) after establishment of two of the three landmarks of subsequent gonadarche (growth spurt and thelarche). • Previously it was presumed that ‘adrenarche’ stimulates the onset of ‘gonadarche’.

Puberty • Currently this hypothesis is not accepted. • After a long gap (from late infancy), pulsatile release of GnRH starts functioning again and hypothalamic-pituitary gonadal axis is reactivated (gonadarche). • Probably the resurgence of HPO axis is in response to metabolic signals from the periphery (nutrition, weight gain, etc.). This is apart from genetic and familial influence. • FSH and LH start rising from around 8 years of age followed by gradual increase of estrogen resulting in development of breast and also participating in growth spurt. • Before ‘thelarche’ and ‘pubarche’ there is rapid increase in growth (growth spurt). • This is primarily due to steroid induced increase of GH and IGF-1 secretion and to a lesser extent sex steroid concentration. • Finally, rising steroid (estrogen) concentration will restrict adult height by stimulating epiphyseal fusion. • Following growth spurt, thelarche, pubarche, and menarche (onset of menses) start. This is the final landmark of onset of puberty. • Initially menses are irregular, infrequent and anovulatory. With maturation of estrogen feedback mechanism, ovulatory cycles increase in frequency and by late puberty become well established.

REFERENCES 1. Seminara SB, Hayes FJ, Crowley WF. Gonadotropinreleasing hormone deficiency in the human (idiopathic hypogonadotropic hypogonadism and Kallmann syndrome): Pathophysiological and genetic considerations. Endocr Rev. 1998; 19(5):521–39. 2. Maggi R, Cariboni AM, Marelli MM, Moretti RM, Andrè V, Marzagalli M, et al. GnRH and GnRH receptors in the pathophysiology of the human female reproductive system. Hum Reprod Update. 2016;22(3):358–81. 3. Costa MA. The endocrine function of human placenta: An overview. Reprod Biomed Online [Internet]. 2016;32(1):14–43. Available from: http://dx.doi.org/10.1016/j.rbmo.2015.10.005 4. Sunil Kumar Kota, Kotni Gayatri SJ, Lalit Kumar Meher SKK, SVS. Krishna KDM. Fetal endocrinology. Indian J Endocrinol Metab. 2013;17 (4):568–79. 5. Kindahl H. Placenta functions with special emphasis on endocrine changes—A comparative overview. Acta Vet Scand. 2007;49(SUPPL.1):1–5.

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6. Tsutsumi R, Webster NJG. GnRH pulsatility, the pituitary response and reproductive dysfunction. Endocr J. 2009;56(6):729–37. 7. Johnson DC. Sexual Differentiation of Gonadotropin Patterns Departments of Obstetrics and Gynecology and Physiology, University of Kansas Medical Center, Kansas City, Kansas 66103. The scientific heritage of Professor Emil Witschi includes the foundations for the c. AM ZOOLOCIST. 1972; 205(12):193–205. 8. Grumbach MM. The neuroendocrinology of human puberty revisited. Horm Res. 2002;57(SUPPL. 2):2–14. 9. Plant TM. Hypothalamic control of the pituitarygonadal axis in higher primates: Key advances over the last two Decades. J Neuroendocrinol. 2008;20(6):719–26. 10. El Majdoubi M, Sahu A, Ramaswamy S, Plant TM. Neuropeptide Y: A hypothalamic brake restraining the onset of puberty in primates. Proc Natl Acad Sci USA. 2000;97(11):6179–84. 11. Gore AC, Mitsushima D, Terasawa E. A possible role of neuropeptide Y in the control of the onset of puberty in female rhesus monkeys. Neuroendocrinology. 1993;58(1):23–34. 12. Brann DW, Mahesh VB. Excitatory amino acids: Evidence for a role in the control of reproduction and anterior pituitary hormone secretion. Endocr Rev. 1997;18(5):678–700. 13. R. Medhamurthy, HL Dichek, TM Plant, I. Bernardini, and GB Cutler J. Stimulation of Gonadotropin secretion in prepubertal monkeys after hypothalamic excitation with aspartate and glutamate. J Clin Endocrinol Metab. 1990;71 (5):1390–2. 14. Plant TM, Gay VL, Marshall GR, Arslan M. Puberty in monkeys is triggered by chemical stimulation of the hypothalamus. Proc Natl Acad Sci U S A. 1989;86(7):2506–10. 15. Seminara SB, Messager S, Chatzidaki EE, Thresher RR, Acierno JS, Shagoury JK, et al. The GPR54 Gene as a Regulator of Puberty. new Engl J Med Orig. 2003;349:1614. 16. Shahab M, Mastronardi C, Seminara SB, Crowley WF, Ojeda SR, Plant TM. Increased hypothalamic GPR54 signaling: A potential mechanism for initiation of puberty in primates. Proc Natl Acad Sci USA. 2005;102(6):2129–34. 17. Rometo AM, Krajewski SJ, Voytko M Lou, Rance NE. Hypertrophy and increased kisspeptin gene expression in the hypothalamic infundibular

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nucleus of postmenopausal women and ovariectomized monkeys. J Clin Endocrinol Metab. 2007;92(7):2744–50. Popa SM, Clifton DK, Steiner RA. The role of kisspeptins and GPR54 in the neuroendocrine regulation of reproduction. Annu Rev Physiol. 2008;70:213–38. Plant TM, Ramaswamy S, DiPietro MJ. Repetitive activation of hypothalamic G protein-coupled receptor 54 with intravenous pulses of kisspeptin in the juvenile monkey (Macaca mulatta) elicits a sustained train of gonadotropin-releasing hormone discharges. Endocrinology. 2006;147(2):1007–13. Wakabayashi Y, Nakada T, Murata K, Ohkura S, Mogi K, Navarro VM, et al. Neurokinin B and dynorphin A in kisspeptin neurons of the arcuate nucleus participate in generation of periodic oscillation of neural activity driving pulsatile gonadotropin-releasing hormone secretion in the goat. J Neurosci. 2010;30(8):3124–32. Shibata M, Friedman RL, Ramaswamy S, Plant TM. Evidence that down regulation of hypothalamic KiSS-1 expression is involved in the negative feedback action of testosterone to regulate luteinising hormone secretion in the adult male rhesus monkey (Macaca mulatta). J Neuroendocrinol. 2007;19(6):432–8. Plant TM, Ramaswamy S. Kisspeptin and the regulation of the hypothalamic-pituitary gonadal axis in the rhesus monkey (Macaca mulatta). Peptides. 2009;30(1):67–75. Frisch R, Revelle R. Variation in body weights and the age of the adolescent growth spurt among Latin American and Asian populations, in relation to calorie supplies. Hum Biol. 1969;41(2):185–212. Simon D. Puberty in chronically diseased patients. Horm Res. 2002;57(SUPPL. 2):53–6. Matkovic V, Ilich JZ, Skugor M, Badenhop NE, Goel P, Clairmont A, et al. Leptin is inversely related to age at menarche in human females. J Clin Endocrinol Metab. 1997;82(10):3239–45. Farooqi IS. Leptin and the onset of puberty: Insights from rodent and human genetics. Semin Reprod Med. 2002;20(2):139–44. EI Terasawa and David l. fernandez. Neurobiological mechanisms of puberty in higher primates. Endocr Rev. 2001;22(1):111–51. B³ogowska A, Rzepka-Górska I, KrzyzanowskaŒwiniarska B. Body composition, dehydroepiandrosterone sulfate and leptin concentrations in girls approaching menarche. J Pediatr Endocrinol Metab. 2005;18(10):975–83.

29. Biason-Lauber A, Zachmann M, Schoenle EJ. Effect of leptin on CYP17 enzymatic activities in human adrenal cells: New insight in the onset of adrenarche. Endocrinology. 2000;141(4):1446– 54. 30. Rosenfield RL. Hirsutism and the variable response of the pilosebaceous unit to androgen. J Investig Dermatol Symp Proc [Internet]. 2005;10(3):205–8. Available from: http://dx.doi.org/10.1111/ j.1087-0024.2005.10106.x 31. Abbassi V. Growth and normal puberty. Pediatrics. 1998;102(2 III):507–11. 32. Bass S, Delmas PD, Pearce G, Hendrich E, Tabensky A, Seeman E. The differing tempo of growth in bone size, mass, and density in girls is region-specific. J Clin Invest. 1999;104(6):795– 804. 33. Taranger J, Engström I, Lichtenstein H, SvennbergRedegren I. Somatic Pubertal Development. Acta Pædiatrica. 1976;65:121–35. 34. Shimon I, Taylor JE, Dong JZ, Bitonte RA, Kim S, Morgan B, et al. Somatostatin receptor subtype specificity in human fetal pituitary cultures. Differential role of SSTR2 and SSTR5 for growth hormone, thyroid-stimulating hormone, and prolactin regulation. J Clin Invest. 1997;99(4): 789–98. 35. M L Hartman, J D Veldhuis MOT. Normal control of growth hormone secretion. Horm Res. 1993; 40(1–3):37–47. 36. Toogood AA, Nass RM, Pezzoli SS, O’Neill PA, Thorner MO, Shalet SM. Preservation of growth hormone pulsatility despite pituitary pathology, surgery, and irradiation. J Clin Endocrinol Metab. 1997;82(7):2215–21. 37. Hlndmarsh PC, Matthews DR, Stratton I, Pringle PJ, Brook CGD. Rate of change (modulation) of serum growth hormone concentrations is a more important factor in determining growth rate than duration of exposure. Clin Endocrinol (Oxf). 1992;36(2):165–70. 38. John R B Perry, Lisette Stolk, Nora Franceschini, Kathryn L Lunetta, Guangju Zhai, Patrick F McArdle, Albert V Smith, Thor Aspelund, Stefania Bandinelli, Eric Boerwinkle, Lynn Cherkas, Gudny Eiriksdottir, Karol Estrada, Luigi Ferrucci, Aaron R Folsom, Mel JMM. Meta-analysis of genome-wide association data identifies two loci influencing age at menarche. Nat Genet. 2009; 41(6):648–50. 39. Sun SS, Schubert CM, Chumlea WC, Roche AF, Kulin HE, Lee PA, et al. National estimates of the timing of sexual maturation and racial differences

Puberty among US children. Pediatrics. 2002;110(5): 911–9. 40. Anderson SE, Must A. Interpreting the continued decline in the average age at menarche: Results from two nationally representative surveys of US girls studied 10 years apart. J Pediatr. 2005; 147(6):753–60. 41. Kaplowitz PB, Slora EJ, Wasserman RC, Pedlow SE, Herman-Giddens ME. Earlier onset of puberty in girls: Relation to increased body mass index and race. Pediatrics. 2001;108(2 II):347–53. 42. Adair LS, Gordon-Larsen P. Maturational timing and overweight prevalence in US adolescent girls. Am J Public Health. 2001;91(4):642–4.

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43. Marshall WA, Tanner JM. Variations in pattern of pubertal changes in girls. Arch Dis Child. 1969; 44(235):291–303. 44. Marshall WA, Tanner JM. Variations in the pattern of pubertal changes in boys. Arch Dis Child. 1970;45(13–23):694–6. 45. Johnston FE, Roche AF, Schell LM, Wettenhall HNB. critical Weight at Menarche. Am J Dis Child. 1975;129(1):19–23. 46. Ma HM, Du ML, Luo XP, Chen SK, Liu L, Chen RM, et al. Onset of breast and pubic hair development and menses in urban Chinese girls. Pediatrics. 2009;124(2).

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2

Precocious Puberty Sabnam Parveen and BN Chakravarty

INTRODUCTION

pubertal changes are observed in girls with a moderate degree of obesity (up to 30% above the average weight for age). It has been observed that in girls over 16 years of age, a minimum of 17% and 22% body fat is required for the initiation of menarche and maintenance of regular periods, respectively.2 Increased body weight correlates with an earlier onset of puberty, however, morbidly obese girls, those with diabetes, or those who practice rigorous exercise (e.g. athletes) but maintain a normal weight and body fat, demonstrate delayed menarche. The girls living in the urban areas or closer to the equator, or at lower altitudes experience an early onset of puberty. Advanced puberty has also been recorded in the girls living with broken families, single parents, or other domestic difficulties.3, 4 Blind girls do exhibit an early onset of puberty, suggesting that light also impacts the pubertal development process.5 Girls applying endocrine-containing chemicals may also develop precocious puberty.4

Puberty is the essential phase of life that marks the transition from childhood to adulthood and eventually leading to the ability of an individual to reproduce. This phase represents a multi-staged event involving the completion of a series of maturational steps spanning through the intrauterine life, proceeding during the neonatal period, and finally culminating at its physiological onset at ages between 8 and 13 years in girls and 9 and 14 years in boys. In humans, the onset of puberty is brought about by a balanced regulation of the hypothalamus, pituitary, and gonadal axis adjusting the positive and negative gonadal steroid feedback loops for pulsatile release of gonadotropins and ovarian steroid hormones. Early activation of the reproductive axis results in the advancement of the timing of puberty, and the condition is termed as precocious puberty. Factors Affecting the Timing of Puberty The timing of the onset of puberty is coaxed by a complex interplay between genetic, nutritional, environmental, and socioeconomic factors. 1 The synchronicity in the age of menarche between the siblings or between the daughters and mothers1 and some populationbased studies provided compelling evidence for the importance of genetic component in determining the puberty timing. Earlier

Precocious Puberty: Controversies on Definition and Indications for Evaluation Precocious puberty is defined as an earlier pubertal development occurring more than 2.5 standard deviations before the average age in the general population. It is much more common in girls than in boys. Conventionally, it is defined when secondary sexual characters 12

Precocious Puberty

develop before 8 years of age in girls and 9 years in boys.6, 7 A Danish study has estimated the prevalence of precocious puberty to be 0.2% in girls and 10:1. The common clinical situation in men, where abnormal ratio is observed, is obesity. In adipose tissues, testosterone is converted to estradiol by aromatase enzyme. With increasing incidence

Azoospermia

of obesity increasing number of men are presenting with abnormal testosterone to estradiol ratio. Liver failure is another cause of estrogen excess. Excess estrogen by negative ‘feedback’ mechanism inhibits pituitary gonadotropin secretion and thereby inhibits intratesticular testosterone production. It has been suggested that estrogen excess in combination with androgen deficiency will result in deficient spermatogenesis.29–31 Aromatase inhibitor by reducing estradiol and increasing androgen level may be effective in improving spermatogenesis in estrogen excess induced oligospermic individuals. The drug is available either in the steroidal (testolactone) or in non-steroidal (anastrozole) formulation. Significant improvement has been reported following daily use of 1 mg dose of non-steroidal aromatase inhibitor anastrozole for a period of three months due to increase in the testosterone to oestradiol ratio. C. Prolactin Excess Hyperprolactinemia may have a significant impact on male infertility. Excess prolactin leads to inhibition of hypothalamic secretion of GnRH which in turn will result in diminished secretion of pituitary FSH and LH leading to diminished synthesis of intratesticular testosterone and impaired spermatogenesis. Low level of testosterone will be responsible for diminished libido, erectile dysfunction and abnormal semen parameters. Hence the overall effect of hyperprolactinemia will lead to diminished male reproductive potential. Hyperprolactinemia is rarely associated with total azoospermia. Treatment of hyperprolactinemia with or without prolactinoma primarily consists of use of dopamine agonists. The commonly used drugs are either bromocriptin or cabergoline. Regarding efficacy, Cabergolin appears to be more effective than bromocriptin specially when prolactinoma has been detected on CT scan or MRI. Resistance to the drug is less compared to bromocriptine. Patients resistant to bromocriptin respond well to cabergoline.

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Cabergolin appears to be the first choice specially when prolactinoma is present. In symptom producing prolactinomas (visual disturbances, persistent headache) transsphenoidal surgical excision or radiotherapy may be necessary. With treatment either medical or surgical, it is expected that there will be reversal of GnRH inhibition of pituitary. But it is essential that patients' serum gonadotropin level should be assessed in the postoperative period. Because patient may require gonadotropin therapy, even after resolution of hyperprolactinemia. In these situations symptoms resulting from intracranial compression require prior attention than the problem of azoospermia. D. Empirical Therapy Empirical therapy is based on theoretical concept and practical experiences with no proven efficacy. It is difficult to predict who will respond to empirical therapy (hormonal, antioxidants, nutraceuticals) and who will not respond but their correct choice and judicious application may be valuable in conjunction with assisted reproductive technology. A few drugs used in general practice as an alternative to ART or sometimes before ART are—clomiphene citrate, low dose testosterone, gonadotropins and antioxidants. Situations in which empirical use of gonadotropin in idiopathic azoospermia has been useful are: a. Patients have normal plasma levels of FSH and inhibin B with hypospermatogenesis detected in seminiferous tubules but without maturation disturbances.32–34 b. In aspirated samples of biopsied testicular tissue containing significant number of apoptotic or immature sperm. FSH has the potentiality to improve sperm microorganelles,35 and improves pregnancy rate following subsequent ICSI.36 c. For the same reason, some cases of nonobstructive azoospermia; use of FSH for the husband before attempting IVF/ICSI has produced better quality embryo and resulted in improved pregnancy rates.37

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Groups of patients likely to respond to surgical treatment are detailed below. Surgical Management of Azoospermic Men Apart from sperm retrieval techniques in ART (ICSI), there are two common surgical approaches for the treatment of azoospermia. These are: a. Repair of varicocele b. Management of vasal or vasoepididymal obstruction. On rare occasions, transurethral excision of müllerian cyst at the point of entry of ejaculatory duct into prostatic urethra may be rewarding.

lining with fibrosis of muscular layer, and therefore surgical canalization will rarely be indicated. Micro-surgical vasovasostomy has significantly improved results compared to those with older techniques.39,40 Patency and pregnancy rates vary directly with the period of obstruction. Results of surgery following obstruction for less than 3 years resulted in patency rate of 97% and pregnancy rate of 96%. 41 Results are better following microsurgical procedures with use of multilayered repair as against single layer repair which was practiced in older technique. Multilayer technique ensures water tight anastomosis which prevents formation of sperm granulomas. Water tight anastomosis is important because there is no constituent of vasal fluid which will help sealing the anastomosis site internally.

Repair of Varicocele This is rarely performed as a surgical treatment of azoospermia. Though several studies have reported favourably in support of varicocele repair, controversy still exists about its utility in the surgical management of male infertility in general—azoospermia in particular. Varicocele has been reported to be associated with 35 to 40% men with primary, and 75 to 80% with secondary infertility. However, this is found only in 15% of general population. The cause of subfertility in varicocele is attributed to venous dilatation. Stasis of blood increases intratesticular temperature which reduces testicular function. It causes testicular damage, decreases testosterone production by interfering with Leydig cell function leading to decreased sperm production. Several studies have demonstrated improved semen parameters, testosterone production and pregnancy outcomes following varicocele repair. Repair of large (Gr. II & Gr. III) varicocele in younger men may have greater beneficial effect on sperm parameters and androgen production than in older men.23,38

Vasoepididymostomy It is a technically challenging micro-surgical procedure. If epididymal obstruction is present, whether primary or secondary chronic vassal obstruction vasoepididymostomy may be required proximal to obstruction to restore continuity of sperm transport. In these situations, the decision is made during the surgery and is based on microscopic examination of the proximal vasal fluid (for sperm) and the duration of obstruction.42,43 These operations for management of azoospermic men are only of historical interest to us.

Surgical Repair of Vasal or Epididymal Obstruction Vas obstruction is more common after vasectomy or secondary to some injury like childhood hernia repair, orchiopexy or following hydrocele operation.39 Obstruction following infection (Gonococcal, chlamydial) causes permanent damage to vasal epithelial

The four specific areas are a. Successful fertilization does not require epididymal transit of sperm. b. ICSI requires a single sperm—may be nonmotile, but should be viable. c. Even in Sertoli cell only syndrome, microscopic foci of spermatogenesis exists in either testis.

Role of ART During last 30 years our knowledge in ART, especially with regard to male infertility has significantly advanced in four specific areas.

Azoospermia

d. With the help of operating microscope— selection and extraction of seminiferous tubules which contain active spermatozoa is possible even in men with Sertoli cell only syndrome. Our knowledge has also advanced in three basic areas related specifically to ICSI i. Sperm selection ii. Sperm extraction for ICSI iii. Sperm protection against reactive oxygen species (ROS). i. Sperm Selection Even in in vivo pregnancies—millions of sperms are deposited in vagina—the competent sperms reach the fallopian tube and the most efficient one will fertilize the oocyte—hence there is a need for efficient sperm selection. The physiological steps of sperm selection in normal in vivo fertilization has been elaborately discussed in Volume 3, Chapter 10 (see page 116) of this series. In IVF following insemination, still there is a scope and need for natural selection of the most efficient sperms, because fertilization is not assisted. But in ICSI, there is no scope for natural sperm selection—because fertilization occurs by ‘assisted sperm injection’. Hence in ICSI sperm to be injected should be carefully selected. How to select normal efficient sperm during ICSI? Sperm selection is performed at three stages of the ICSI procedure. For non-azoospermic subjects sperm assessment is done prior to actual ICSI procedure and also at the time of semen preparation. In azoospermic husbands, sperm selection is carried out during sperm preparation (when adequate number of sperm has been retrieved) or during actual ICSI procedure (when less amount of sperm is available for injection). Hence in azoospermic husbands sperm extraction is performed primarily and if possible sperm selection is carried out subsequently as a secondary procedure.

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ii. Sperm Extraction Sperm extraction is necessary in azoospermia followed by ICSI. Azoospermia may be of two types—obstructive azoospermia (OA) and non-obstructive azoospermia (NOA). In obstructive azoospermia, obstruction may exist in five different areas of seminal pathway, namely (a) rete-testis, (b) epididymis, (c) vas deferens, (d) seminal vesicle, (e) ejaculatory duct. Occasionally vas deferens and seminal vesicle may be absent. Diagnosis is not difficult except in retetestis obstruction. The salient diagnostic parameters of OA (except rete-testis obstruction) are: a. Epididymis is distended b. Serum FSH is normal c. Fructose in seminal plasma is absent in ejaculatory duct and seminal vesicle obstruction d. Azoospermia on semen analysis Sperm extraction in OA is not difficult as epididymis is almost always distended. Microsurgical epididymal sperm aspiration (MESA) involves the aspiration of spermcontaining fluid from individual epididymal tubules and the use of the sperm to fertilize oocytes in vitro. Use of this technique, first employed in the treatment of men with congenital vasal agenesis, has since been extended to include the treatment of men with other forms of vasal and epididymal obstruction. Another approach to epididymal sperm harvesting is the use of percutaneous epididymal sperm aspiration (PESA).44 This procedure is somewhat less invasive than MESA, but the yield of motile sperm is lower than that achieved with MESA. Details of the procedure have been described in chapter on ICSI. Genetic defect may co-exist. Hence, counseling is essential—vertical transmission in male child is a risk factor. The etiology of non-obstructive azoospermia (NOA) may be chromosomal defect (numerical or structural)—as in Klinefelter syndrome or in 46XX males. The acquired causes are— undescended testis, systemic chemotherapy, excessive scrotal thermal exposure. The clinical

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diagnostic parameters include—finding of small testis and flat empty epididymis. Diagnosis is confirmed by low serum FSH level and testicular biopsy reveals evidence of hypospermatogenesis, maturation arrest and Sertoli cell only syndrome. The actual procedures of sperm extraction are as follows: A. TESE with testicular biopsy under local or general anesthesia—not commonly performed nowadays. B. Fine needle or wide bore aspiration biopsy— commonly performed and currently used with 23–19 gauge needle. This will allow aspiration of not only testicular tissue but seminiferous tubules as well C. Under special situation and in some cases of Sertoli cell only syndrome ‘microdissection testicular sperm extraction (TESE) may be useful. This is usually difficult but a more rewarding technique. Advantages of Micro-dissection TESE over Conventional Techniques • Selective extraction of tubules containing mature spermatozoa. • Microscopic vessels can be avoided or cauterized, avoiding over cauterizing to spare devascularization by hematoma formation. • Avoids unnecessary trauma to already compromised testis.

Disadvantages • Time consuming • Surgeon has to be specially trained Cumulative delivery rates in couples where testicular sperm was used are low.45 Men suffering from NOA undergoing TESE are to be counseled that not only are the sperm recovery rates limited but also the fertilization, implantation and conception rates are decreased compared to men with normal spermatogenesis.46 iii. Sperm Protection Against Damage by ROS Aerobic metabolism generates reactive oxygen species—hydroxyl radicals, superoxide anion,

hydrogen peroxide and nitric oxide. They are small and highly reactive due to unpaired electrons that are capable of initiating an uncontrolled cascade of chain reactions, which cause damage to healthy cells. Sperms are vulnerable to damage by ROS attackbecause: • Sperm head plasma membrane is made up of polyunsaturated fatty acid (PUFA), which is an unstable fatty acid. • Premature fusion and disintegration of membranes lead to exocytosis of acrosin before sperm head reaches zona pellucida. • During maturation of spermatozoa, extrusion of cytoplasm allows more place for nuclear DNA—resulting in loss of repair and defense mechanism. • In ICSI (PESA-ICSI)—presence of abnormal and immature sperms enhance ROS production. But there are compensatory mechanisms also in seminal plasma. The compensatory plasma antioxidants are superoxide dismutase (SOD), catalase and glutathione. But, there is no seminal plasma around spermatogonia in azoospermic sample which are retrieved by PESA-TESE. Moreover, these defense mechanisms are disrupted during sperm preparation for IVF or ICSI. For example, high speed centrifugation adds more damage to sperm membrane. Sperm freezing and thawing increase ROS concentration. Strategies to Reduce ROS Damage to Sperm in ART Treatment The following procedures are practiced to reduce ROS damage: a. Antioxidant use in vivo—treating individuals with antioxidants for 3 months before ART; results are not very encouraging b. Antioxidant use in vitro—adding antioxidants (vitamin E, vitamin C and COQ, etc.) to culture media, results are controversial c. Adding human serum albumin to culture media—this is believed to be more protective against oxidative stress (OS). The previous practice for reducing OS and to enhance sperm motility by adding either

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pentoxifylline (PTX) or platelet activating factor has been abandoned. This is because the procedure enhances sperm motility in vitro but their functional potentiality for increasing pregnancy rate is still doubtful. Take Home Message • 50% of infertile couples have male partner defect. • 20% male factor defect is due to azoospermia. • Though classification of azoospermic men has been based primarily on areas affected like (a) pretesticular, (b) testicular and (c) post-testicular or volume of semen ejaculated (low or normal). • But the practical approach from management point of view should be—obstructive (OA) and non-obstructive azoospermia (NOA). • Semen volume to some extent may differentiate between OA and NOA groups. • In all types of classification, there is some overlapping, but volume of ejaculated semen may have significant demarcation. • Low volume semen is more frequently associated with obstructive variety, e.g. absence of seminal vesicles (CBAVD), obstruction of ejaculatory duct and less commonly with—ejaculatory failure or retrograde ejaculation. • Whereas normal volume semen is more commonly associated with NOA—like hypogonadotropic hypogonadism, testicular failure (Sertoli cell only syndrome) and less commonly associated with obstructive features (obstruction in epididymis, testes, etc.) but • Differentiation is possible by estimation of FSH, LH, size of testes, presence/absence of fructose in semen. • Very high or very low level of FSH, LH and testosterone signifies either testicular failure, hyper- or hypogonadotropic azoospermia. In these cases size of testes is also small. • Normal FSH, LH may be present both in NOA and OA. • Testicular biopsy with or without evidence of spermatogenesis will solve the problem. • Even in NOA, there may be areas of focal spermatogenesis (normally in this group there should be absence of spermatogenesis). • Identification of such area will require special expertise and experience (facilities for micro TESE and if possible vaso-epididymal surgery at the same sitting). • From enhancement of fertility point of view, more than 90% of azoospermic men are currently

•

•

•

•

treated by ART—IUI (in case of retrograde ejaculation), IVF (PESA—obstructive azoospermia) or TESE/Micro TESE (NOA)—and results are rewarding. Surgical treatment consists of vasoepididymostomy and varicocelectomy when indicated—in expert hands these treatments are still considered worth attempting. Medical treatment is rarely successful except i n very rare cases like hypogonadotropic hypogonadism (prolonged treatment with hMG/ FSH/hCG) and with Pseudoephedrine (alpha agonist) for retrograde ejaculation. Obstructive azoospermia (CBAVD) is frequently associated with CF gene mutation (25–81%); chromosome (47XXY, 46XX male) defect and microdeletion of Y chromosome is 14% and 5– 30% of azoospermic men respectively. Chromosome analysis and genetic counseling are of utmost importance before attempting ART using husband's sperm through PESA-TESE/ ICSI.

REFERENCES 1. Cadman SM, Kim SH, Hu Y, González-Martínez D, Bouloux PM. Molecular pathogenesis of Kallmann's syndrome. Horm Res. 2007;67(5):231–42. 2. Kulkarni ML, Balaji MD, Kulkarni AM, Sushanth S, Kulkarni BM. Kallmann’s syndrome. Indian J Pediatr. 2007;74:1113–5. 3. Vasilev V, Daly A, Beckers A. Familial pituitary adenomas: An overview. Tumors Cent Nerv Syst Pineal, Pituitary, Spinal Tumors. 2013;10:103–12. 4. Masarani M, Wazait H, Dinneen M. Mumps orchitis. J R Soc Med. 2006;99(11):573–5. 5. Steven Rubin, Michael Eckhaus, Linda J Rennick, Connor GG Bamford and WPD. Molecular biology, pathogenesis and pathology of mumps virus. J Pathol [Internet]. 2015;235(2):242–252. Available from: https://www.ncbi.nlm.nih.gov/pmc/ articles/PMC3624763/pdf/nihms412728.pdf 6. C o c u z z a M , A l v a r e n g a C , P a g a n i R . T h e epidemiology and etiology of azoospermia. Clinics. 2013;68(SUPPL. 1):15–26. 7. Meistrich ML. The Effects of Chemotherapy and Radiotherapy on Spermatogenesis in Humans. Fertil Steril. 2013;100(5):1–14. 8. Durairajanayagam D, Sharma RK, du Plessis SS, Agarwal A. Testicular Heat Stress and Sperm Quality. Male Infertil. 2014;(June):105–25.

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9. Jensen TK, Bonde JP, Joffe M. The influence of occupational exposure on male reproductive function. Occup Med (Chic Ill). 2006;56(8):544– 53. 10. Agarwal SS du P and A. Environmental Insults on Spermatogenesis. Bienn Rev Infertil. 2011; 2:133–54. 11. Mohammed A, Chinegwundoh F. Testicular varicocele: An overview. Urol Int. 2009;82(4):373–9. 12. Niedzielski JK, Oszukowska E, S OwikowskaHilczer J. Undescended testis—Current trends and guidelines: A review of the literature. Arch Med Sci. 2016;12(3):667–77. 13. Goel P, Rawat JD, Wakhlu A, Kureel SN. Undescended testicle: An update on fertility in cryptorchid men. Indian J Med Res. 2015;141:163–71. 14. Mierla D, Jardan D, Stoian V. Chromosomal abnormality in men with impaired spermatogenesis. Int J Fertil Steril. 2014;8(1):35–42. 15. Denise Andréa Silva de Souza, , Fábio Rueda Faucz C, Lilian Pereira-Ferrari V, Santos Sotomaior and SR. Congenital Bilateral Absence of the vas deferens as an Atypical Form of Cystic Fibrosis: Reproductive Implications and Genetic Counseling. Andrology. 2018;6(1):127–35. 16. Bieth E, Hamdi SM, Mieusset R. Genetics of the congenital absence of the vas deferens. Hum Genet [Internet]. 2020;(0123456789). Available from: https://doi.org/10.1007/s00439-02002122-w 17. Khanna K, Liu DR. Epididymitis and orchitis. Fleisher Ludwig's 5-Minute Pediatr Emerg Med Consult. 2012;79(7):583–7. 18. Taylor SN. Epididymitis. Clin Infect Dis. 2015; 61(April 2013):S770-3. 19. Aziz N. The importance of semen analysis in the context of azoospermia. Clinics. 2013;68(SUPPL. 1): 35–8. 20. Irvine DS. Epidemiology and aetiology of male infertility. Hum Reprod. 1998;13(SUPPL. 1):33–44. 21. Matthew R. Macey, Ryan C. Owen SSR and RMC. Best practice in the diagnosis and treatment of varicocele in children and adolescents. Ther Adv Urol. 2018;10(9):273–82. 22. Agarwal A, Sharma R, Harlev A, Esteves S. Effect of varicocele on semen characteristics according to the new 2010 World Health Organization criteria: A systematic review and meta-analysis. Asian J Androl. 2016;18(2):163–70. 23. Kantartzi PD, Goulis CD, Goulis GD, Papadimas I. Male infertility and varicocele: Myths and reality. Hippokratia. 2007;11(3):99–104.

24. Tiseo BC, Esteves SC, Cocuzza MS. Summary evidence on the effects of varicocele treatment to improve natural fertility in subfertile men. Asian J Androl. 2016;18(2):239–45. 25. Cocuzza M, Cocuzza MA, Bragais FMP, Agarwal A. The role of varicocele repair in the new era of assisted reproductive technology. Clinics. 2008; 63(3):395–404. 26. Inci K, Gunay LM. The role of varicocele treatment in the management of non-obstructive azoospermia. Clinics. 2013;68(SUPPL. 1):89–98. 27. Miyagawa Y, Tsujimura A, Matsumiya K, Takao T, Tohda A, Koga M, et al. Outcome of gonadotropin therapy for male hypogonadotropic hypogonadism at university affiliated male infertility centers: A 30-year retrospective study. J Urol. 2005;173 (6):2072–5. 28. Whitten SJ, Nangia AK, Kolettis PN. Select patients with hypogonadotropic hypogonadism may respond to treatment with clomiphene citrate. Fertil Steril. 2006;86(6):1664–8. 29. Carreau S, Hess RA. Oestrogens and spermatogenesis. Philos Trans R Soc B Biol Sci. 2010;365(1546):1517–35. 30. J. handelsman D, Wishart S, Conway AJ. Oestradiol enhances testosterone-induced suppression of human spermatogenesis. Hum Reprod. 2000; 15(3):672–9. 31. Smith LB, Walker WH. The regulation of spermatogenesis by androgens. Semin Cell Dev Biol. 2014;30:1–29. 32. Foresta C, Bettella A, Rossato M, Sala G La, De Paoli M, Plebani M. Inhibin B plasma concentrations in oligozoospermic subjects before and after therapy with follicle stimulating hormone. Hum Reprod. 1999;14(4):906–12. 33. Foresta C, Bettella A, Spolaore D, Merico M, Rossato M, Ferlin A. Suppression of the high endogenous levels of plasma FSH in infertile men are associated with improved Sertoli cell function as reflected by elevated levels of plasma inhibin B. Hum Reprod. 2004;19(6):1431–7. 34. Demyashkin GA. Inhibin B in seminiferous tubules of human testes in normal spermatogenesis and in idiopathic infertility. Syst Biol Reprod Med [Internet]. 2019;65(1):20–8. Available from: https://doi.org/10.1080/19396368.2018.1478470 35. Santi D, Crépieux P, Reiter E, Spaggiari G, Brigante G, Casarini L, et al. Follicle-Stimulating Hormone (FSH) Action on Spermatogenesis: A Focus on Physiological and Therapeutic Roles. J Clin Med. 2020;9(4):1014.

Azoospermia 36. Lintsen AME, Eijkemans MJC, Hunault CC, Bouwmans CAM, Hakkaart L, Habbema JDF, et al. Predicting ongoing pregnancy chances after IVF and ICSI: A national prospective study. Hum Reprod. 2007;22(9):2455–62. 37. Kumar R. Medical management of non-obstructive azoospermia. Clinics. 2013;68(SUPPL. 1):75–9. 38. Miyaoka R, Esteves SC. A critical appraisal on the role of varicocele in male infertility. Adv Urol. 2012;2012:4–5. 39. Hendry WF. Testicular, epididymal and vasal injuries. BJU Int. 2000;86(3):344–8. 40. Sheynkin YR, Hendin BN, Schlegel PN, Goldstein M. Microsurgical repair of iatrogenic injury to the vas deferens. J Urol. 1998;159(1):139–41. 41. Hayden RP, Li PS, Goldstein M. Microsurgical vasectomy reversal: contemporary techniques, intraoperative decision making, and surgical training for the next generation. Fertil Steril [Internet]. 2019;111(3):444-53. Available from: https://doi.org/10.1016/j.fertnstert.2019.01.004

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42. Baker K, Sabanegh E. Obstructive azoospermia: Reconstructive techniques and results. Clinics. 2013;68(SUPPL.1):61–73. 43. Saitz TR, Ostrowski KA, Acevedo AM, Bash JC, Klimek J, Fuchs EF, et al. The vasal fluid proteomic profile and microscopic sperm presence at time of vasectomy reversal. Transl Androl Urol. 2020; 9(5):2000–6. 44. Esteves SC. Percutaneous epididymal sperm aspiration as a method for sperm retrieval in men with obstructive azoospermia seeking fertility: Operative and laboratory aspects. Int Braz J Urol. 2015;41(4):817. 45. Osmanagaoglu K, Vernaeve V, Kolibianakis E, Tournaye H, Camus M, Van Steirteghem A, et al. Cumulative delivery rates after ICSI treatment cycles with freshly retrieved testicular sperm: A 7-year follow-up study. Hum Reprod. 2003;18 (9):1836–40. 46. Chiba K, Enatsu N, Fujisawa M. Management of non-obstructive azoospermia. Reprod Med Biol. 2016;15(3):165–73.

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5 Unexplained Spontaneous Miscarriage BN Chakravarty and Pratip Chakraborty

INTRODUCTION

Apart from ‘known’ causes (like genetic, structural, infective, endocrine and immune causes), evidence suggest that in women with unexplained recurrent fetal loss, ‘depth’ and ‘quality’ of implantation play a vital role on adverse outcome in different trimesters of pregnancy. The depth and quality will depend on two important e v e n t s occurring during implantation (endometrium)—immunomodulatory changes in the endometrium and subsequently the placental vascularization. The delicately tuned and meticulously balanced immunomodulatory changes in the endometrium during the process of implantation is perturbed in URSM by sudden failure to synchronize the preparatory programme of implantation of both the embryo and the endometrium. Because during this vital period, prior to implantation, incoming embryo through ‘cross-talk’ sends signal to the endometrium to complete preparatory changes at each molecular level for its implantation. In URSM, even if embryo (conceptus) is normal, malfunction of decidualization or remodeling of endometrium may result due to dyssychrony of maternal factors at the endocrine and the molecular level. The significant dysfunction observed at these two levels involve abnormal endocrine function and dysregulated production in

Apparently unexplained spontaneous miscarriage (USM) is frustrating to the couple and perplexing to the clinician as well. Moreover, if the background etiology is not explored and treated at the outset, similar mishaps are likely to recur, ultimately leading to well recognized clinical situation of unexplained recurrent spontaneous miscarriage (URSM). There are two types of miscarriage— sporadic and recurrent. Recurrent miscarriage affects about 1% of couples globally.1 On the other hand, 50% of all women must have experienced one or more sporadic miscarriages during their child bearing period; the incidence usually increases with increasing maternal age.2 Reproductive loss is an independent predictor of future pregnancy outcome. Several studies3,4 have shown that the risk of further miscarriage multiplies after each successive pregnancy loss reaching 45% after three consecutive miscarriages. The commonest cause of sporadic miscarriage is chromosomal anomaly in the conceptus. In contrast, in the recurrent unexplained miscarriage group, women will tend to lose pregnancy even when the conceptus in subsequent pregnancy will have no ‘abnormal’ chromosomal set up. 60

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imprecise interaction of implantation related molecules. The specific hormones and implantation molecules involved in endometrial remodeling are—estrogen, progesterone, thyroid, interleukin-1B (IL-B), pinopode, leukemia inhibitory factor (LIF), integrins and their ligands, adhesion molecules, proteolytic enzymes like matrix metalloproteinases and their inhibitors. The synchronization of their function is primarily controlled by maternally produced hormone progesterone. The second abnormal endometrial event observed during implantation may involve placental vascularization. Over the last decade, evidence have accumulated to suggest that some cases of URSM and later pregnancy complications, e.g. PET, IUGR PTL, etc. are due to exaggerated hemostatic response (hypercoagulable state) during pregnancy leading to placental thrombosis and infarction. Increased thrombosis may be due to hyperhomocysteinemia (HHcy) resulting in microthrombi formation in the vascular bed of the placenta which can impair sustained placental function. Apart from the thrombogenic effect of elevated Hcy on pregnancy, few recent studies have also implicated the adverse effect of high serum or follicular fluid Hcy levels on defect in folliculogenesis, oocyte number, maturity and embryo quality t h a t m a y h a v e f u t u r e bearings o n the establishment and maintenance of pregnancy. Based on these concepts, many clinicians treat patients with history of previous unexplained spontaneous miscarriage (not necessarily recurrent) with prophylactic use of progesterone and low molecular weight heparin (LMWH).

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Summarizing, it appears that the probable causes of unexplained miscarriage (both sporadic and recurrent) are: 1. Defective gametogenesis 2. Defective embryogenesis 3. Defective implantation 4. Defective placental vascularization Sporadic miscarriage is always ‘unexplained’ whereas in about 50% women with recurrent miscarriage—the exact cause remains unknown (Fig. 5.1). The Objective of the Presentation • Attempt to identify etiology following first unexplained spontaneous miscarriage (USM). • If explored, similar miscarriages which are likely to recur may be prevented. • In addition, identification of correct or nearly correct etiology could avoid misuse of two most commonly used drugs— progesterone and thromboprophylactic drugs (aspirin and LMWH). PROBABLE CAUSES OF UNKNOWN ETIOLOGY AT EACH LEVEL

Mechanism involved and possible events leading to unexplained miscarriage (both sporadic and recurrent) under each etiological factor are briefly outlined below: 1. Defective Gametogenesis The female meiotic process is highly error prone, especially in humans. Errors in chromosome segregation during meiosis result in the production of sperm (Fig. 5.2a) or eggs (Fig. 5.2b) with abnormal numbers of chromosomes,

Fig. 5.1: Percentage (%) wise different causes of miscarriage. In approximately 50% women presenting with the history of recurrent spontaneous miscarriage the cause remains unknown

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Fig. 5.2a: Meiotic division during gametogenesis in sperm

rather than the normal complement. When these gametes undergo fertilization, they produce aneuploid embryos. Aneuploids are cells missing chromosomes or having extra chromosomes, e.g. 2n –1 (monosomics) or 2n +1 (trisomics). About 20% of human conceptions are lost as a result of such meiotic errors. This happens because in females meiosis is arrested at the dictyate stage for days/months/years. Meiotic segregation errors escalate with age in females, increasing the incidence of conceptions

with developmental abnormalities such as Down syndrome (trisomy 21) and aneuploidyrelated miscarriages. Each gamete (oocyte or sperm) originally “diploid” has to undergo two times reduction division followed by reassortment/realignment of genes to become a mature “haploid” sperm or oocyte after fertilization. During this process, especially during the process of ‘crossing over’ and realignment, formation of abnormal gamete is not uncommon. Fertilization with

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Fig. 5.2b: Meiotic division during gametogenesis in oocyte

such gametes will lead to formation of abnormal conceptus—ultimately leading to miscarriage. 2. Defective Embryogenesis Errors during the processes of gametogenesis and fertilization have a major impact in fertility until implantation. An apparently normal gamete could turn “abnormal” after the random ‘realignment’ of genetic material between male and female pronuclei during nuclear syngamy. It is to be remembered that the transfer of a normally formed centriole to the oocyte by the spermatozoa is essential for proper fertilization (Fig. 5.3). Malformation of the centrosome is associated with detrimental phenomenon at fertilization such as abnormal sperm ‘aster’ formation which leads to the

failure or lack of syngamy resulting in cleavage arrest.5 Improper centrosome function may also lead to numerical chromosomal abnormalities causing aneuploidy or mosaicism in the embryo resulting in an apparently unexplained miscarriage.6,7 Even if gamete is normal but thereafter during fertilization, pronuclear formation occurs followed by dissolution and exchange of genetic material between male and female pronuclei is completed (nuclear syngamy). The procedure occurs through random ‘realignment’ of genes which may again be abnormal resulting in abnormal zygote (embryo) formation. If embryogenesis continues and finally implantation occurs—pregnancy will invariably terminate into an apparently unexplained miscarriage.

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Fig. 5.3: Schematic representation of zygote formation

Are these abnormalities recurrent? Or Are they linked to parental genetic or chromosomal defect? They are not recurrent. They are simply sporadic and co-incidental. Therefore, chromosome analysis of the conceptus which is very often recommended in these types of unexplained miscarriage is not very significant or predictive for the outcome of future pregnancies. In addition, this abnormality has no link with parental chromosomal anomaly. In summary, defective gametogenesis and embryogenesis are two very significant causes of spontaneous sporadic miscarriage. This type of miscarriage usually occurs in the first trimester of pregnancy (around 8–10 weeks of gestation) and usually remains unexplained. The typical clinical presentation is as follows— 7–10 weeks of pregnancy is followed by onset of bleeding, nonappearance of fetal pole (anembryonic pregnancy)—or absence or disappearance of cardiac pulsation, etc. (NB: Conventional definition of miscarriage is interruption in the continuation of pregnancy after the onset of clinical pregnancy {appearance of gestational sac} up to 24 weeks of gestation. In IVF pregnancy this definition remains confusing. Still now many studies consider onset of pregnancy by the date of positive hCG in blood and include them in reporting as pregnancy {even before gestational sac is visible on USG scan} commonly known as biochemical pregnancy).

3. Defective Implantation Even if chromosomal anomaly does not exist in the developing embryo, unexplained miscarriage may occur if there is error in implantation either in ‘depth’ or in ‘quality’. Implantation consists of three stages: (a) The blastocyst contacts the implantation site of the endometrium (apposition); (b) trophoblast cells of the blastocyst attach to the receptive endometrial epithelium (adhesion); and ( c ) invasive trophoblast cells cross the endometrial epithelial basement membrane and invade the endometrial stroma (invasion). The crosstalk between a receptive endometrium and a competent blastocyst can only occur during a limited time span, known as the “window of implantation”. The risk of spontaneous abortion significantly increases with implantation beyond this window period. The desynchronized molecular dialogue mediated by cytokines, a variety of growth factors, prostaglandins, matrix degrading enzymes and their inhibitors, and adhesion molecules result in specific changes. The changes consist of (a) pinopode formation; (b) ‘integrin’ expression (these two changes occur above the endometrial surface epithelium); (c) endometrial remodeling and; (d) vascularization of sub-epithelium stromal cells and extracellular matrix (ECM) (the last two changes occur below the surface of epithelium). Hence, implantation should

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neither be too deep nor too superficial to allow a firm blastocyst attachment and anchorage.

progesterone may rescue. Hence, this forms the basis of exogenous progesterone management in USM.

Remodeling or Decidualization This is the vital step of implantation. Decidualization of the human endometrium involves a dramatic morphological and functional differentiation of human endometrial stromal cells. Decidualization occurs as a consequence of immunomodulation (progesterone induced) leading to ‘helpful’ cytokine migration, production of prostaglandin (COX-1 and COX-2) and two significant proteolytic enzyme production [plasminogen activator (PA) and matrix metalloproteinases (MMP). Progesterone, together with proteins that are regulated by progesterone and/or cyclic adenosine monophosphate (cAMP), including heart and neural crest derivatives expressed transcript 2 (HAND 2), forkhead box O1 (FOXO1), homeobox A10 (HOXA10), and signal transducers and activators of transcription (STAT), forms a critical network for the decidualization of human endometrial stromal cells through the signal received from implanting blastocyst (Fig. 5.4). Sequence of these events may not be completed in the absence of efficient corpus luteum (progesterone) and a normal efficient embryo. This is an area where exogenous

Progesterone: An Immunomodulatory Hormone It is known that conceptus is an allograft to the mother because it contains paternal antigen and is likely to be rejected. But it is not rejected because of the following immunomodulatory changes induced in the mother by the conceptus. Physiological pregnancy requires the maternal immune system to recognize and tolerate embryonic antigens. The semiallograft conceptus bearing paternal classical MHC class I antigens (HLA-C) are processed with self-major histocompatibility complex (MHC) class II, to specific maternal CD4 + T helper cells by maternal antigen presenting cells (APCs) for production of various cytokines resulting in two different types of immune response/s: (a) Humoral (response is generated through human leukocyte antigen (HLA)) and (b) cellular (response is generated through peripheral blood mononuclear cells (PBMC) through ‘T’ lymphocytes). a. HLA Mediated Immunomodulation Paternal contribution in the conceptus through HLA induces two types of responses in the mother—(a) Type I response: The response is

Fig. 5.4: Remodeling of molecular structure for implantation. The process is controlled by progesterone

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through antibody which is ‘aggressive’ which may reject the conceptus; (b) Type II response: This is also through another type of antibody, discriminatory in nature and will protect the conceptus. This means that this a ‘protective’ antibody. Why and how Type II Antigen Produces Protective Antibodies? In an outbred population like humans, no two individuals are genetically and, therefore, anti-genetically similar in their genetic setup. This means that husband and wife are seldom similar in their genetic and, therefore, antigenic makeup. This dissimilarity in the gene distribution allows production of protective antibody in the mother by the conceptus. This is because the gene in the father and, therefore, in the conceptus is different from gene existing in the mother. This allows production of protective antibodies. Therefore, in majority of cases, the conceptus is protected and pregnancy continues. But in small number of cases, where genes and the antigens of the mother and father are similar (and gene in the conceptus is also similar) the protective antibody will not develop and the conceptus will be rejected. The commonly observed dissimilarity of gene between two individual is because of ‘random’ replication of nuclear DNA and ‘random’ realignment of genes which occurs five times before a zygote or an individual is created in the maternal environment of uterus of gametogenesis and nuclear syngamy (Fig. 5.2). b. Alloimmune Cell Mediated Immunomodulation (PBMC—‘T’ Lymphocytes) The decidua plays a fundamental role in ensuring immune tolerance toward the semiallogenic conceptus, protecting it from the mother’s immune system. Regulatory T cells (Tregs) are CD4+, CD25+ T cells, having the role to suppress the immune response. During early pregnancy, in the decidua there is an increase in T regs, which produce immunosuppressive cytokines, such as IL-10, for inducing immune tolerance. Other cells involved

in maternal immune tolerance are the uterine natural killer (uNK), a particular type of NK cells, which lose their cytotoxic functions during pregnancy and play a supportive role by enhancing angiogenesis. uNK cells induce immune tolerance by reducing inflammation through interferon- (IFN-). Thus a helpful environment in the endometrium (Th2 bias) is created through migration of favorable cytokines, preimplantation prostaglandins and proteolytic enzymes and is induced by trophoblastic antigens through conversion of Th 1 bias to Th 2 bias generated through ‘T’ lymphocytes. Normally endometrium remains nonreceptive under the influence of Th 1 bias. Progesterone stimulated induction of progesterone induced blocking factor (PIBF) blocks Th 1 and stimulates Th 2 bias in the endometrium (favourable for implantation). Therefore, during immunomodulatory phase of implantation progesterone plays a vital role (Fig. 5.5). Immunological recognition of pregnancy results in up-regulation of progesterone receptors on activated lymphocytes.8,9 In the presence of progesterone, lymphocytes of pregnant women synthesize a 34-kDa protein known as progesterone-induced blocking factor (PIBF), 8,9 which mediates both the immunomodulatory, 8,9 and anti-abortive 8,9 properties of progesterone. It has been found that PIBF expression of maternal T-lymphocytes increases as a result of pregnancy and that the stimulus for PIBF induction occurs immediately after implantation. Progesterone probably acts as an immunological suppressant blocking T-helper (Th1) activity and inducing release of Th2 cytokines (protective antibodies). 4. Defective Placental Vascularization Inadequate, placental blood flow has been suggested as one of the possible contributing causes in this subgroup. Apart from deficient nitric oxide synthesis due to progesterone inadequacy, another significant cause of compromised placental blood flow during early pregnancy is placental thrombosis due to thrombophilic disorders. It should be emphasized that pregnancy itself

Unexplained Spontaneous Miscarriage

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Fig. 5.5: Role of progesterone for the stabilization of blastocyst implantation

induced a hypercoagulable hematological environment. Identifiable causes of placental thrombosis are: 1. Already identified—APL syndrome 2. In the process of identification—inherited thrombophilic disorders. What are Probable Causes of Thrombophilic Disorders? Thrombophilia is defined as a tendency to form thrombosis. Individuals prone to develop thrombosis may be classified into three subgroups depending on etiological factors. Genetic Three genetic mutations have been identified— which if present in an individual may predispose thrombus formation. These three factors are—factor V (Leiden), factor II (prothrombin) and gene controlling regulations of methylenetetrahydrofolate reductase (MTHFR) enzyme activity. Mutation of MTHFR causes elevation of homocysteine—a by-product of methionine metabolism. In Caucasian women, one of the common causes of unexplained miscarriage is mutation of factor V (Leiden) but in south-east Asian women, incidence of hyperhomocysteinemia as a cause of

spontaneous miscarriage due to placental microthrombosis is more common. In these cases thromboprophylaxis with aspirin or LMWH is a rational treatment.10,11 Mechanism of miscarriage due to hyperhomocysteinemia a. Placental microthrombus may develop due to generalized vascular atherosclerosis. These women may also have a risk of coronary artery disease (CAD) in their mid-forties for the same reason, i.e. hyperhomocysteinemia.12–14 They usually have a background of family history of hyperinsulinemia—features of obesity, PCOS, etc. (thromboprophylaxis with Aspirin and LMWH may help).

b. Defective chorionic villous vascularization: A defective vascularization of the terminal villious may be due to thrombus formation (hyperhomocysteinemia) may be due to deficient NO (nitric oxide) generation as a consequence of progesterone deficiency (deficient eNOS)—endothelial nitric oxide synthase deficiency15—progesterone may help in these cases. c. Elevated plasminogen activator inhibitor (PA-I): This phenomenon may also be due to

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decreased response to NO vasodilation15,16 which may cause vascular endothelial damage,17 leading to placental inadequacy. d. Direct embryo toxicity: Embryo may also be damaged by direct toxic effect of homocysteine.18,19 Take Home Message • Spontaneous miscarriage is defined as interruption in the continuation of clinical pregnancy till the age of fetal viability, i.e. < 24 weeks of gestation). • In around 50% women with history of one or two previous miscarriage no cause is apparently detected. They have been designated as unexplained miscarriage. • With each pregnancy loss, the prospect of future successful pregnancy gradually declines and the possibility is reduced to 45% after 3 consecutive loss. • According to conventional definition, recurrent miscarriage is defined in a woman when she suffers 3 consecutive pregnancy losses (incidence 1%; but few clinicians will prefer to accept two consecutive losses (incidence 5%) as the more acceptable criteria for defining recurrent miscarriage. • In clinical practice, two broad groups of miscarriages are identified; sporadic and recurrent. • Majority of sporadic losses are only by ‘chance’, and remains ‘unexplained’ while in recurrent miscarriage in about 50% women, the cause remains unexplained. In remaining 50% women cause may be identified (infective, structural, endocrinal, parental chromosomal, immunological, etc.). • In unexplained group (mostly sporadic and partly recurrent) possible defects are being speculated in 4 areas—defective gametogenesis, defective embryogenesis, defective implantation, defective placental vascularization. • Defects in gametes and embryos are common causes of early pregnancy loss; they are difficult to identify and not treatable. However, most of the sporadic and very few recurrent miscarriages are in this group. • Other than the defective gamete-embryo related causes of implantation failure, ‘depth’ and ‘quality; of implantation are also equally significant factors for the remaining groups of women presenting with recurrent miscarriage. These two factors will mostly depend on dysfunction of endometrial immunomodulation and placental vascularization.

• Till now, the exact diagnosis of these two defects in women with unexplained miscarriage have not been adequately explored. • However, available evidence expressed in this chapter, suggest prophylactic progesterone and thromboprophylactic drugs (aspirin and LMWH) might appear to be rational treatment in these group of women.

REFERENCES 1. Raj Rai LR. Recurrent miscarriage. Lancet. 2006; 368:601–11. 2. Hyde KJ, Schust DJ. Genetic considerations in recurrent pregnancy loss. Cold Spring Harb Perspect Med. 2015;5(3):1–18. 3. El Hachem H, Crepaux V, May-Panloup P, Descamps P, Legendre G, Bouet PE. Recurrent pregnancy loss: Current perspectives. Int J Womens Health. 2017;9:331–45. 4. Jeve YB, Davies W. Evidence-based management of recurrent miscarriages. J Hum Reprod Sci. 2014;7(3):159–69. 5. Avidor-Reiss T, Mazur M, Fishman EL, Sindhwani P. The Role of Sperm Centrioles in Human Reproduction—The Known and the Unknown. Front Cell Dev Biol. 2019;7(October):1–15. 6. McCoy RC. Mosaicism in Preimplantation Human Embryos: When Chromosomal Abnormalities are the Norm. Trends Genet. 2017;33(7):448–63. 7. Viotti M. Preimplantation genetic testing for chromosomal abnormalities: Aneuploidy, mosaicism, and structural rearrangements. Genes (Basel). 2020;11(6). 8. Szekeres-Bartho J. The Role of Progesterone in Feto-Maternal Immunological Cross Talk. Med Princ Pract. 2018;27(4):301–7. 9. Shah NM, Lai PF, Imami N, Johnson MR. Progesterone-related immune modulation of pregnancy and labor. Front Endocrinol (Lausanne). 2019;10 (MAR):1–19. 10. Wijeyaratne CN, Nirantharakumar K, Balen AH, Barth JH, Sheriff R, Belchetz PE. Plasma homocysteine in polycystic ovary syndrome: Does it correlate with insulin resistance and ethnicity? Clin Endocrinol (Oxf). 2004;60(5):560–7. 11. Chakraborty P, Goswami SK, Rajani S, Sharma S, Kabir SN, Chakravarty B, et al. Recurrent Pregnancy Loss in Polycystic Ovary Syndrome: Role of Hyperhomocysteinemia and Insulin Resistance. PLoS One. 2013;8(5):1–6. 12. Faria-Neto JR, Chagas ACP, Bydlowski SP, Lemos Neto PA, Chamone DA, Ramirez JAF, et al.

Unexplained Spontaneous Miscarriage Hyperhomocystinemia in patients with coronary artery disease. Brazilian J Med Biol Res. 2006; 39(4):455–63. 13. Tinelli C, Di Pino A, Ficulle E, Marcelli S, Feligioni M. Hyperhomocysteinemia as a risk factor and potential nutraceutical target for certain pathologies. Front Nutr. 2019;6(April):1–13. 14. Bozkurt A, Toyaksi H, Acartürk E, Tuli A, Çayli M. The effects of hyperhomocysteinemia on the presence, extent, and severity of coronary artery disease. Jpn Heart J. 2003;44(3):357–68. 15. Dayal S, Blokhin IO, Erger RA, Jensen M, Arning E, Stevens JW, et al. Protective vascular and cardiac effects of inducible nitric oxide synthase in mice with hyperhomocysteinemia. PLoS One. 2014;9(9):1–8.

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16. M a t t e o C e s a r i , M a r c o P a h o r a n d R A I . Plasminogen activator inhibitor-1 (PAI-1): A key factor linking fibrinolysis and age-related subclinical and clinical conditions. Cardiovasc Ther. 2011;28(5):1–28. 17. Sabuncu T, Vural H, Harma M, Harma M. Oxidative stress in polycystic ovary syndrome and its contribution to the risk of cardiovascular disease. Clin Biochem. 2001;34(5):407–13. 18. Greene NDE, Dunlevy LPE, Copp AJ. Homocysteine is embryotoxic but does not cause neural tube defects in mouse embryos. Anat Embryol (Berl). 2003;206(3):185–91. 19. Garlick PJ. Pathophysiological Consequences of Homocysteine Excess. Rev Rhum. 2006;(22): 1722–5.

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6 Obesity: Definition and Etiology BN Chakravarty

INTRODUCTION

resulting in excessive accumulation of fat in the body. From biochemical definition point of view, obesity is defined as excess storage of triglycerides in adipose cells. There is also a difference between obesity and overweight. Obesity is an excess of body fat whereas overweight combines body weight including muscles, bone, fat, organs and body fluid in excess of ideal weight. The following formula gives ideal body weight in pounds: Women: 100 + {4 × (height in inches minus 60)} Men: 120 + {4 × (height in inches minus 60)} In an overweight individual, a weight very close to ideal weight, the individual is overweight but not over fat. Estimation of body fat is more helpful than measurement of height and weight. Though other complex methods are available, it is more simple to utilize ‘Body mass index nomogram’ (BMI); that defines both ‘overweight’ and ‘obesity’. The body mass index (BMI), the quetelet index is the ratio of weight divided by the height square (in metric unit):

Obesity is defined as abnormal or excessive fat accumulation that impairs health.1 The commonest cause of obesity is excessive calorie intake combined with lack of physical activity.2 Nearly every system of the body is affected due to this imbalance. The commonest ones are cardiovascular diseases, diabetes, musculoskeletal disorders, sleep apnea and increased risk of certain malignancies of breast, endometrium and colon.3,4 With these complications the life expectancy of young adults with BMI more than 45 is reduced by as much as 5 to 20 years.5 In addition to its impact on general, metabolic and possible malignant disorders, obesity also has profound adverse consequences on reproductive health of a woman. 6 The complications are mainly centered around ovulatory—menstrual dysfunction, pregnancy related complications which affect both mother and the child. These aspects of obesity specially related to reproductive health will be discussed in the subsequent chapter. Definition of Different Terminologies Related to Obesity There are two different definitions of obesity; clinical and biochemical.

BMI =

Weight (w) Height 2 (h)

Where, w = mass in kg. h = height in meters BMI = body mass index

Clinically obesity is defined as imbalance between energy intake and energy expenditure 70

Obesity: Definition and Etiology

A woman with BMI 25 or more is defined as ‘overweight’ whereas a women with BMI with 30 or more is defined as ‘obese’. Of course this definition relates to standard as observed in an average American women. In our country, the ‘cut-off’ point for overweight women can be reduced to 22; whereas for obese patients, it can also be reduced to 27. Risk factor for obesity will be lowest in middle aged women when the body mass index is 19. Hence, apart from individual risk of obesity either due to overweight or over fat, the measurement of BMI is more predictive for the possible complication, morbidity and mortality for being overweight and over fat (Fig. 6.1). Components for Height, Overweight and Over Fat (Obesity) Overweight Body weight depends on the following components:

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• Muscles and lean tissue 35% • Bones 12% • Fat 27% Rest of the body weight: Skin, organs, blood, etc. Women are More Prone to be Over Fat than Men: Why? Since men have more muscles and less body fat compared to women and muscle have high metabolic rate and consequently burns more calorie. Therefore, males are relatively less obese compared to females.

Significance of Amount and Abnormal Location of Storage Abdominal visceral fat are dangerous to human health. This abnormal storage is strong link to syndrome ‘X’ and is also a marker of deadly quartet of metabolic

Fig. 6.1: Terms used to describe various levels of body fat

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syndrome; high insulin surge, high cholesterol and also high blood pressure. Clinical markers (Fig. 6.2) are: • BMI • Waist: Hip circumference ratio

finally leads to obesity. In this context, it is worth remembering that women have more deficient leptin receptors in hypothalamus than in men. Consequently, the risk of obesity due to leptin receptor deficiency is more in women than that in men. However, a physiological level of leptin is essential for onset of puberty. The entire set of mechanism is represented in Fig. 6.3.

Fig. 6.2: Clinical “markers” of abnormal body fat and their location

Leptin in Onset of Puberty and Initiation of Reproductive Function There is physiological weight gain during onset of puberty. Increased weight gain is primarily due to onset of secretion of growth hormone (GH), IGF-1 and physiological hyperinsulinemia. Further weight gain is due to increased amount of leptin. Increased amount of leptin will suppress production of hypothalamic neuropeptide Y. Suppressed production of neuropeptide Y will lead to withdrawal of its inhibitory effects on hypothalamic release of pulsatile GnRH. As a consequence, the entire hypothalamic-pituitary axis is switched on to be activated resulting in onset of puberty.

Etiological Factors of Obesity Genetic and environmental factors are the two most significant components of obesity. Metabolic and endocrine disorders like hypothyroidism and Cushing’s syndrome also contribute a small role for obesity. In this chapter, we are considering obesity in general and their impact on reproduction in women not associated with any specific disease. In this context, two common genes need specific attention: a. Leptin and its receptor gene: Mutated in obese women b. In patients with insulin resistance and obesity; the specific gene involved is postreceptor insulin gene defect. Leptin and Obesity Leptin is a fat cell hormone regulated by leptin gene in fat cell only. Leptin regulates appetite and physical activity (BMI) through a neurotransmitter; known as neuropeptide-Y (NPY) located in the hypothalamus. Obese women have leptin deficiency or leptin resistance. Therefore, NPY activity in the hypothalamus cannot be controlled. Increased hypothalamic NPY activity increases appetite and reduces desire for physical activity. The net consequence is accumulation of fat which

Fig. 6.3: Link between leptin and obesity. Women compared to men have deficient leptin receptors in hypothalamus

Obesity: Definition and Etiology

Body weight has direct relationship to onset of puberty. The girl with anorexia nervosa will have amenorrhea because of absence of leptin or reduced number of fat cells. There is another explanation which also suggests that obesity may have an adverse impact on reproduction. Obesity is a common association of PCOS. Insulin resistance (IR) and noninsulin dependent diabetes mellitus (NIDDM) are also commonly associated with PCOS. Contrary to the previous concept, it is now accepted that obesity causes insulin resistance. It has already been suggested that in majority, specially in women, pathological obesity is central in location. Central obesity refers to abdominal and visceral obesity. Abdominal obesity includes accumulation of fat in peripheral abdominal wall whereas visceral obesity includes fat in four different areas; omental, mesenteric, retroperitoneal and perinephric fat. Visceral obesity causes hyperinsulinemia in two ways (Fig. 6.4): 1. By generating cytokines which causes insulin resistance, hyperinsulinemia, NIDDM, PCOS 2. The second pathway consists of the following sequence of events:

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Visceral fat is more susceptible for lipolysis, leading to production of free fatty acids (FFA). FFA is transported to portal circulation. In liver, it produces low density lipoprotein (LDL) leading to decreased insulin sensitivity ultimately to hyperinsulinemia—PCOS-NIDDM (Fig. 6.4). Environmental Factors for Obesity They have a big role in obesity by unmasking genetic or metabolic susceptibility. One of the examples is intake of ‘high glycemic’ index food. High glycemic index (GI) foods are those that cause rapid and short period release of sugar and lipid in the blood. This causes more desire for frequent snacking whereas low glycemic index food cause slow release of sugar in the blood (Fig. 6.5). Consequently there is less drive for frequent snacking. Some examples (Table 6.1) of food with ‘high glycemic index’ are listed in Table 6.1. Stress and Weight Gain Stress is also an environmental factor which may be responsible for weight gain. During stress, concentration of glucocorticoid increases. This has preference for high protein fat

Fig. 6.4: Link between central obesity and insulin resistance

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Fig. 6.5: Blood sugar response curves for high GI and low GI food

Table 6.1: Some examples of glycemic index Corn flakes

112

Spaghetti

50–60

All bran

55

Rice

110–120

Oatmeal

70

Potato, cooked

80–120

Whole wheat bread

50–70

Potato, mashed

101

Pizza

85

Milk

40–50

Waffles

110

Cola

97

food which increases abdominal fat storage. Abdominal fat increases insulin resistance leading to non-insulin dependent diabetes mellitus. Take Home Message • Obesity is increasing globally specially among men and women in the reproductive age group. • Although still not a great concern from Indian reproductive health point of view, but since last few decades, incidence has increased considerably amongst affluent class of population even in India. • Obesity and overweight have detrimental influences on several human body functions including reproductive health. • Obesity has been defined in two ways; clinically and biochemically. • Clinically obesity means storage of fat resulting from imbalance between energy intake and energy expenditure. • Biochemically obesity means storage of triglycerides in adipose cells. • There is also difference between overweight and obesity.

• Obesity means excess body fat whereas overweight includes muscles, bones, fat, organs and body fluid which are in excess of ideal weight. • In an overweight individual, very close to ideal weight, the individual is overweight but not over fat. • Estimation of body fat and its location is more important than measurement of height and weight. • Besides other methods, body mass index nomogram (BMI) is more simple which defines both ‘overweight and obesity’. • Women are prone to be more fat than men. • Because men have more muscles than body fat compared to women. • Muscles set our metabolic rate. Therefore, men with more muscles will lead to higher metabolism and thereby will lose more fat. • BMI indicates both the states, ‘overweight and over fat’ • From weight and obesity point of view, all individuals may be classified in five groups related to their individual BMI. • Normal weight, non-obese (BMI 18.5 to 24.9); overweight non-obese (BMI 25 to 29.9); obese (BMI 30 to 34.9); severely obese (BMI 35 to 39.9); morbidly obese (BMI 40 or more). • Apart from BMI, location of fat specially abdominal and visceral fat are dangerous for human health. • Abdominal storage of fat has strong link to ‘metabolic syndrome’. • This is also clinically evident, as women with this syndrome will have increased ‘waist-hip’

Obesity: Definition and Etiology

• • • •

• • • •

• •

•

• • •

• •

circumference ratio (< 0.8) apart from visceral and abdominal storage of fat. The two most important etiological factors of obesity are—genetic and environmental. Specific genes involved are—leptin gene and gene involved with insulin resistance and obesity. Leptin is a fat cell hormone present in fat cell only. Leptin regulates appetite and desire for physical activity through a neurotransmitter, located in hypothalamus known as neuropeptide-Y (NPY). Obese women have leptin deficiency and leptin resistance. Therefore, activity of hypothalamic NPY cannot be controlled. Increased NPY activity increases appetite and decreases desire for physical activity. At the onset of puberty, there is increased amount of leptin because of puberty associated physiological weight gain. Increased leptin concentration suppresses production of hypothalamic neuropeptide-Y. Suppressed NPY will lead to withdrawal of its inhibitory effect of hypothalamic release of pulsatile GnRH. This explains the relationship of body weight to onset of puberty with regard to leptin concentration in serum. The second defect is mutation of post-receptor insulin gene. Contrary to our previous belief, it is now known obesity causes insulin resistance. Environmental factors may cause obesity by unmasking genetic and metabolic susceptibility to obesity. One of the examples of environmental factors is intake of ‘high glycemic’ index food. High glycemic index foods are those which can cause rapid and short period of release of sugar in blood.

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• These cause more desire for frequent snacking whereas low glycemic index food will cause low release of sugar in the blood. • Stress of modern society is also considered as an environmental factor for obesity. • During stress, concentration of glucocorticoid level increases which augments preference of high protein and fat food, increasing abdominal fat storage.

REFERENCES 1. Mahutte N, Kamga-Ngande C, Sharma A, Sylvestre C. Obesity and Reproduction. J Obstet Gynaecol Canada [Internet]. 2018;40(7):950–66. Available from: https://doi.org/10.1016/j.jogc.2018.04. 030 2. Redman LM, Heilbronn LK, Martin CK, Alfonso A, Smith SR, Ravussin E. Effect of calorie restriction with or without exercise on body composition and fat distribution. J Clin Endocrinol Metab. 2007;92(3):865–72. 3. Aune D, Sen A, Prasad M, Norat T, Janszky I, Tonstad S, et al. BMI and all cause mortality: Systematic review and non-linear dose-response meta-analysis of 230 cohort studies with 3.74 million deaths among 30.3 million participants. BMJ. 2016;353. 4. Fong P, Boss D, Yap T, Tutt A, Wu P, MerguiRoelvink M. Overweight, Obesity, and Mortality from Cancer in a Prospectively Studied Cohort of U.S. Adults. N Engl J Med. 2003;348:1625–38. 5. Fontaine KR, Redden DT, Wang C, Westfall AO, Allison DB. Years of life lost due to obesity. J Am Med Assoc. 2003;289(2):187–93. 6. Chandrasekaran S, Neal-Perry G. Long-term consequences of obesity on female fertility and the health of the offspring. Curr Opin Obstet Gynecol. 2017;29(3):180–7.

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7 Obesity and Reproductive Health: Molecular and Clinical Aspect BN Chakravarty

INTRODUCTION

and molecular pathways. Endocrinological link through GnRH pulse generator, androgen, insulin, IGF-1 have already been discussed in detail in Chapter 3 of this book (PCOS and Infertility). The following paragraphs will briefly outline a few molecular links between reproductive outcome and obesity during child bearing period. The clinical impact will be discussed in the final section of this chapter.

During past few decades, incidence of obesity (defined as BMI over 30 kg/m2) is increasing rapidly both in the developed as well as in developing areas of the world.1 In 2014, the number had reached to 641 million adults (266 million in men and 375 million in women) as against 105 million adults in 1975 (34 million men and 71 million women).2 Moreover, if this trend continues, worldwide obesity prevalence will rise to 18% in men and surpass 21% in women by 2025.2 To highlight the importance, World Health Organization (WHO) has declared obesity as a global epidemic; and simultaneously stressed the idea that it still remains an under-recognized world health problem.3 It is now well recognized that depending on the degree and duration of weight gain, obesity can cause exacerbation of a number of co-morbidities including cardiovascular diseases, diabetes, musculoskeletal disorders, sleep apnea, increased rise of certain malignancies, such as breast, endometrial and colon cancer.4 Obesity also has an increased impact on reproductive health. Obese women have increased risk of menstrual dysfunction, anovulatory infertility and pregnancy related complications.5 All these adverse reproductive impacts of obesity are mediated through endocrinological

Molecular Link between Obesity and Reproductive Health As far as molecular pathways are concerned, the adverse impact of obesity is mediated through certain inflammatory products. The important bioactive molecules in the adipose tissue are ‘adipokines’. Adipokines interact through multiple molecular pathways of insulin resistance, inflammation, hypertension, cardiovascular risk, oocyte development, maturation, and embryonic implantation. Other associated adverse reproductive outcomes of obesity are related to delayed conception, increased miscarriage rates and poor success rate following assisted reproduction. The major role for these adverse impacts is played by adipokines which exist as ‘major fat tissue soluble products’. 76

Obesity and Reproductive Health: Molecular and Clinical Aspect

What are Adipokines? Adipokines are constituents of adipose tissue. Adipose tissue is now considered as an endocrine organ which will perform significant roles in coordinating many physiological events such as reproduction, immune response, glucose and lipid metabolism through release of a number of bioactive cytokines collectively known as adipokines, which commonly control metabolic regulation and inflammatory process. Very recently adipokines, the important constituents of adipose tissue have b e e n found to be intimately linked with pathophysiology of reproductive health of obese infertile women seeking treatment. The information gathered about adipokines in obese women and their impact on reproductive outcomes are briefly outlined below:

The family of adipokines includes two groups of molecules: • Adipose specific cytokines • Non-adipose specific cytokines

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Adipose specific cytokines include; leptin, adiponectin (APN), resistin, visfatin and omentin. Non-adipose specific cytokines are—retinol binding protein (RBP4), lipocalin 2 (LCN2), chemerin, interleukin 6 (IL6), interleukin 1 (IL1) and tumor necrosis factor  (TNF-). These molecules representing family of adipokines have both harmful and helpful effects on reproductive health of obese women. For example, abnormal serum levels of most of these molecules have been shown to be strongly associated with both insulin resistance (IR), type-2 diabetes mellitus (T2DM) and PCOS. Severe dysfunction of adipose tissue molecules may lead to production of certain harmful cytokines TNF- (non-adipose specific cytokines) and at the same time overproduction of some cytokines such as adipose specific cytokines (APN) may also be beneficial (Table 7.1). Apart from generating molecules directly involved with reproduction, activated adipocytes also generates molecules which may accelerate comorbidity associated with reproduction in obese women. The possible comorbidities are presented in Fig. 7.1.

Table 7.1: Beneficial and harmful effects of family of adipokines on reproductive outcome in obese women Name of adipokines

Serum levels

Beneficial or harmful effect on reproduction in obese women

Leptin

Elevated

Reduce insulin induced ovarian sterioidogenesis and LH induced E2 production in granulosa cells

Adiponectin

Suppressed

Plasma insulin increases

Resistin

Elevated

Insulin resistance +

Visfatin

Elevated

Increases insulin sensitivity

Omentin

Suppressed

Increases insulin sensitivity

Chemerin

Elevated

Increases insulin sensitivity

Fig. 7.1: The possible comorbidities of activated adipocytes which may accelerate comorbidity associated with reproduction in obese women

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Take Home Message • It is already known that obesity has adverse impact on cardiovascular disease, diabetes, musculoskeletal disorder, sleep apnea, increased risk of certain malignancy such as breast, endometrial and colon cancer. • In addition, obesity has an increased adverse impact on reproductive health. • All these adverse impacts of obesity are mediated through endocrinological and molecular pathways. • Regarding molecular link, the adverse impacts are the consequences of production of certain inflammatory products. • The important bioactive molecules in the adipose tissue are known as ‘adipokines’. • The major endocrinological and clinical impacts of obesity are mediated through ‘adipokines’ which exist as ‘major fat tissue soluble products’. • Adipose tissue is now considered as an endocrine organ. • Adipose tissue through adipokines perform many significant functions for coordinating various physiological events such as reproduction, immune response, glucose and lipid metabolism through release of a number of bioactive cytokines, the different component of adipokines which commonly control metabolic regulation and inflammatory processes. • Family of adipokines consist of two main groups; adipose specific cytokines and non-adipose specific cytokines. • Adipose specific cytokines are leptin, adiponectin (APN), resistin, visfatin and omentin. • Non-adipose cytokines are: Retinal binding protein (RBP4), lipocalin 2 (LCN2), chemerin, interleukin 6 (IL6), interleukin 1 (IL1) and tumor necrosis factor  (TNF ). • These molecules may have both harmful and helpful reproductive impact in obese women. • For example, abnormal serum levels of most of the molecules are strongly associated with both insulin resistance (IR), and type-2 diabetes mellitus (T2DM). • Similarly severe dysfunction of some adipose tissue molecules may lead to production of some harmful cytokines like TNF  (non-adipose specific cytokines). • But at the same time, overproduction of some other cytokines such as APN may also be beneficial (adipose specific cytokines).

• Apart from generating molecules directly involved with reproduction, activated adipocytes also generate molecules which may increase comorbidity associated with reproduction. • The specific co-morbidities likely to be accelerated through activated adipocytes are—hypertension, atherosclerosis, thrombophilia, diabetes and dyslipidemia.

Clinical Impact of Obesity on Fertility It is not a fact that obese women do not become pregnant. But certainly obesity reduces chances of fecundity.6, 7 Both hormonal and mechanical factors play their role. The related hormonal factors associated with obesity and reproduction are well known. Interplay of these hormones, namely insulin resistance with consequent hyperinsulinemia, low sex hormone binding globulin (SHBG), elevated androgen, increased peripheral conversion of androgen to estrogen by aromatase, increased insulin like growth factor 1 and high leptin levels8,9 lead to both menstrual and ovulatory irregularities. Because combined effects of these hormonal abnormalities induce hypothalamic dysfunction, abnormal gonadotropin secretion, reduced folliculogenesis, dysovulatory or anovulatory menstrual cycles and lower progesterone levels in the luteal phase,10–12 are the other consequences. It is accepted that anovulatory infertility is more common in obese than in non-obese women.13, 14 But it is also interesting to note that even in obese women the incidence of infertility is related to distribution of location of body fat. It has been shown that women with higher waist circumference (excess abdominal fat) are more likely to suffer from anovulatory infertility than similar obese women with same BMI who have less abdominal fat.15,16 On the other hand, even ovulatory women with obesity have less fecundity and take prolonged period for conception.17–20 The discrepancy may be due to decreased frequency of intercourse. In male obesity one of the important factors for decreased frequency of intercourse is erectile dysfunction and decreased desire. However, in obese women

Obesity and Reproductive Health: Molecular and Clinical Aspect

waist circumference, cycle irregularities and decreased frequency of intercourse increases the time to pregnancy (cycle fecundity) both in nulliparous and parous woman when BMI increases from 25 to 30 to more than 35.21

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Obesity and Male Infertility If both partners are obese, sexual relationship may be affected. Scrotal temperature may be high because of closer contact with surrounding tissues.22,23 Male with obesity have decreased testosterone level, which correlate negatively with both their fasting insulin and leptin levels. 24,25 In spite of these hormonal aberrations, there is a great deal of controversy regarding abnormalities of semen profile in obese men. Although there is no doubt that obese couples suffer from higher incidence of infertility, but it is not very clear that to what extent the sperm quality contributes to this association. Though infertility in obese man could not be precisely associated with sperm quality or function, but literature survey shows a distinct relationship between male obesity and erectile dysfunction. 26–28 Improvement of erectile dysfunction through any method, e.g. change of lifestyle, diet and exercise, pharmacological interventions or even through bariatric surgery29,30 has helped to improve the situation.

fluctuating levels) cannot produce a positive feedback effect on hypothalamus to induce LH surge hence leading to anovulation. In these cases overall suppression of static oestrogen with aromatase inhibitors (letrozole) will be better alternative than clomiphene. With regard to gonadotropin, which is more frequently used with IUI and IVF rather than with simple ovulation induction, in patients with BMI more than 30, the dose requirement will be higher and duration of administration prolonged with lower peak estradiol level and less number of larger and medium sized follicles.31 The lower response and larger dose requirement are possibly due to increased volume of distribution. Regarding hCG, absorption has been reported to be more with intramuscular rather than with subcutaneous injection. With regard to pregnancy outcome, IUIgonadotropin regime is more favorable than with simple ovulation induction. 31–34 The rational explanations are correction o f anovulation, compensation for erectile dysfunction and decreased frequency of intercourse. So far the impact of obesity on IVF outcome is concerned, the results reported by society for Assisted Reproductive Technology (SART) registry 35 is given in Table 7.2.

Response of Obese Women to Fertility Treatment Though obesity as such is not an independent indication for fertility enhancing treatment but incidentally obesity may be an intermediate co-factor which may modify the drug dose, protocol and outcome of treatment. Three common types of infertility treatments are ovulation induction, intrauterine insemination and IVF. Similarly three commonly used drugs used in these treatment protocols are—clomiphene citrate, letrozole and gonadotropins. Between clomiphene and letrozole, letrozole is preferred in obese woman. Because fat cells are rich in aromatase. Aromatase helps in conversion of testosterone to oestrogen. Excessive “static” level of estrogen (not

The results published by SART study are briefly outlined below: • There is slight decline in the number of oocytes retrieved and also in the number of high quality embryos produced as the BMI rises over 40. • Implantation, clinical pregnancy and live birth rates decline gradually with increasing severity of obesity. • However, the absolute decline in pregnancy rate is small. • Based on the data presented, implantation rate declines by 0.3 to 0.4% for each 1 kg/m2 over 25 BMI. • Thus the overall possibility of a live birth per each start cycle declines from 31.4% with a normal BMI to 28% in women with BMI 30–34.9.

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Table 7.2: The impact of female obesity of IVF outcomes (Provost MP et at.)35

Number of IVF cycles

BMI 18.5–24.9

BMI 25–29.9

BMI 30–34.9

BMI 35–39.9

BMI 40–44.9

BMI 45–49.9

BMI >50

134,588

54,822

24,922

11,747

4084

1292

463

Oocytes retrieved

12.4

12.3

12.3

12.1

11.6

11.2

10.5

Cancellation rate

10.3%

11.3%

11.3%

12.2%

13.3%

14.2%

11.7%

Embryos transferred

2.4

2.4

2.4

2.4

2.4

2.5

2.3

Implantation rate

29.5%

28.3%

26.9%

25.8%

23.6%

22.9%

20.3%

Clinical pregnancy rate

37.9%

36.8%

35.7%

33.7%

32%

30.6%

30%

Pregnancy loss rate

11.3%

12.7%

14.6%

15.3%

14.8%

17.6%

20.3%

Live birth rate

31.4%

29.8%

28%

26.3%

24.3%

22.8%

21.2%

• Further decline to 24.3% was observed in women with BMI between 40 and 44.5. • And declined birth rate was 21.2% in women with BMI more than 50. • The cause of low success rate may be due to poor embryo quality or alternatively with defective endometrial receptivity.36 • Miscarriage rate is higher possibly, again due to poor endometrial receptivity. • Even with donor egg, live birth rate per cycle start is lower compared to non-obese recipient again suggesting the cause being poor endometrial receptivity. • Obesity may create problem in the technologies of ART. Following COH; the ovaries may shift to higher position in the pelvis, making them more difficult to visualize with transvaginal scanning. • Obesity increases the risk of complications of oocyte retrieval, such as bleeding, infection and injury to surrounding structures. Anesthesia may create problem. Impact of Obesity on Maternal Risks of Pregnancy Miscarriage Miscarriage rate increases with increasing level of obesity. The incidence is similar for all types of pregnancies; either spontaneous, induced or IVF pregnancies. The hazard ratio is 1.23 in obese women compared to nonobese control. 37 Miscarriage rate in these women is higher both in sporadic as well as in recurrent miscarriage groups. In women with

history of recurrent miscarriage, the risk of subsequent miscarriage is elevated 3–4 fold in obese compared to non-obese control.38,39 Apart from miscarriage, the risk of gestational diabetes, pre-eclampsia (PE), preterm labor (PTL), premature rupture of membranes (PROM), risk of macrosomia, shoulder dystocia, etc. are all increased. Incidence of increased risk of PROM and PTL in obese patients may be due to increased circulating adipokines and inflammatory proteins. Increased risk of sleep apnea and antenatal depression has also been reported.40–43 Additionally, following operative delivery, there is increased incidence of wound infection, wound dehiscence, postpartum hemorrhage and deep venous thrombosis. Fetal and Neonatal Risks Incidence of fetal macrosomia in patients with obesity is not directly related to her fat volume but indirectly associated through association of gestational diabetes. The incidence of macrosomia has been reported to be higher 7.7% in obese diabetic compared to non-obese diabetic woman.44 Maternal obesity associated with fetal macrosomia increases the incidence of obstetric intervention. In addition, child born of obese mother has higher risk of obesity, diabetes and cardiovascular diseases in later life.44 The risk of congenital anomaly in the offspring is higher suggesting that maternal obesity alters fetal development during the embryonic period. 45 Neural tube, oral and

Obesity and Reproductive Health: Molecular and Clinical Aspect

gastrointestinal defects are also common. Other commonly observed defects consist of spina bifida, hydrocephaly, anorectal atresia and oral clefts, etc. One of the causes of these congenital anomalies is possibly poor glycemic control. Another problem in relation to fetal anomaly may be a relative difficulty in the detection of the anomaly because maternal obesity may obstruct visualisation of the defect which may be responsible for error of prenatal detection. A retrospective study has reported 20% less detection of anomalous fetus in women with high BMI compared to normal BMI. Screening and Advice to Obese Woman Seeking Fertility Care Comorbidities with obesity are well known. It has already been discussed in the earlier part of this chapter that obesity increases insulin resistance and type 2 diabetes mellitus. In addition obesity induces an inflammatory state which also accelerates the risk of hypertension, dyslipidemia and cardiovascular diseases.45–47 Central obesity in particular is associated with greater risk of cardiovascular diseases. 48–50 Obesity is also associated with two other co-morbidities which may affect a woman in her reproductive years. One is a risk of endometrial hyperplasia and endometrial carcinoma in premenopausal years.50–53 The other one is obstructive sleep apnea.54 Plenty of literature demonstrate that increased risk of endometrial carcinoma has a positive correlation with increasing level of BMI. It has been reported that compared to non-obese controls, women with BMI 40 or more have a relative risk of 2.1 dying from breast cancer and relative risk of 6.3 dying from endometrial cancer. Sleep apnea also in the long run will lead to cardiovascular dysfunction. About 35% of woman more than 35 BMI report problem of sleep apnea.55 Sleep apnea may be associated with oxygen desaturation, episodic hypercapnia, negative intrathoracic pressure, all of which will cause repeated arousal from sleep. Sleep apnea itself may be an independent prognostic

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marker for aggravating cardiovascular risk in obese woman during pregnancy. Effective Treatment of Obese Infertile Woman for Weight Reduction Depending on degree of obesity, three types of treatment are advocated; in India morbidly obese patient seeking infertility treatment are not very frequently seen as they are in USA or in Europe. The recommended treatments are: • Lifestyle modification, diet and exercise • Pharmacotherapy • Bariatric surgery In general, treatment outcome to some extent is negatively influenced due to two of the following limitations A. It is to be realised that body weight is tightly regulated by a complex homeostatic system and a strong neuroendocrine mechanism which defend the body against weight loss accounting for 95% “weight regain” (RECIDIVISM).56, 57 This is more commonly associated with behavior and obesity management. This means that unless behavioral management is continued for a longer period there is always a risk of weight gain. The same phenomenon is also common following discontinuation of pharmacologic treatment or if bariatric surgery is reversed. B. The second problem is associated with poor body image and low self-esteem. These will lead to depression, anxiety and an inferiority complex. These may have an influence on binge eating (frequent snacking, a craze for high glycemic food).58,59 These are the two challenges of the therapy advocated for the weight loss. Specific therapy will now be briefly outlined: a. Lifestyle modification, diet and exercise: This is the first and acceptable line of treatment for the woman with low or modest obesity (BMI 10 µIU/ml merit treatment with LT4.

1b. Pregnancy Related Complications and Outcomes in Subclinical Maternal Hypothyroidism Unlike overt maternal hypothyroidism, the effects of SCH on pregnancy outcomes are conflicting. SCH in first trimester increases rate of spontaneous miscarriage, irrespective of thyroid antibody status. The higher risk may be evident even in mothers with TSH

above 2.5 µIU/ml. 27 A graded increase in miscarriage risk has been demonstrated as maternal TSH concentrations increase from 2.5 µIU/ml and the effect is further augmented by the presence of anti-TPO antibody positivity.27, 28 The risk was relatively higher in antibody negative women with SCH, i.e. with TSH > 4 and < 10 µIU/ml (OR 3.40; 95% CI: 1.62, 7.15), but even more so in antibody positive women with TSH between 2.5–4 µIU/ml (OR 4.96; 95%CI: 2.76, 8.90) and highest in antibody positive SCH (OR 9.56; 95%CI: 3.76, 24.28). 28 Strong associations between anti-TPO antibody positivity and pregnancy complications in OH and anti-TPO antibody positivity and fetal outcomes in SCH have also been identified in a more recent study; this study failed to identify any association of anti-Tg antibody on any of the outcomes.29 Some studies have even reported significantly higher pregnancy loss in antiTPO antibody negative women with TSH concentrations between 2.5 and 5.0 µIU/ml compared to those with TSH concentrations below 2.5 µIU/ml.30 In summary, while the risk of miscarriage is apparent in antibody positive women with TSH above 2.5 µIU/ml, such an adverse effect in antibody negaive women has not consistently been apparent until maternal TSH exceeds 4 µIU/ml. TSH elevation above trimester specific reference ranges in first and second trimesters

Thyroid and Reproduction: Endocrinological and Clinical Aspects

is also associated with pregnancy induced hypertension and pre-eclampsia.31 Preterm birth, prematurity and respiratory distress syndrome are likely to be higher in SCH in pregnancy. SCH, when detected in third trimester, is more likely to result in small for gestational age (SGA) and LBW babies. 32 Maternal hypothyroidism of any degree has also been associated with higher pediatric endocrine morbidity (hypoglycemia and a composite of thyroid disease, diabetes, hypoglycemia and obesity), respiratory tract infections, asthma and epilepsy in the offspring.33 Treatment of SCH when maternal TSH is more than 4 µIU/ml, particularly if instituted in first trimester, reduces adverse obstetric outcomes and preterm delivery irrespective of antibody status.34, 35 Pregnant women with positive anti-TPO antibody might benefit from LT4 therapy even is initiated in the second trimester. 34 Treating pregnant women with TSH between 2.5 and 4 µIU/ml, particularly if antibody negative, likely has no benefit and may be associated with increased gestational hypertension and higher gestational diabetes instead.36 Two RCTs, the controlled antenatal thyroid screening (CATS) study and the randomized trial of thyroxine therapy for subclinical hypothyroidism or hypothyroxinemia diagnosed during pregnancy have evaluated the effect of LT4 therapy, instituted after f i r s t trimester, and found no effect on neurocognitive outcomes in offspring of mothers with SCH at 3 years and 5 years of age, respectively.37, 38 No significant effect of LT4 therapy on IQ of children at 9.5 years of age was also evident in the follow-up of the CATS trial, the CATS II study.39 A further analysis of the study has recently been published wherein 475 participants from the original study completed a series of questionnaires [strengths and difficulties questionnaire (SDQ), child attention-deficit/hyperactivity disorder (ADHD) questionnaire, and the social communication questionnaire (SCQ), used as a screen for autism spectrum disorder (ASD)] about their children’s behavior

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between 7 and 10 years of age (average age 9.5 years). No significant group differences were observed between children born to mothers with normal TFT, mothers with treated SCH and untreated SCH.40 Practice Pearl • SCH in pregnancy (TSH between upper cutoff for trimester specific reference range (or 4 µIU/ml) and 10 µIU/ml) should be treated in pregnancy, particularly if TPO antibody positive • LT4 therapy may be considered in SCH in pregnancy even when anti-thyroid antibodies are negative.

1c. Pregnancy Related Complications and Outcomes in Euthyroid Mothers with Underlying AITD Anti-TPO and anti-Tg antibody are the two markers commonly used to identify underlying AITD in clinical practice. Most studies investigating AITD and clinical outcomes, however, measured anti-TPO antibody only. Available evidence supports 2–2.5 times increased risk of spontaneous pregnancy loss, sporadic or recurrent, in women with thyroid autoimmunity vs women without thyroid antibody. The association, however, is not uniform; some studies failed to prove a positive correlation, while others found an almost two-fold increased risk of miscarriage in antiTPO positive euthyroid women, particularly those with PCOS or unexplained infertility and having a TSH of more than 2.5 µIU/ml, but not in those with TSH lower than this.41, 42 Though the association between preterm delivery and thyroid autoimmunity in women with normal TFT is not uniform, a significant bulk of available evidence suggests thyroid autoantibody positivity (anti-TPO and/or anti-Tg) is associated with 1.5–2 times increased risk for preterm delivery, even when the TSH is less than 2.5 µIU/ml. Interestingly, sub-group analysis revealed a significant association with anti-TPO antibody, but, not with antiTg antibody.43 There are some evidences to suggest that underlying thyroid autoimmunity is also associated with placental abruption,

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neonatal respiratory distress syndrome, perinatal death, subtle intellectual impairment during childhood, attention deficit/hyperactivity problems and autism spectrum disorders after adjustment for maternal TSH levels. The underlying mechanisms for such an association remain unclear and a number of mechanistic hypotheses have been proposed that include mild thyroid dysfunction, crossreactivity of antibodies with hCG receptors on the zona pellucida, the presence of concurrent non-organ specific autoimmunity, increased levels of endometrial cytokines in women with thyroid autoimmunity and immune mediated fetal resorption.44 Thyroid functions change during pregnancy and a linear association of hCG with FT4 and, an inverse association of hCG with TSH levels is observed during normal pregnancy. Underlying AITD alters these relationships as the positive association between FT4 and hCG is blunted and the negative association of TSH with hCG is significantly attenuated in anti-TPO antibody and/or anti-Tg antibody positive pregnant women compared to antibody negative ones.45 Moreover, antibody positive women with these hormonal alterations, suggesting inappropriate thyroid gland response to hCG, are at increased risk of preterm delivery. Recently TPO expression has been documented on cumulus granulosa cells of ovarian follicles.46 Interaction between the antigen (TPO) and antibody (anti-TPO) might be another explanation of higher prevalence of infertility, spontaneous pregnancy loss and prematurity in AITD. Thyroid antibodies and levothyroxine (TABLET) trial, is a recently published placebo controlled randomized controlled trial, that included euthyroid anti-TPO antibody positive women with a history of infertility or miscarriage and evaluated the effect of LT4 on live birth.47 It was found that a fixed dose of 50 µg of LT4, started preconception and continued throughout pregnancy, did not alter the rates of miscarriage or live birth. In a another prospective interventional trial, LT4 treatment of euthyroid anti-TPO antibody positive pregnant women significantly reduced the

rate of preterm delivery than the untreated group. There was also a significant decrease in miscarriage rates (from 13.8 to 3.5%) in LT4 treated euthyroid anti-TPO-positive women.48 The dose of LT4 in that particular study was higher and was adjusted according to TSH levels to achieve a level, identical to that of antibody negative controls during pregnancy. However, the mean estimated gestational age at which LT4 was started was ten weeks, and all but one of the eight losses in the untreated group had occurred before 11 weeks. So, the favourable impact of the intervention upon the primary endpoint remains questionable. Another ongoing multicenter randomized, double blind placebo controlled trial (T4LIFE), is investigating the effect of LT4 administration pre-pregnancy on live birth rate in euthyroid anti-TPO antibody positive women with recurrent miscarriage. As the design of the T4-LIFE is very similar to the TABLET study, it will be interesting to see if the TABLET’s results will be confirmed. In contrast to association studies, interventional studies of LT4 therapy for prevention of preterm delivery are sparse. Preterm birth, a secondary outcome of the TABLET trial, was not different between the LT4-treated and placebo groups. The discrepancy between association studies and intervention studies probably suggest multiple pathogenic mechanisms and an interesting model has recently been proposed (Fig. 11.4). In the early stage of AITD, concentrations of circulating anti-thyroid antibodies are relatively low. At this stage, the thyroidal response to hCG is unimpaired and the main impact is through TPO-anti-TPO antibody interaction in ovarian tissue. These patients are unlikely to be benefit from LT4 supplementation and agents that reduce antibody titres like selenium, glucocorticoid and intravenous immunoglobulin might be effective. In fact, these agents have been found to be effective in some studies. On the other hand, with increasing concentrations of antibody level, the thyroidal response to hCG is impaired and patients develop relative thyroid hormone insufficiency during the

Thyroid and Reproduction: Endocrinological and Clinical Aspects

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Fig. 11.4: Proposed model linking thyroid autoimmunity and infertility (Dosiou C49)

stress of pregnancy. A portion of patients of this particular subgroup may benefit from LT4 therapy. Euthyroid women with underlying AITD are more likely to manifest thyroid dysfunction during pregnancy due to stress of pregnancy on a thyroid gland with compromised reserve, necessitating increased surveillance throughout pregnancy. ATA suggests TSH estimation every 4 weeks till mid-pregnancy (16– 20 weeks) and at least once around week 30. According to the 2017 American Thyroid Association (ATA) pregnancy guidelines, LT4 treatment may be considered in antibody positive pregnant women having TSH between 2.5 µIU/ml and the upper limit of the trimesterspecific reference range. Practice Pearl LT4 therapy in pregnancy if TSH is between 2.5 and 4 µIU/ml and thyroid antibody is positive may be considered after discussion with the woman; though, not practised by the authors.

1d. TSH Target in Pregnancy for LT4 Started Before or, in Current Pregnancy Once LT4 therapy is initiated in pregnancy, the TSH needs to be kept in the lower half of

trimester specific reference range or less than 2.5 µIU/ml, if the former is not available.22 If a lady, who was biochemically euthyroid on LT4, becomes pregnant a TSH should be checked at the earliest and the daily dose of LT4 should be increased by 25–30% pending consultation. This is particularly important if the pre-conception TSH was more than 1.5 µIU/ml, to avoid TSH elevation in first trimester. Alternatively, she may be advised to take 2 additional tablets of the same dose weekly, meaning thereby taking 9 tablets a week, as suggested by the ‘THERAPY’ trial; this corresponds to 29% increase in LT4 dosage. 50 In this process, sub-normal TSH may be encountered in some patients suggesting over replacement. Though erring on the side of overdosing is preferred over underdosing, over-treatment is better be avoided as both low and high FT4 concentrations have been found to be associated with decrease in child IQ and MRI evidence of reduced cerebral grey matter volume.51 In addition, the CATS II trial has shown that worse behavior problems are seen in children born to mothers, who had been over-treated (as defined by high FT4)

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with LT4 during their pregnancy. 40 To summarize, LT4 therapy in mother should be adjusted to keep FT4 in the upper half and TSH in the lower half of trimester specific reference range. Table 11.3 denotes the indications of LT4 therapy in pregnancy. Practice Pearl Table 11.3: Treatment of hypothyroidism in pregnancy, including TSH targets TSH (µIU/ml) in any trimester

Anti-TPO antibody

LT4 therapy

 10

Not required

Yes

4–10

Positive

Yes

4–10

Negative

Probably yes

2.5–4

Positive

Consider, particularly in high risk groups

2.5–4

Negative

No

3x upper reference for the assay) at or after 18–22 weeks the fetus should be carefully monitored for development of fetal hyperthyroidism throughout the rest of pregnancy. The sonological evidences of fetal hyperthyroidism are fetal goitre, fetal tachycardia (heart rate >170/min persistent for over 10 minutes and moderate variability exhibiting acceleration without deceleration), IUGR, accelerated bone maturation ( d i s t a l femoral ossification before 31 wk), signs of congestive heart failure, and fetal hydrops. Practice Pearl • Thyrotoxicosis in pregnancy, especially in the first half, needs differentiation between gestational transient thyrotoxicosis and Graves’ disease

• Graves’ disease in pregnancy may be treated with small doses of carbimazole/ methimazole, with discussion regarding small risk of birth defect in the newborn. • Women with active or, even inactive Graves’ disease in pregnancy should have measurement of TSH—receptor antibody as this may predict fetal hyperthyroidism.

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A. Thyroid Disorders in Postpartum Period The rebound of immune system after the relatively immunosuppressed state of pregnancy may herald new onset GD or postpartum thyroiditis (PPT) in women with underlying AITD. PPT is an inflammatory autoimmune condition, wherein preformed THs are released due to destruction of the thyroid follicular cells. In its classic form, PPT is characterized by transient thyrotoxicosis that usually appears between 2–6 months following delivery, followed by transient hypothyroidism and ultimately a state of euthyroidism by the end of one year post-partum. However, permanent hypothyroidism following spontaneous resolution of thyrotoxicosis is encountered in 10–20% of such cases. The clinical course of PPT varies, with approximately one quarter of patients present with the classical form, one quarter with isolated thyrotoxicosis phase and the remaining one-half present with isolated hypothyroidism.55 Postpartum GD and PPT needs to be differentiated from each other as these two disorders have different natural course and management strategies. Early onset of thyrotoxicosis (within 3 months of delivery) and TT3: TT4 (ng: µg) 20, positive TRAb, increased thyroidal vascularity on doppler USG suggest GD. In difficult cases, thyroid scintigraphy with technetium, done during thyrotoxic phase, reliably differentiates the two conditions as the uptake is low in PPT while normal/high in GD. During the thyrotoxic phase of PPT, symptomatic women may be treated with beta-blockers, and ATD is not required. Practice Pearl • Thyrotoxicosis in postpartum period needs differentiation between postpartum thyroiditis and Graves’ disease.

• The latter may present with permanent primary hypothyroidism without a clinically recognizable preceding thyrotoxic phase.

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B. Thyroid Disorders and Assisted Reproduction Controlled ovarian hyperstimulation during assisted reproduction technique (ART) significantly elevates serum estradiol level to values seen during late pregnancy. Rapid elevation of serum estradiol increases TBG, which in turn reduces FT4 and thus raises TSH by negative feedback. The elevation in TSH is unpredictable, but is higher in women with positive anti-thyroid antibodies and/or higher baseline TSH. Thyroid function testing should be performed either before or 1 – 2 weeks after controlled ovarian hyperstimulation, since results obtained during this period may be difficult to interpret. In nonpregnant women with mild TSH elevations following controlled ovarian stimulation, serum TSH measurements should be repeated in 2–4 weeks, since levels may normalize. Findings from different retrospective studies suggest better pregnancy outcomes (increased clinical pregnancy rates, decreased prematurity and decreased incidence of LBW delivery) following in vitro fertilization (IVF), if TSH is less than 2.5 µIU/ml. Adverse effects have been observed more consistently when TSH is more than 4.5 µIU/ml. Beneficial effect (like higher pregnancy rates, lower pregnancy loss and higher live birth rates) of LT4 is clearly observed if treatment is started at TSH levels more than 4.5 µIU/ml. Though data are conflicting, LT4 treatment in women undergoing ART seems to improve pregnancy rates even with TSH concentrations above 2.5 µIU/ml. Quality of embryo during IVF is reportedly poor in euthyroid antibody positive women compared with antibody negative controls. Despite above, thyroid autoimmunity probably does not alter miscarriage rate in euthyroid women undergoing intracytoplasmic sperm injection (ICSI). Not surprising that LT4 does not alter clinical pregnancy rate, miscarriage rate, preterm birth rate or, live birth rate in euthyroid antibody-positive women undergoing ART.56–59 Two small studies, however, found a beneficial effect of glucocorticoids on overall pregnancy rates, when used before intrauterine insemination (IUI) or embryo transfer

in antibody positive women with TSH less than 2.5 µIU/ml.60, 61 A meta-analysis looking at effect of LT4 on obstetric outcomes in patients with subclinical hypothyroidism or thyroid autoimmunity undergoing IVF after achieving euthyroid state concluded that though the miscarriage rates decrease by about 50%, live birth remained unchanged.62 Practice Pearl • Thyroid function testing, if needed, should be conducted before or, 1–2 weeks after controlled ovarian hyperstimulation. • For women undergoing ART LT4 is indicated when TSH > 4.5 µIU/ml; benefit in those with TSH > 2.5 µIU/ml and < 4.5 µIU/ml, with or without antibody positivity is equivocal • Glucocorticoid therapy for euthyroid, autoantibody positive women undergoing ART is not based on solid evidence. Take Home Message • Thyroid hormones play important role in the normal function of hypothalamo-pituitarygonadal axis in both sexes. Thyroid hormone receptors are present in the hypothalamus, pituitary, testes, ovarian epithelial cells, oocytes, ovarian granulosa cells, uterine endometrium, placenta and fetal tissues; hence it is not difficult to surmise that human reproduction should be dependent on normal thyroid function. • Both hypothyroidism and hyperthyroidism are associated with disorders in puberty, menstrual irregularities, altered sexual behavior, infertility and unfavourable pregnancy outcomes. • Thyroid autoimmunity per se, irrespective of maternal thyroid status, has also been implicated in female infertility, spontaneous miscarriage and unwanted maternal and fetal morbidities. • Thyroid function tests (TFT) are altered significantly by the hormonal changes in uncomplicated pregnancy; hence these need to be interpreted with caution in pregnant women. If available, trimester specific reference ranges for FT4, TT4 and TSH should be used to interpret TFT in pregnancy. • Overt hypothyroidism (OH) in pregnancy, as defined by TSH > 10 µIU/ml irrespective of FT4/ TT4 value, is associated with adverse pregnancy

Thyroid and Reproduction: Endocrinological and Clinical Aspects outcomes like gestational hypertension, miscarriage, fetal death, prematurity and unfavorable fetal consequences like LBW and lower IQ. Hence, OH in pregnancy needs to be treated. • Sub clinical hypothyroidism (SCH) (normal FT4/ TT4 and TSH more than reference range, but less than 10 µIU/ml) in pregnancy is associated with spontaneous miscarriage and the risk probably starts increasing when maternal TSH concentration exceeds 2.5 µIU/ml. The risk is directly proportional to degree of TSH elevation and further augmented by underlying thyroid autoimmunity. Maternal SCH is also associated with pregnancy induced hypertension, preterm birth, and SGA. Effect of SCH in pregnancy on IQ of the offspring is unclear. a. Thyroid antibody positive pregnant women with SCH should be treated with LT4. b. LT4 therapy may be considered in SCH in pregnancy, even when anti-thyroid antibodies are negative. c. Role of LT4 in pregnant women with thyroid autoimmunity and TSH between 2.5–4 µIU/ml is doubtful. • Two common causes of thyrotoxicosis in pregnancy are Gestational Thyrotoxicosis and Graves’ Disease (GD), which are often differentiated by thorough history and examination. The former is treated symptomatically, while the later may require anti-thyroid drugs. • Thyroid function may be altered in postpartum period owing to the rebound autoimmunity after the relative immunosuppression of pregnancy. Postpartum thyroiditis and new onset or relapse of GD are not infrequent in postpartum period; the former usually needs symptomatic and the latter specific treatment. • Hormonal changes associated with controlled ovarian hyperstimulation during assisted reproduction techniques have an impact on thyroid function, particularly in presence of underlying autoimmunity or compromised thyroid gland reserve. TFT should be monitored in the context of controlled ovarian hyperstimulation.

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of response of treatment for hypothyroidism on infertility. Int J Appl Basic Med Res. 2012; 2(1):17–9. Yoshioka W, Amino N, Ide A, et al. Thyroxine treatment may be useful for subclinical hypothyroidism in patients with female infertility. Endocr J. 2015; 62:87–92. Chen CW, Huang YL, Tzeng CR, et al. Idiopathic low ovarian reserve is associated with more frequent positive thyroid peroxidase antibodies. Thyroid. 2017; 27:1194–1200. Korevaar TIM, Mý´nguez-Alarco´n L, Messerlian C, et al. Association of Thyroid Function and autoimmunity with ovarian reserve in women seeking infertility care. Thyroid. 2018; 28:1349– 58. Poppe K, Glinoer D, Van Steirteghem A, et al. Thyroid dysfunction and autoimmunity in infertile women. Thyroid. 2002;12(11):997–1001. Monteleone P, Parrini D, Faviana P, et al. Female infertility related to thyroid autoimmunity: the ovarian folliclehypothesis. Am J ReprodImmunol. 2011; 66(2):108–14. Van Voorhis BJ, Stovall DW. Autoantibodies and infertility: a review of the literature. J Reprod Immunol. 1997;33(3):239–56. Alexander EK, Pearce EN, Brent GA, et al. 2017 Guidelines of the American Thyroid Association for the Diagnosis and Management of Thyroid Disease During Pregnancy and the Postpartum. Thyroid. 2017;27(3):315–89. Carani C, Isidori AM, Granata A, et al. Multicenter study on the prevalence of sexual symptoms in male hypo- and hyperthyroid patients. J Clin Endocrinol Metab. 2005;90(12):6472–9. Pramanik S, Mukhopadhyay P, Ghosh S. Total T4 rise in pregnancy: a relook?. Thyroid Res. 2020; 13:14. Pramanik S, Mukhopadhyay P, Bhattacharjee K, et al. Trimester-specific reference intervals for thyroid function parameters in Indian pregnant women during final phase of transition to iodine sufficiency. Indian J Endocr Metab 2020;24: 160–4. Berbara TMBL, Morais NS, Saraiva DA, et al. Selective case finding versus universal screening for detecting hypothyroidism in the first trimester of pregnancy: a comparative evaluation of a group of pregnant women from Rio de Janeiro. Arch EndocrinolMetab. 2020;64(2):159–64. Kianpour M, Aminorroaya A, Amini M, et al. Thyroid-stimulating hormone (TSH) serum levels and risk of spontaneous abortion: A prospective population-based cohort study. ClinEndocrinol (Oxf). 2019;91(1):163–9.

28. Liu H, Shan Z, Li C, et al. Maternal subclinical hypothyroidism, thyroid autoimmunity, and the risk of miscarriage: a prospective cohort study. Thyroid. 2014; 24(11):1642–9. 29. CigrovskiBerkoviæ M, Herman Maheèiæ D, MarinkoviæRadoševiæ J, et al. Hypothyroidism and pregnancy: still a controversial issue. Gynecol Endocrinol. 2020;1–5. 30. Negro R, Schwartz A, Gismondi R, et al. Increased pregnancy loss rate in thyroid antibody negative women with TSH levels between 2.5 and 5.0 in the first trimester of pregnancy. J Clin Endocrinol Metab. 2010; 95:E44–E48. 31. Wu MQ, Liu J, Wang YQ, et al. The Impact of Subclinical Hypothyroidism on Adverse Perinatal Outcomes and the Role of Thyroid Screening in Pregnancy. Front Endocrinol (Lausanne). 2019; 10:522. 32. Derakhshan A, Peeters RP, Taylor PN, et al. Association of maternal thyroid function with birthweight: a systematic review and individualparticipant data meta-analysis. Lancet Diabetes Endocrinol. 2020;8(6):501–10. 33. Eshkoli T, Wainstock T, Sheiner E, et al. Maternal Hypothyroidism during Pregnancy and the Risk of Pediatric Endocrine Morbidity in the Offspring. Am J Perinatol. 2019;36(9):975–80. 34. Zhao L, Jiang G, Tian X, et al. Initiation timing effect of levothyroxine treatment on subclinical hypothyroidism in pregnancy. Gynecol Endocrinol. 2018;34(10):845–8. 35. Nazarpour S, Ramezani Tehrani F, Simbar M, et al. Effects of Levothyroxine on Pregnant Women With Subclinical Hypothyroidism, Negative for Thyroid Peroxidase Antibodies. J Clin Endocrinol Metab. 2018;103(3):926–35. 36. Maraka S, Mwangi R, McCoy RG, et al. Thyroid hormone treatment among pregnant women with subclinical hypothyroidism: US national assessment. BMJ. 2017;356:i6865. 37. Lazarus JH, Bestwick JP, Channon S, et al. Antenatal thyroid screening and childhood cognitive function. N Engl J Med. 2012;366(6):493–501. 38. Casey BM, Thom EA, Peaceman AM, et al. Treatment of Subclinical Hypothyroidism or Hypothyroxinemia in Pregnancy. N Engl J Med. 2017;376(9):815–25. 39. Hales C, Taylor PN, Channon S, et al. Controlled Antenatal Thyroid Screening II: Effect of Treating Maternal Suboptimal Thyroid Function on Child Cognition. J ClinEndocrinolMetab. 2018;103(4): 1583–91.

Thyroid and Reproduction: Endocrinological and Clinical Aspects 40. Hales C, Taylor PN, Channon S, et al. Controlled Antenatal Thyroid Screening II: Effect of Treating Maternal Suboptimal Thyroid Function on Child Behavior. J ClinEndocrinolMetab. 2020;105(3): dgz098. 41. Unuane D, Velkeniers B, Bravenboer B, et al. Impact of thyroid autoimmunity in euthyroid women on live birth rate after IUI. Hum Reprod. 2017; 32:915–22. 42. Seungdamrong A, Steiner AZ, Gracia CR, et al. for the Eunice Kennedy Shriver National Institute of Child Health and Human Development Reproductive Medicine Network. Preconceptionalantithyroid peroxidase antibodies, but not thyroid-stimulating hormone, are associated with decreased live birth rates in infertile women. FertilSteril. 2017; 108: 843–50. 43. He X, Wang P, Wang Z, et al. Thyroid antibodies and risk of preterm delivery: a meta-analysis of prospective cohort studies. Eur J Endocrinol. 2012; 167(4):455–64. 44. Twig G, Shina A, Amital H, et al.. Pathogenesis of infertility and recurrentpregnancy loss in thyroid autoimmunity. J Autoimmun. 2012; 38(2–3):J275-J281. 45. Korevaar TI, Steegers EA, Pop VJ, et al. Thyroid autoimmunity impairs the thyroidal response to human chorionic gonadotropin: two populationbased prospective cohort studies. J Clin Endocrinol Metab. 2017; 102:69–77. 46. Monteleone P, Faviana P, Artini PG. Thyroid peroxidase identified in human granulosa cells: another piece to the thyroid-ovary puzzle? Gynecol Endocrinol. 2017; 33:574–6. 47. Dhillon-Smith RK, Middleton LJ, Sunner KK, et al. Levothyroxine in women with thyroid peroxidase antibodies before conception. N Engl J Med. 2019; 380:1316–25. 48. Negro R, Formoso G, Mangieri T, et al. Levothyroxine treatment in euthyroid pregnant women with autoimmune thyroid disease: effects on obstetrical complications. J ClinEndocrinolMetab. 2006; 91:2587–91. 49. Dosiou C. Thyroid and Fertility: Recent Advances. Thyroid, 2020;30(4):479–86. 50. Yassa L, Marqusee E, Fawcett R, et al. Thyroid hormone early adjustment in pregnancy (the Therapy) trial. J Clin Endocrinol Metab. 2010;95 (7):3234–41. 51. Korevaar TI, Muetzel R, Medici M, et al. Association of maternal thyroid function during early pregnancy with offspring IQ and brain morphology in childhood: a population-based prospective cohort study. Lancet Diabetes Endocrinol. 2016; 4(1): 35–43.

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52. Millar LK, Wing DA, Leung AS, et al. Low birth weight and pre-eclampsia in pregnancies complicated by hyperthyroidism. Obstet Gynecol. 1994; 84: 946–9. 53. Andersen SL, Olsen J, Laurberg P. Foetal programming by maternal thyroid disease. Clin Endocrinol (Oxf). 2015; 83(6):751–8. 54. Andersen SL, Olsen J, Wu CS, et al. Birth defects after early pregnancy use of antithyroid drugs: a Danish nationwide study. J ClinEndocrinol Metab. 2013; 98(11):4373–81. 55. Stagnaro-Green A. Approach to the patient with postpartum thyroiditis. J ClinEndocrinolMetab. 2012 ; 97(2):334–42. 56. Poppe K, Autin C, Veltri F, et al. Thyroid autoimmunity and intracytoplasmic sperm injection outcome: a systematic review and meta-analysis. J Clin Endocrinol Metab. 2018; 103:1755–66. 57. R a o M , Z e n g Z , Z h o u F , e t a l . E f f e c t o f levothyroxine supplementation on pregnancy loss and preterm birth in women with subclinical hypothyroidism and thyroid autoimmunity: a systematic review and metaanalysis. Hum Reprod Update. 2019; 25:344–61. 58. WangH, GaoH, ChiH, et al. Effect of levothyroxine on miscarriage among women with normal thyroid function and thyroid autoimmunity undergoing in vitro fertilization and embryo transfer: a randomized clinical trial. JAMA. 2017; 318:2190–98. 59. N e g r o R , M a n g i e r i T , C o p p o l a L , e t a l . Levothyroxine treatment in thyroid peroxidase antibody-positive women undergoing assisted reproduction technologies: a prospective study. Hum Re-prod. 2005; 20:1529–33. 60. Turi A, Giannubilo SR, Zanconi S, et al. Preconception steroid treatment in infertile women with antithyroid autoimmunity undergoing ovarian stimulation and intrauterine insemination: a double-blind, randomized, prospective cohort study. ClinTher. 2010;32(14):2415–21. 61. Michael AE, Papageorghiou AT. Potential significance of physiological and pharmacological glucocorticoids in early pregnancy. Hum Reprod Update. 2008;14(5):497–517. 62. Rao M, Zeng Z, Zhao S, et al. Effect of levothyroxine supplementation on pregnancy outcomes in women with subclinical hypothyroidism and thyroid autoimmuneity undergoing in vitro fertilization/intracytoplasmic sperm injection: an updated meta-analysis of randomized controlled trials. ReprodBiolEndocrinol. 2018; 16:92– 100.

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12 Müllerian Anomalies: A New Clinical Classification and Overview BN Chakravarty

INTRODUCTION

Abnormalities of müllerian ducts may involve different segments of female genital tract either individually or collectively. In these women, phenotypes and external genitalia are typically feminine and gonads are histologically and functionally normal ovaries. Disorders of müllerian duct development may adversely affect three distinct areas intimately involved in reproduction. The anatomical and physiological functions of reproduction which are closely related to development of müllerian ducts are: a. Cyclic menstruation b. Potential for reproduction c. Normal sexual function This background allows us an opportunity to classify müllerian anomalies based on the broad subjective and objective findings, with a view for planning the rational treatment protocol for specific group of anomalies. For example, müllerian anomalies with presence of a functioning uterus will have a specific type of approach of treatment which is not similar to those applicable for müllerian anomalies with absence of a functioning uterus (MRKH). This is because müllerian anomalies with a functioning uterus will have different sub-groups of abnormal anatomical presentation leading to total outflow obstruction

(total cryptomenorrhea), partial outflow obstruction (partial cryptomenorrhea) or no outflow obstruction. The last group comprising normally menstruating women, may remain undiagnosed unless they have the subjective symptoms of infertility and/or recurrent pregnancy loss. Moreover, some o f these anomalies are easily treatable while in others, treatment may be extremely challenging. Based on this concept, we have proposed a new therapeutic classification of müllerian anomalies with a view to categorize them in groups likely or unlikely to be benefited with available measures of surgical or semi surgical correction procedures. Group A: Müllerian anomalies with absence of functioning uterus: True amenorrhea  typical MRKH syndrome Group B: Müllerian anomalies with presence of a functioning uterus but with: • (B1) Complete outflow obstruction • (B2) Partial outflow obstruction • (B3) No outflow obstruction Group C: Rare müllerian anomalies; for example, segmental absence of fallopian tubes; stenotic narrow introitus associated with either complete bladder and urethral exstrophy (ectopia vesicae), etc. (few case reports have been published by the author).

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ANATOMIC AND CLINICAL PRESENTATION OF THESE DISORDERS

Group A: Müllerian Anomalies with Absence of Functioning Uterus; Typical Meyer-Rokitansky-Küster-Hauser (MRKH) Syndrome—True Amenorrhea) Incidence Literature survey provides a wide range of prevalence varying between 1 and 26%.1 It has been reported that müllerian anomalies have a mean prevalence of 3–4%2 worldwide. Of all müllerian anomalies, the incidence of MRKH syndrome has been reported to be varying between 1 in 4000 and 1 in 10000.3 While some studies attribute gonadal dysgenesis and müllerian anomalies to be the two most common causes of primary amenorrhea,4, 5 other reports specially from India6 and Thailand7 suggest MRKH syndrome to be the most common cause of primary amenorrhea. Clinical Presentation They usually present during adolescence with symptoms of primary amenorrhea and ‘blind’ vagina. Vagina may be absolutely blind or there may be a vaginal pouch (about 2.5 cm depth). Reared up as females, they have typical female phenotype with normal development of breast, female body proportion, hair distribution, external genitalia and 46XX chromosome. They may have associated skeletal

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and other abnormalities specially related to kidney, urethral opening and spine. Occasionally Turner’s features may be associated with MRKH syndrome. Some of the commonly observed features of external genitalia are presented in Fig. 12.1. Internal Pelvic Organs in MRKH Syndrome Two solid müllerian bulbs (embryologic precursors of uterus) are seen on either side of the pelvis connected by a thick solid fibromuscular band running underneath the vesicorectal fold of peritoneum just behind the bladder. The downward and medial-ward migrating two müllerian ducts unite in the midline behind the bladder. The small knob behind the bladder detected on ultrasound may sometimes be reported as a small hypoplastic uterus. Ovaries look normal often polycystic. Fallopian tubes appear normal with normal patency though occasionally they are hypoplastic and blocked. In less than 10% of cases a functioning uterus (often bicornuate) may exist with hematometra and hematosalpinx. Even when the knobs are non-functioning small islets of hematometra may lead to periodic pelvic pain. Association of endometriosis and fibroid are not uncommon with MRKH syndrome. A few photographs (both schematic and operative) of internal pelvic organs of MRKH syndrome are presented below (Fig. 12.2).

Fig. 12.1: (A) Blind vagina with normal feminine appearance in MRKH syndrome (a small vaginal pouch is visible), (B) MRKH syndrome: Appearance of external genitalia; Big urethral opening in the central part of introitus with blind vagina, (C) This figure represents a case of blind vagina with absent introitus. The two big openings above and below the thin strip of perineal skin are the dilated urethra and dilated rectum. Photograph of external genital organ is from a married girl. She used to have satisfactory sexual relation either through rectal or urethral opening

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Fig. 12.2: (A) Internal pelvic organs (schematic) and internal pelvic organs (laparotomy), (B) MRKH-big müllerian knob, big ovaries (PCOS), (C) MRKH-bladder peritoneum pulled up cervical knob visible; Cervical knob behind the bladder is also seen. The müllerian knobs are also visible on either side of pelvis connected by a fibromuscular band running underneath the vesicorectal fold of peritoneum

Diagnosis Clinical features, USG scan provide sufficient evidences for diagnosis. MRI may confirm but not mandatory and expensive as well. Management of Blind Vagina (MRKH Syndrome) The treatment is to create a new vagina (neovaginoplasty) for restoration of sexual function. Menstrual and reproductive function cannot be restored. Surrogacy used to be the only choice for achieving a genetic motherhood. Recently, delivery of viable baby has been reported8 following uterine transplantation in women with MRKH syndrome. Even if uterine transplantation is possible in near future, vaginoplasty is pre-requisite for restoration of complete reproductive function.

Vaginoplasty There are two methods used for creating a new vagina: (a) Non-surgical—pressure technique and (b) surgical—neovaginoplasty. Though majority of opinion is in favor of surgical correction, many gynecologists including American Congress of Obstetrician and Gynecologists have recommended nonsurgical pressure method as the first therapy since 2006.9 Various methods of creating a new vagina are listed below. A. Non-surgical methods Dilatation: Non-surgical method of creating vagina by simple use of dilatation was first designed in 1938 by Frank and subsequently modified by Ingram10,11 (Fig. 12.3).

Fig.12.3: Vaginal moulds designed by author

Müllerian Anomalies: A New Clinical Classification and Overview

Use of Moulds It has been reported that vagina formed by non-surgical pressure method remained permanent in depth and caliber even if the patient has neglected the procedure for more than a year. Though success has been achieved following non-surgical method using Frank’s or Ingram’s technique but consensus of opinion is more in favor of surgical canalization of a blind vagina. If properly and correctly performed it provides satisfactory results both anatomically and functionally. B. Surgical methods Three broad principles of currently used surgical methods (vaginoplasty): a. Creation of a space between bladder and rectum. Dissection of an avascular space up to the peritoneum of POD is the primary step of a successful vaginoplasty. b. A graft is essential to cover the raw area of the newly dissected space—commonly used for graft is split thickness autologous skin graft obtained from buttocks of the patient. Other types of graft have also been used (e.g. peritoneum, amnion, bowel, etc.). c. The most important step is to prevent constriction and closure of the space during the process of healing. This is achieved by continuous and prolonged dilatation by use of mould for a variable period of time during the postoperative phase. The idea of modern vaginoplasty started following combined effort of Abbe–Wharton and McIndoe.12–14 Primarily Abbe–Wharton showed the way by creating a space between bladder and rectum, necessity of placing an inlay graft and in addition, importance of continuous prolonged dilatation of the dissected space by placing a ‘balsa form’ during the contractile phase of healing. McIndoe only popularized the method by giving it a substantial trial. The work was published in late 1940s.

Types of Vaginoplasty • Abbe-McIndoe method (1938):12,13 inner wall of the cavity is lined with split thickness skin graft held in place with mould/forms

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• Wharton (1938):14 Placed a condom-covered mould instead of skin graft in the neovagina. • Different modifications of McIndoe (using different types of graft): – Full thickness skin graft – Amnion15,16 – Peritoneum17 – Interceed – Bowel vaginoplasty—Ileum18, Sigmoid.19, 20 Now these methods are obsolete. Occasionally these techniques are performed for vaginal augmentation or creation of vaginal pouch after exenteration operation. Recent Laparoscopic Approach Only the basic approach of some of the commonly used procedures are being enumerated. • Vecchietti approach:21 Vaginal dilatation with ovoid bead or mould in contact with vestibular area and attached to the abdominal wall by wires which are threaded retroperitoneally via laparoscope. Vesicorectal space may or may not be dissected for negotiation of the wire. There is a risk of bladder and/or rectal injury by this procedure (Fig. 12.4). • Davydov’s abdomino (laparoscope) perineal approach:22 Creating neovagina (by perineal dissection) and laparoscopic approach for dissection and fixation of peritoneal graft (through combined abdomino (laparoscope) perineal approach) and fixing peritoneal graft over the raw area of neovagina.

Fig. 12.4: Laparoscopic Vecchietti procedure

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Differential Diagnosis of Absence of Vaginal Opening (Blind Vagina) An anxious mother may bring her girl child before puberty when vaginal opening is not clearly visible (confusion with blind vagina). The causes of such abnormality in the child may be—(a) Labial adhesion, due to low grade chronic vulvitis, (b) labia fusion, may be due to congeital adrenal hyperplasia (CAH) or (c) incomplete variety of testicular feminizing syndrome, (d) imperforate hymen or (e) complete absence of vagina (Fig. 12. 5).

Clinical markers, USG scan, laparoscope, MRI, RIA and chromosomal analysis may identify the exact diagnosis. Previusly, the provisional diagnosis was made by clinical landmarks including external examination which was corroborated by sudsequent laparotomy. The approach to clinical diagnosis for differentation of these defects have been presented in the following table formate (Table 12.1) and photographic presentations (Fig. 12.5).

Table 12.1: Difference between imperforate hymen and complete absence of vagina Imperforate hymen

Complete absence of vagina

Color of the obstructing membrane

Pearly white, smooth

Pinkish with rugosity of obstructing membrane

Concavity or convexity of the obstructing membrane

Convex outwards

Concave inwards

Syringing and needle puncture test

Saline introduced through obstructing membrane can be re-aspirated Microscopic examination reveals squamous epithelium

Saline injected cannot be aspirated

Rectal examination

Uterus palpable

Uterus not palpable

Fig. 12.5A to E: (A) Labial adhesion, (B) congenital adrenal hyperplasia (girl aged 14 yr), (C) incomplete variety of testicular feminizing syndrome in girl aged 12 yr (absence of pubic hair), (D) imperforate hymen and (E) MRKH-obstructing membrane—concave inwards, pinkish in color—rugosity

Müllerian Anomalies: A New Clinical Classification and Overview

Group B: Müllerian Anomalies with a Functioning Uterus Group B1: Müllerian Anomalies with a Functioning Uterus but with Complete Outflow Obstruction Women with this group of anomaly may have three types of defect with a normally functioning uterus—(a) cervical atresia, (b) vaginal atresia and (c) cervicovaginal atresia. Internal pelvic organ are normal and sometimes malformed (bicornuate and unicornuate) (Fig. 12.6). These

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patients present with primary amenorrhea (cryptomenorrhea) during adolescence. If they are not treated correctly and rationally during adolescence, their fertility potential will be lost. They exhibit obstructions at different levels of genital tract offering different grades of treatment complexities during surgical correction. Obstruction may be segmental or total, and usually involve either vagina, cervix or both. Segmental or total obstructive partition may exist at various levels of vagina, cervix or both (Fig. 12.7).

Fig. 12.6A to C: Complete outflow obstruction—hematometra with or without hematocolpos; (A and B) clinical photograph and (C) schematic diagram

Fig. 12.7A to F: Level of obstruction and operation complexities: (A) Cervicovaginal atresia—complete, (B) cervicovaginal atresia—incomplete only hematometra, (C) only mid-segment vaginal atresia—hematometra and hematocolpos, (D) atresia of lower third of vagina hematometra and hematocolpos, (E) isolated cervical atresia only hematometra, (F) schematic diagram of obstruction in different segments of vagina (leading to hematometra)

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Operative Complexities a. Complete cervicovaginal atresia: • May be found in 5–10% of MRKH syndrome • Only hematometra with or without hematosalpinx may exist (Fig. 12.7A); • Operative difficulty—most complex b. Upto 1/3rd of vaginal atresia: • With complete/incomplete cervical atresia • Only hematometra with or without hematosalpinx may co-exist (Fig. 12. 7B); • Operative difficulty—complex c. Midsegment vaginal atresia: • Hematometra and hematocolpos may exist together (Fig. 12.7C); • Operative difficulty—less complex than level shown in Fig. 12.7A and B d. Lower 1/3rd vaginal atresia without cervical atresia: • Leading to hematometra and hematocolpos (Fig. 12.7D); • Operative difficulty—least complex e. Isolated cervical atresia: • Hematometra is present (Fig. 12.7E); • Operative difficulty—complex but less than level shown in Fig. 12.7A and B f. Isolated transverse septum at various levels of lower genital tract (cervix and vagina) leading either to hematometra or to hematocolpos (Fig. 12.7F) The approach is abdominoperineal/vaginal (except perhaps in Group d)—currently laparoscopy is being used, previously laparotomy was the only choice. The levels of existence of transverse septum at different level of lower genital tract leading to total outflow obstruction has a significant impact on outcome of therapeutic intervention. With higher level obstruction, the success following recanalization is likely to be inferior than those with lower level of obstruction. Obstruction at the cervical level (cervical atresia) with or without vaginal malformation carries the worst prognosis following recanalization. Cervical Atresia Either only cervical or combined cervicovaginal atresia may be encountered in clinical

practice. Both are difficult to be corrected. However, only vaginal rather than cervicovaginal atresia is relatively easier. There are two broad groups of cervical atresia—total and partial. Total Cervical Atresia (Fig. 12.8) Total absence of cervix is very rare, partial atresia is more common. In total atresia, corpus containing hematometra narrows down to a peritoneal fold to be connected with vaginal vault. Torsion of the corpus containing hematometra may occur with such abnormality.

Fig.12.8: Total cervical atresia

Partial Cervical Atresia The second variety, called partial cervical atresia may be sub-classified into four groups: a. Upper 2/3rd cervical atresia (Fig. 12.9A) b. Lower 1/3rd cervical atresia (Fig. 12.9B) c. Stenosis/constriction around center of cervix (Fig. 12.9C) d. Total stenosis (but not absence) of cervix— cervical fragments encountered—occasionally on histology, cervical glands are seen (Fig. 12.9D)

Clinical Presentation These patients also present during adolescence with primary amenorrhea (cryptomenorrhea). Diagnosis is based on clinical features, USG

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Fig. 12.9A to D: (A) Upper cervical atresia, (B) lower cervical atresia, (C) stenosis around center of cervix and (D) total stenosis of cervix

and MRI. Exact site and extent of obstruction cannot always be precisely identified. Treatment Options (Group B1) There are Two Treatment Options Vaginoplasty or cervicovaginoplasty with surgical canalization. This is also known as ‘function restoring surgery’. If properly performed, adverse effects of retrograde menstrual blood flow following cryptomenorrhea may be prevented. However, there may be three objections against function restoring surgery (conservative surgery): a. Risk of restenosis b. Endometriosis is not uncommon in these women c. Absence of cervical epithelium and glands in cervical atresia compromising chances of pregnancy. The selection criteria for surgical canalization are: a. Age 12–20 years b. No endometriosis c. Not more than one previous attempt of surgical canalization is acceptable for repeat surgery . Surgical canalization either cervical or cervicovaginal atresia has been attempted since 1980s. Previously this was performed through abdominoperineal approach. Since 1990s, laparoscopy has been introduced for surgical correction of cervical and cervicovaginal atresia.

Restenosis and recurrence of outflow obstruction is the commonest problem which spoils this entire effort. This is common with cervical and cervicovaginal atresia when compared with only vaginal atresia with a functioning uterus. Various techniques and their modifications have been suggested to prevent restenosis. Some of them are mentioned below: i. Kriplani23 attributes use of a silicone stent in the neocervix until the resolution of inflammation is complete, which was the main reason for reduction of restenosis in their cases. ii. We have reported24 two stage procedure (vaginoplasty followed by cervicoplasty) through laparoscopic procedure and thereafter covering raw area of neocervix with vaginal flap—the same way as we cover raw areas following amputation of cervix with Bonney’s stitches (Fig. 12.10). iii. Acien 25 described a more complicated procedure of excision of atretic area of cervix and reanastomosis of uterine corpus with neovagina (Fig. 12.11). Apart from these technologies—the basic points which influence or help to prevent restenosis are the following—(a) size of channel (neocervix), (b) duration of stenting the channel, (c) presence of a natural vagina adjacent to newly created channel and more importantly, the number of menses occurring before the stent is taken out or displaced should be minimum ten. Out of 28 cases treated and reported by us26 we achieved 2 viable pregnancies delivered

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Fig. 12.10A and B: Operative steps of preventing restenosis by covering raw area of cervix with dissected vaginal flap by Bonney’s stitch (by author)

by caesarean section in cervicovaginal atresia (total vaginal and total cervical).

Fig. 12.11: Vagino-corporeal reanastomosis (by Acien)

Group B2: Müllerian Anomalies with Presence of a Functioning Uterus with Partial Outflow Obstruction In this group, one müllerian duct is normally developed and canalized resulting in normal menstrual outflow, adequate potential for reproduction and usually no problem with sexual function. Whereas, the contralateral müllerian duct though partially developed and functioning as a normal uterine horn with non-dissolution or partial dissolution of the partition formed

by fusion of two müllerian ducts or failure of vertical fusion or non-canalization with urogenital sinus will lead to following types of anatomical and clinical disorders. Anatomical defect: Obstruction at the junction of upper 1/3rd and lower 2/3rd of vagina clinical consequence—hematocolpos, hematometra, hematosalpinx (Fig. 12.12A). a. Anatomic disorder: Obstruction at the level of cervix  clinical consequence— hematometra and hematosalpinx (Fig. 12.12B) b. Anatomic defect: Obstruction at the level of upper 2/3rd and lower 1/3rd of vagina. There may or may not exist a communicating channel between the two upper horns but collected blood of hematometra does not drain out completely unless the communicating gap is sufficiently big (Fig. 12.12C). Clinical consequences– hematometra and hematocolpos c. Anatomical defect: A rudimentary müllerian knob, pedunculated or sometimes sessile, attached to normally developed and normally functioning müllerian horn (Fig. 12.12D and E). Clinical consequence–The rudimentary horn may be solid or canalized—containing hematometra when canalized. In pedunculated rudimentary horn the connecting stalk is usually not canalized and solid but occasionally may be canalized.

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Fig. 12.12: (A) Partial obstruction at the junction of upper 1/3rd and lower 2/3rd of vagina, (B) partial obstruction at the level of cervix, (C) partial obstruction at the level of upper 2/3rd and lower 1/3rd of vagina. There may exist a communicating channel at the level of cervix or lower part of body—may be insufficient in diameter for complete drainage of hematometra and (D and E) rudimentary müllerian knob pedunculated or sometimes sessile attached to normally developed and normally functioning müllerian horn (well developed contralateral horn uterus)

Even with noncanalised stalk of functioning rudimentary horn ectopic pregnancy has been reported. Transperitoneal migration of spermatozoa may explain such type of unusual abnormal pregnancy. Rupture of horn due to ectopic pregnancy may sometimes be life threatening. Clinical consequences of a rudimentary müllerian horn are: • Hematometra • Ectopic (cornual pregnancy) • Torsion • Impaction (in the pelvis) during labour (when rudimentary müllerian knob remains unrecognized) Management Excision of the obstructed segment, if properly performed at the correct age may restore all functions essential for reproduction. Presence of hematocolpos (low level obstruction) may create problem during surgical correction.

Group B3: Müllerian Anomalies with Functioning Uterus but with no Outflow Obstruction This may be the commonest non-symptom producing müllerian anomaly (seen in clinical practice). Anatomical variations of this group have been demonstrated in the following diagrams (Fig. 12.13). Often these müllerian anomalies are diagnosed during routine investigation of infertility and recurrent miscarriage. Septate and subseptate uterus sometimes may have to be corrected by septoplasty. Cervical cerclage is an alternate and effective procedure for treating women with history of recurrent miscarriage associated with these müllerian anomalies. Unification of uterus, didelphys or bicornuate uterus is rarely performed nowadays. Uterine cavity augmentation by lateral metroplasty in unicornuate uterus though often suggested and reported to be a satisfactory procedure for the treatment of infertility and recurrent miscarriage, has still remained controversial.

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Fig. 12.13: Types of müllerian anomalies. (A) Unicornuate, (B) arcuate, (C) bicornuate, (D) septate, (E) didelphys, (F) complete vaginal septum

RARE MÜLLERIAN ANOMALIES

Two groups of rare müllerian anomalies have been reported by us: The first group (group a) consisted of two similar cases but with gross different anatomical presentation.27 First case (group a1)—17-year-old girl had history of repair of anterior abdominal wall with autologous skin graft with colonic transplantation of ureters in childhood. On examination she had divergent labia, mons pubis, pubic rami and bifid clitoris. In addition, the introitus was stenotic and was displaced in the region of mons pubis. She had normal menarche, normal menstrual cycle and no electrolyte imbalance as a consequence of ureterocolic anastomosis (Fig. 12.14). 2nd case (group a2) in the same group was a 19-year-old girl with minimal grades of urethral exstrophy, with similar types of displaced stenotic introitus with divergent labia, mons pubis and pubic rami. She had intact abdominal wall skin but with totally deficient abdominal muscles. She had no

history of previous surgery and was having normal menstrual cycle (Fig. 12.15). Both of them had surgical reconstruction with introitoplasty and replacement of vagina in the perineal region. Both delivered viable babies within one year of surgery. In the second group (group b), 3 cases of isolated congenital tubal defect with absent ampullary segment and blocked fimbria were recorded. Ampullary fimbrial anastomosis was performed. Blocked ampullary end was excised. ‘Fish-mouth’ opening was created on the ‘opened’ ampullary end of the tube. The tips of the ‘fish-mouth’ opening of the tubes were anchored with two separate vicryl stitches. Through stab incision at the center of blind fimbria, the ends of anchoring stitches were pulled through the newly created fimbrial opening and fixed by everting lips of the fish-mouth on the outer surface of fimbria. Out of three, one patient achieved a spontaneous pregnancy. The steps of surgical correction of patients in group b have been demonstrated in Fig. 12.16A to D.

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Fig. 12.14: Group a1: (A) Appearance of external genitalia, (B) division of perineum, (C) dissection of vaginal tube, (D) fixation of dissected vagina

Fig.12.15: Group a2: (A) Appearance of external genitalia, scaphoid abdominal wall, (B) vaginal tube dissected, perineum dissected, (C) vaginal mucosa fixed to perineal skin

Embryologic explanation of the first two cases (groups a1 and a2): Around 6th week of gestation, infra-umbilical mesoderm intercedes between the bladder and cloacal membrane, giving rise to origin of genital tubercle, lower abdominal wall and pubic rami. Failure of mesodermal invasion may lead to breakdown of cloacal membrane. This accident may result in exstrophy of bladder, deficient lower abdominal wall and pubic rami (group a1). In group a2, there was minimal degree of urethral exstrophy with total deficiency of abdominal muscles but skin was intact.

Special Feature Group B Currently IVF is the conventional treatment in these types of cases. But no case has been reported so far to achieve pregnancy by surgical procedure as reported in these patients. Take Home Message • Patients with müllerian anomalies usually present with three major clinical problems; abnormal or absent menstrual cyclicity, problem in sexual function and impaired reproductive potential • Surprisingly some of them remain asymptomatic (as in Gr-B3) and undiagnosed and are detected

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Fig. 12.16A to D: ‘Pull through technique’—ampullary fimbrial anastomosis

•

• •

•

•

•

only during routine investigation of infertility and recurrent miscarriage Numerous classifications of müllerian anomalies have been suggested; some are too simple while others are very complex. Based on clinical presentation and anatomic defects, we have suggested a new classification. Broad classification grouping consists of: (a) Müllerian anomalies with a nonfunctioning uterus, (b) müllerian anomaly with functioning uterus. The anomalies with a functioning uterus have been further classified in relation to their anatomic varieties and degree of severity of clinical symptoms into three further sub-groups—(i) müllerian anomaly with a functioning uterus with complete outflow obstruction, (ii) müllerian anomaly with a functioning uterus with partial outflow obstruction, (iii) müllerian anomaly with a functioning uterus but without outflow obstruction. Some of these anomalies are easily treatable or may not require treatment at all, while in others, treatment may be extremely challenging. Lastly in addition to these commonly encountered groups of anomalies in clinical practice, a few rare forms of müllerian defects have been observed with their treatment protocol and have been illustrated in this chapter.

REFERENCES 1. Rackow BW, Arici A. Reproductive performance of women with müllerian anomalies. Curr Opin Obstet Gynecol 2007;19(3):229–37. 2. Acien P, Acien M. The presentation and management of complex female genital malformations. Hum Reprod Update 2016;22(1):48–69. 3. Counseller vs. Congenital absence of the vagina. JAMA 1948; 136:861. 4. Dietrich JE, Millar DM, Quint EH. Non-Obstructive Müllerian Anomalies. J Pediatr Adolesc Gynecol [Internet] 2014;27(6):386–95. 5. Tran ND, Hunter SK, Yankowitz J. CME review article. Ginecol Obstet Mex 2004;59(6):456–63. 6. Parikh R, Nakum K, Kadikar G, Gokhle A. Müllerian anomalies: a cause of primary amenorrhea. Int J Reprod Contraception, Obstet Gynecol [Internet] 2013;2(3):393–7. Available from: http://www. ijrcog.org/index.php/ijrcog/article/view/115 7. T a n m a h a s a m u t P , R a t t a n a c h a i y a n o n t M , Dangrat C, Indhavivadhana S, Angsuwattana S, Techatraisak K. Causes of primary amenorrhea: A report of 295 cases in Thailand. J Obstet Gynaecol Res 2012;38(1):297–301. 8. Mats Brännström, Liza Johannesson, Hans Bokström, Niclas Kvarnström, Johan Mölne, Pernilla Dahm-Kähler, Anders Enskog, Milan

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10. 11. 12. 13.

14. 15.

16.

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Milenkovic, Jana Ekberg, Cesar Diaz-Garcia, Markus Gäbel, Ash Hanafy, Henrik Hagberg, Michael Olausson, Lars Nilsson, Livebirth after uterus transplantation, The Lancet, Volume 385, Issue 9968, 2015: 607–16 American Congress of Obstericians and Gynecologists, Müllerian agenesis: diagnosis, management and treatment. Committee Opinion No. 562. Obstet Gynecol 2013; 121: 1134. Frank RT. The formation of an artificial vagina without operation. Am J Obstet Gynecol 1938; 35:1053. Ingram JM. The bicycle seat stool in the treatment of vaginal agenesis and stenosis: a preliminary report. Am J Obstet Gynecol 1981;140:867. Abbe R. New method of creating a vagina in a case of congenital absence. Med Rec 1898;54:836. McIndoe AH, Banister JB. An operation for the cure of congenital absence of the vagina. J Obstet Gynaecol Br Emp 1938;45:490 Wharton IR. A simple method of constructing a vagina. Ann Surg 1938;107:842. Chakravarty B N. Congenital absence of the vagina and uterus—simultaneous vaginoplasty and hysteroplasty, J Obstet Gynecol India 1977; 27:627. Chakravarty BN, Gun KM, Sarkar K. Congenital absence of vagina: anatomico-physiological consideration. J Obstet Gynecol India 1977; 27:621. Rothman D. The use of peritoneum in the construction of a vagina, Obstet Gynecol, 1972, vol. 40 (pg. 835–838) Baldwin JF. The formation of an artificial vagina by intestinal transplantation. Ann Surg 1904; 40: 398 Goligher JC: The use of pedicled transplants of sigmoid or other parts of the intestinal tract for

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vaginal construction. Ann R Coll Surg Engl. 1983; 65: 353–5. Shirodkar VN. Contribution stoobstetrics and gynaecology. Edinburgh E. and S. Livingstone, 1960:84–108. Vecchietti G. Neovagina nella syndrome di in Rokitanski Kuster-Hauser. Attualita Obstet Ginecol. 1965;11:131–47 Davydov SN. 12 Jahre erfahrung mit der Kolpopoesis unter Verwendung von Peritoneum. Gynakologe. 1980;13:120–1 Kriplani A, Kachhawa G, Awasthi D, et al. Laparoscopic-assisted uterovaginal anastomosis in congenital atresia of uterine cervix: follow-up study. J Minim Invasive Gynecol 2012;19:477. B. Chakravarty , R. Sankar Roy , S C. Halder and A. Ganesh; Newer surgical approach to prevent restenosis following canalisation of cervicovaginal atresia, Journal of Obstetrics and Gynaecology, 2015; Early Online: 1–2. P. Acién, MI. Acién, F. Quereda and T. Santoyo; Cervicovaginal agenesis: spontaneous gestation at term after previous reimplantation of the uterine corpus in a neovagina: Case Report; Human Reproduction Vol.23, No.3 pp. 548–553, 2008. Baidyanath Chakravarty, Hiralal Konar, and Nagendra N. Roy Chowdhury; Pregnancies after reconstructive surgery for congenital cervicovaginal atresia. Am J Obstet Gynecol 2000;183:421–3. Ashalatha Ganesh, Nalini J. Gupta and Baidyanath Chakravarty; Surgical correction of rare Müllerian anomalies and spontaneous pregnancies: a case report; Fertility and Sterility_Vol. 89, No. 3, March 2008.

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13 Ultrasound Imaging in Female Infertility Management Sunita Sharma, Sanghamitra Ghosh, Kamal Oswal and BN Chakravarty

Infertility affects about 10–15% couples of reproductive age and represents a significant part of clinical part in evaluation and management in clinical practice.1 In contrary to the popular perception, overall prevalence of infertility does not seem to have increased over the past couple of decades. 2 However, evaluation and management of infertility have changed drastically during the same period. Clinical investigation should be offered when couple fails to achieve a pregnancy after one year of unprotected vaginal intercourse. It should be done after 6 months, if woman is older than 35 years or if there are indicators for underlying problems like irregular menstrual cycle, uterine or tubal pathology, presence of endometriosis and male factor.3 Infertility is associated with emotional stress, and any delay in diagnosis of the cause of infertility is quite stressful specially in women of advanced age where treatment has to be started as early as possible. For the last several decades, evaluation of the probable cause of female infertility and demand for assisted reproductive technology (ART) procedures has evolved considerably. Imaging plays a crucial role in determining the probable factors involved in female infertility. Available diagnostic modalities are complex and some are invasive involving ionizing radiation with or without contrast like hysterosalpingogram (HSG), hysteroscopy,

laparoscopy, magnetic resonance imaging (MRI) and computed tomography. Advancements in ultrasound technology have made it an important diagnostic tool for both evaluation and management of female infertility. It has unique advantages in assessment of female pelvis over other modalities due to its safe and non-invasive nature, ease of use, inexpensive, free from radiation, and easily repeatable and reproducible, thus making it a primary diagnostic tool for assessment of infertility.4 Campbell, et al. in 2001, was first to introduce the ‘‘one-stop shopping’’ concept in diagnostic assessment of infertile females. 4 According to this, a single comprehensive ultrasound imaging in the clinician’s office can provide entire assessment of an infertile female. 4 It provides information regarding structure of pelvis, pelvic pathology and anatomic abnormalities of female reproductive tract, thus avoiding multiple diagnostic tests. This chapter features the role of ultrasound in evaluation of pelvic morphology and its abnormalities and how sonographic imaging can improve management of infertile couples. Two-dimensional (2D) ultrasound is considered as the primary tool for preliminary investigation of the female pelvis 5 and three-dimensional (3D) ultrasound captures volumes of information that can be reconstructed and displayed in any plane.6 Uterus, tubes, ovaries and peritoneal factors causing infertility

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can be imaged by ultrasound in different menstrual phases—proliferative, periovulatory and secretory phase to get relevant information related to infertility. ULTRASOUND IMAGING OF UTERUS

The sonographic appearance of uterus and endometrium is dynamic and keeps changing with different phases of menstrual cycle and during each point in a woman’s life. Understanding of normal physiology and its influence on sonographic appearance of female reproductive tract changes helps a clinician to differentiate between normal and pathological changes in the pelvis. The uterus can be examined by transabdominal (TAS) or transvesical and transvaginal sonography (TVS). Other techniques such as transrectal and translabial approach are rarely used and are generally reserved in patients where neither TAS nor TVS are feasible. TAS is advantageous in assessing the upper pelvis in patients with larger uterus that appears greater than 12 weeks (comparable gestational age) in size. However, TAS requires a full bladder which can be uncomfortable in some patients and provides a limited image resolution in patients with high BMI as well as with lower abdominal scars from previous surgery(s). Uterus is best examined transvaginally (TVS) with a high-frequency transducer which provides better resolution images with greater accessibility. TVS is usually performed after the patient has emptied her bladder, and between day 5 and day 9 of the cycle.7 During the reproductive years, uterus is usually pear shaped and size of the body of uterus is double that of the cervix. In postmenopausal age group and before puberty, uterus is tubular shaped with equally sized uterine body and cervix.2 If such uterus is observed in a woman during her reproductive age, it may indicate deficiency of sex hormones as seen in hypogonadotropic hypogonadism or ovarian failure. In anteverted uterus, the fundus is observed on the right side of the screen, whereas in retroverted uterus, the fundus is observed on the left. It is difficult to scan the uterus if it is in the same

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axis as the cervix and vagina as the ultrasound beam and position of uterus are not perpendicular. It is common to observe the uterus to be slightly deviated to the left or right and more deviations can be seen when there are pathological lesions in the pelvis, such as pelvic masses or peritoneal adhesions. It is important to define the position of the uterus specially if the patient had undergone various invasive procedures (curettage, hysteroscopy, insertion of an intrauterine contraceptive device or embryo transfer). Three major portions of the uterus: Endometrium, myometrium and serosa have different echo-texture on scanning. The myometrium is homogeneously echodense while its inner layer can be a little less echogenic, but the echo-texture of the junctional zone with the endometrium is smooth and well demarcated. Typically, the endometrial cavity appears as slit-like in sagittal section with the anterior and posterior endometrial wall, apposing one another. The size of uterus should be measured on mid sagittal plane from the tip of serosal surface of fundus to external orifice (os) of the cervix. If uterus is angulated, measurements of uterus and cervix are taken separately and added. Maximal anteroposterior (AP) diameter is also measured in the same sagittal plane from outer anterior wall to outer posterior wallperpendicular to the sagittal plane and uterine width is the maximal diameter on each side of the uterus in the transverse plane (Fig. 13.1A). Endometrial thickness (ET) measurement is best done by TVS. Early proliferative endometrium (just after menstruation) is usually homogenous, brightly echogenic and 25% in recurrent late miscarriages and preterm deliveries.9,10 HSG followed by MRI was the method of choice to detect uterine anomalies before widespread application of 3D ultrasound. More conventional 2D ultrasound imaging has also been demonstrated as a good screening tool for diagnosing congenital uterine anomalies.11 2D ultrasounds can suggest MDA but cannot accurately differentiate between a bicornuate uterus and a septate uterus. 3D ultrasound is superior to 2D and demonstrated to be as precise as MRI and can be a first choice to diagnose MDA (Fig. 13.6D and F).2 It is more acceptable than MRI, being less expensive, more interactive and moreover one can perform it in their own clinic. MRI is indicated in those cases when difficulty is faced in the ultrasound examination. 3D ultrasound can also detect intracavitary lesions (Fig. 13.2B and D), more accurately when combined with saline distension (Fig. 13.3C). 3D pelvic ultrasound assesses both external and internal contour of the uterus and provides an accurate image of uterine cavity, serosal surface and uterine myometrium when reconstructed in coronal

view. Renal tract anomalies should be excluded in presence of suspected MDA because renal abnormalities and fusion defects of MDA can exist together especially when it is unicornuate uterus.12 Evaluation of Fallopian Tubes Normal fallopian tubes are not visualized b y standard 2D ultrasound. Tubes are visible when there is free fluid in adnexal region which outlines the tubes or if there is a paratubal cyst. When fallopian tubes are obstructed, intraluminal secretion distends the tubes and forms hydrosalpinx which can be visualized on sonography (Fig. 13.7). Hydrosalpinx classically appears as tubular fluid filled ‘C’ or ‘S’ shaped structure adjoining the uterus with incomplete septa as the tube folds on itself. When thickened endosalpingeal fold projects into tubal lumen, it appears as echogenic mass of 2–3 mm size creating the “cogwheel sign” or beads on “string sign” as sonographic marker of hydrosalpinx. Dilated tubes may be confused with an ovarian cyst or peritoneal inclusions cyst but combination of these sonographic features like tubular shape

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Fig. 13.7A and B: 2D ultrasound images showing different hydrosalpinges

of cyst with waist sign and visualization of ovary separately, indicates abnormal dilation of fallopian tube (Fig. 13.7). Internal echoes or debris inside hydrosalpinx may indicate an infection. 3D volume and inverse mode can precisely differentiate ovarian cyst and tubal distension. The entire hydrosalpingeal tube with fluid-filled portions appears opaque, forming a cast of the cystic tube by 3D volume and in inverse mode by 2D appears as multiple noncommunicating fluid collection. Hydrosalpinx adversely influences pregnancy outcome in an IVF cycle and also increases the risk of ectopic pregnancy. Studies have reported that removal of hydrosalpinx before IVF improves success rate.13 Assessment of tubal patency is most commonly done by HSG which uses iodine containing contrast and exposes the patient to radiation. This method is fairly accurate in detecting proximal tubal disease having limitation of not detecting anatomical abnormality, which require further diagnosis by laparoscopy or MRI. Hysterosalpingo-contrast sonographyHYCoSy (normal saline agitated with air is injected into the uterine cavity) has been proposed as test for tubal patency. Tubal patency is determined on visualizing the agitated saline in the peritoneal cavity. HyCoSy for tubal patency test is reasonable, accurate, better tolerated, and cheaper compared to HSG.14,15 SIS for evaluation of

endometrial cavitary lesions are able to detect tubes simultaneously if enough pressure is given while injecting saline mixed with air so that tubes are filled and allowed to evaluate. 3D ultrasound along with injection of contrast media further improves accuracy of fallopian tube assessment. Advantage is that, clinician can do imaging of whole pelvis and can a l s o exclude uterine or endometrial pathology in the same sitting along with patency test. Evaluation of Ovary TVS imaging of ovary plays an important role in the evaluation and management of infertility. Ovarian reserve and functions are usually assessed by history, clinical evaluation, and hormonal assay. However, normal antral follicular count (AFC) and/or dominant follicle in the mid cycle and good endometrial thickness visualized by ultrasound are indicators of normal ovarian function. A nulliparous ovary is ellipsoid and situated in ovarian fossa adjacent to the lateral pelvic wall near the bifurcation of common iliac artery. Ovarian size varies with age, menstrual phase, menstrual status and body habitus. Ovarian volume of premenstrual women varies from 2.5 to 21.9 ml. Postmenopausal ovaries decreas in size and correlate with hormonal status and age of menopause.

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Ovarian Reserve

During reproductive age, a normal ovary has both immature and developing follicles throughout the entire menstrual cycle. Ovarian reserve correlates with the number of follicles (2–9 mm, anechoic, sharply marginated) in the ovaries that can potentially grow into mature follicles and hence a marker for the fertility potential of the women. AFC is well accepted as an indicator of ovarian reserve. Both AMH and AFC are accurate in predicting ovarian reserve and providing information about response to ovarian stimulation. The mean AFC and AMH levels have a linear relationship with age until 30 years, followed by a gentle decrease till 35 years and a steep

decrease thereafter.2,16 Lack of international standardization of AMH assessment and higher costs are some of the associated limitations of AMH as an ovarian reserve test. Assessing AFC has an advantage as the clinician can also detect uterine and ovarian pathology simultaneously. It can be done by real-time 2D, stored 2D-ultrasound ‘cine-loops’. A recent introduction of a 3D automated technique allows for semi-automated assessment of antral follicle using sonography-based automated volume count software (Sono AVC) (Fig. 13.8). Real-time 2D-ultrasound imaging can adequately assess AFC in clinical practice but 3D-ultrasound has certain advantages like lower interobserver variability, shorter examination time and offline data analysis.

Fig. 13.8A to D: Automated follicle counting using Sono AVC software

Ultrasound Imaging in Female Infertility Management

Other ultrasound markers for predicting ovarian reserve like ovarian volume, vascularity, are not of much importance. The basal stromal peak systolic velocity (PSV) is also considered to be associated with ovarian reserve. The mean baseline ovarian stromal PSV of 10 cm/s indicates normal response while a poor or high response is indicated by a mean PSV of 5 cm/s and 15 cm/s, respectively.17 A normal ovary (Fig. 13.9A) can be distinguished from polycystic ovaries (PCO) (Fig. 13.9B) by the following sonographic markers according to Rotterdam criteria such as 12 follicles in each ovary, measuring 2–9 mm in diameter and/ or increased volume (>10 cm 3). However, recent modification of the Rotterdam criteria has been proposed for diagnosis of PCOS only when >25 follicles/ovary are observed.18 This is to limit over diagnosis of PCOS, while adhering to Rotterdam criteria of 2003. Ovarian Cyst Functional follicular cyst, cystic corpus luteum, or cyst due to unruptured follicles, endometriomas, dermoids, hemorrhagic cysts (Fig. 13.9D to G) are the most commonly observed cysts in the reproductive age group during ultrasound imaging. 2 Functional (Fig. 13.9D) or luteal cysts usually disappear

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within a few months. Cysts of size 4 cm to avoid rupture or torsion.23 Additional advantage of ultrasound is that it can display vascularity using Doppler ultrasound without using any contrast media. Corpus luteal or hemorrhagic cysts typically show peripheral blood flow while poly cystic ovaries display increased central stromal blood flow using pulsed Doppler and color ultrasound. Pelvic mass with central blood flow may indicate malignancy. Role of Ultrasound in Assisted Reproduction Ultrasound has become an important tool for evaluation and management of women undergoing assisted reproduction. It provides vital information and overview of both the structural and physiological assessment of the reproductive organs. Following steps are guided by ultrasound to improve ART outcome: • Screening of women prior to treatment • Prediction of ovarian reserve • Monitoring of response to controlled ovarian stimulation • Assessment of endometrial receptivity • Facilitates oocyte retrieval • Facilitates embryo transfer • Detection of post-treatment complications

Screening of Women Prior to Treatment Assessment of pelvis before ART is mandatory to exclude any pelvic pathologies that may have a detrimental effect on fertility and early pregnancy outcome. Ovarian pathologies like PCOS, endometrioma, and other types of ovarian cysts can be evaluated by ultrasound imaging and need to be treated accordingly. Using ultrasound imaging, hydrosalpinx can be diagnosed with a high degree of confidence, which otherwise decreases IVF pregnancy and if treated prior to IVF, can significantly improve fertility outcome. Intracavitary lesions like submucous fibroids, polyps, adhesions and congenital uterine anomalies that affect reproductive outcome also need to be excluded along with myometrial pathologies like intramural fibroid and adenomyosis which also influence ART success. Accessibility of ovaries is also very important especially in severe endometriosis and enlarged uterus (fibroid and adenomyosis) so that proper planning for oocyte retrieval can be done. Prediction of Ovarian Reserve Prediction of ovarian reserve has become increasingly important before ART as it helps to counsel the couples and allows personalization of the stimulation protocols prior to ART. 2 AFC can be correlated with fertility status and regarded as the best predictor for the number of mature oocytes collected during oocyte retrieval. If ultrasound performed on day 2/3 of the cycles shows follicles >6 mm in size, it indicates that these AFs may grow more rapidly following a controlled ovarian stimulation leading to poor-quality oocytes or even atresia of follicles.2 Other sonographic markers for prediction of ovarian response are assessment of ovarian volume and ovarian blood flow which are considered to be inferior than AFC in predicting ovarian response.24 Monitoring of Response to Controlled Ovarian Stimulation and Oocyte Retrieval Controlled ovarian stimulation is the key step in ART and is useful to increase the number of oocytes and thereby increasing the chances of good quality embryos. Exogenous gonadotropins

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develop multiple follicular growth and is monitored by TVS (Fig. 13.10A and B). Serial ultrasonography is performed to determine and track the number and size of developing follicles in follicular phase, so that the optimal time of oocyte retrieval can be predicted. Simultaneously, endometrium is also evaluated for its thickness, morphology, pattern and vascularity. Follicular growth rate is between 1.1 and 1.4 mm/day in natural cycles but it grows at a faster pace of 1.7 mm/day in stimulated cycles. During early follicular phase, ultrasound monitoring is done at an interval of 3–4 days and when follicles are near maturation, monitoring is done at an interval of 1–2 days. Ultrasound monitoring ensures that stimulation is adequate and identifies if the patient is at risk for ovarian hyperstimulation (OHSS) (Fig. 13.10D). If necessary, it helps to adjust the gonadotropin

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dosage and/or ‘coasting’ can be done when required. Mature oocytes can be retrieved from follicles of the size of at least 14 mm. Studies have correlated size of follicle and morphological quality of embryos. It has been observed that follicles of 16 and 22 mm in size contain oocytes of similar quality.25 Ovulation trigger is administered when there are three or more follicles measuring 17–18 mm. Follicular volume estimation by 3D ultrasound and time of ovulation trigger are also correlated with oocyte quality. 26 Rodriguez-Fuentes, et al. observed a higher probability of getting mature oocytes with follicular volume of 0.6 ml.26 Fertilization rates are also reported to be higher for an oocyte from a follicle with volume between 5 and 7 ml when compared to follicular volume, between 1 and 5 ml. Demonstration of perifollicular blood flow with high grade vascularity are also makers

Fig. 13.10: (A) Follicular monitoring following ovulation induction of (A) stimulated ovary, (B) a hyperstimulated ovary after 5 days of gonadotropin stimulation and (C) OPU under USG

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for increased good quality oocyte, fertilization rate and eventually pregnancy rates. 27,28 Oocyte retrieval is done under TVS guidance safely and injury to vital structure can be avoided (Fig. 13.10C). Assessment of Endometrial Receptivity A receptive endometrium is crucial for embryo implantation. Ultrasound imaging can determine thickness, morphology and volume of the endometrium. Endometrial thickness (ET) of 14 mm is also associated with increased miscarriage rate. Endometrial patterns under ultrasound evaluation have been classified into three phases of menstrual cycle. In early follicular phase, triple-line is seen as a central hyperechoic line and two hypoechoic layers surrounding it. During periovulatory period, a triple line pattern is observed. An intermediate isoechogenic pattern with the same echogenicity as the surrounding myometrium is also observed with a central echogenic line. Luteal phase endometrium does not show any triple line pattern but is homogeneous and hyperechogenic under ultrasound evaluation. The best pattern is the triple line pattern with a central echogenic line, inner hypoechoic regions, and hyperechogenic outer walls which usuallygives more conception cycles. Endometrial waves which originate from the sub-endometrial myometrium and are hormone sensitive, can be observed by TVS. Supraphysiological E2 during multi-follicular development can increase the uterine contractility which is reported to be detrimental for implantation. Sub-endometrial contractility monitoring is not routinely advised but can be considered cause of repeated implantation failure specially those complaining of cramps around the time of embryo transfer (ET). Endometrial and sub-endometrial flow increases in the follicular phase and reaches maximum during preovulatory period. Color Doppler combined with pulsed Doppler and endometrial volume assessment can assess receptive endometrium and improved

implantation to some extent, but has limited value in clinical practice. Oocyte Retrieval Laparoscopy was the technique of oocyte retrieval (OR) before 1980. Danish group first described the ultrasound guided oocyte retrieval with the help of transabdominal probe. 29 Current standard is TVS guided. This reduces incidences of complications like injury to bladder, bowel, or bleeding from blood vessels. It is usually done in lithotomy or semi-lithotomy position under sedation. After cleaning vagina with normal saline, oocytes are retrieved with the help of a 17gauge needle. The echogenic tip of the needle is visible throughout the procedure and is aligned with the ultrasound beam. Follicle flushing is not done routinely as it is not advantageous in terms of oocyte yield and takes more time. It can be done when numbers of follicles are very few. Embryo Transfer (ET) ET is a crucial step in ART program and can be done using “clinical touch” or under “ultrasound guidance”. Recent Cochrane reviews reported improved pregnancy rate when ultrasound-guided ET is performed and it is now the current standard procedure. Advantage of ultrasound guided ET is that we can visualize the tip of catheter and can avoid touching the fundus. During difficult transfer, ultrasound helps in negotiating the catheter and ensuring the proper placement of embryos. Ultrasound for the Management of ART Complications and Outcome OHSS is a serious iatrogenic complication that arises due to stimulation with fertility drugs and after hCG trigger. It is suspected in women with PCOS, thin built women, women undergoing GnRH agonist protocol who grow a higher number of developing follicles with high E2 level. Sonographic markers of OHSS are ovarian enlargement (>10 cm) with multiple follicles (>11 mm on day 11 more than 11 follicles) in both ovaries and free fluid in the peritoneal cavity. In rare occasions, these big ovaries may undergo torsion or

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rupture. In severe cases, pleural effusion can be seen. Ultrasound guided (TAS or TVS) paracentesis or thoracocentesis is needed in case of large volume of peritoneal or pleural fluid accumulation. Other complications like hemorrhage, infection and tubo-ovarian abscess are not very common. Pre-existing hydrosalpinx or puncture of endometrioma are risk factors for infection. Intraperitoneal hemorrhage can be seen under ultrasound as complex echogenic free fluid in POD and peritoneal cavity. Take Home Message • Ultrasound has become an essential component for both diagnosis and management of female infertility. This modality is reliable, minimally invasive, and can be done at clinic while saving time of the couple. Integrating sonographic data with clinical findings will allow improved management of infertile couple. • One comprehensive sonographic assessment performed around day 5 to 9 of the cycle provides most of the information about the morphology o f r e p r o d u c t i v e o r g a n s a n d i ts a n a t o m i c abnormalities. It also helps in physiological assessment related to reproductive potential of women. • 2D and 3D ultrasound provides information about shape and appearance of endometrium, endometrial cavity, endomyometrial junction. Sonohysterography can also provide information about uterine cavitary lesions and tubal patency. • 3D ultrasound with advanced Doppler technique facilitates better understanding to evaluate uterine and endometrial blood flow for assessment of endometrial receptivity. • It helps in monitoring of follicular development during ovarian stimulation, guides oocyte retrieval and embryo transfer in IVF. • Post treatment complications of IVF can also be monitored using ultrasound.

REFERENCES 1. Thoma ME, McLain AC, Louis JF, King RB, Trumble AC, Sundaram R, et al. Prevalence of infertility in the United States as estimated by the current duration approach and a traditional constructed approach. Fertil Steril. 2013;99(5):1324–31. 2. Centers for disease control and prevention. Report, Natl Surv Fam growth Fertil. 2018.

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3. Committee P, Society A. Diagnostic evaluation of the infertile female: A committee opinion. Fertil Steril [Internet]. 2015;103(6):e44—e50. Available from: http://dx.doi.org/10.1016/j.fertnstert. 2015.03.019 4. Kelly SM, Sladkevicius P, Campbell S, Nargund G. DEBATE—continued Investigation of the infertile couple/: a one-stop ultrasound-based approach. Hum Reprod. 2001;16(12):2481–4. 5. In Medicine AI of U. AIUM practice guideline for the performance of peripheral venous ultrasound examinations. J Ultrasound Med. 2010;29(1): 166–72. 6. Sakhel K, Benson CB, Platt LD, Goldstein SR, Benacerraf BR. Begin With the Basics. J Ultrasound Med. 2013;32:381–8. 7. Groszmann YS, Benacerraf BR. Complete evaluation of anatomy and morphology of the infertile patient in a single visit; the modern infertility pelvic ultrasound examination. Fertil Steril [Internet]. 2016;105(6):1381–93. Available from: http:// dx.doi.org/10.1016/j.fertnstert.2016.03.026 8. Shwayder J, Sakhel K. Imaging for Uterine Myomas and Adenomyosis. Vol. 21, Journal of Minimally Invasive Gynecology. Elsevier; 2014. p. 362–76. 9. P Á. Incidence of Müllerian defects in fertile and infertile women. Hum Reprod. 1997;12(7):9262259. 10. Saravelos SH, Cocksedge KA, Li TC. Prevalence and diagnosis of congenital uterine anomalies in women with reproductive failure: A critical appraisal. Hum Reprod Update. 2008;14(5):415–29. 11. Valdes C, Malini S, Malinak LR. Ultrasound evaluation of female genital tract anomalies/: A review of 64 cases. 1984;285–92. 12. Campbell S. Ultrasound Evaluation in Female Infertility: Part 2, the Uterus and Implantation of the Embryo. Obstet Gynecol Clin North Am. 2019;46(4):697–713. 13. Strandell A, Lindhard A, Waldenström U, Thorburn J, Janson PO, Hamberger L. Hydrosalpinx and IVF outcome: A prospective, randomized multicentre trial in Scandinavia on salpingectomy prior to IVF. Hum Reprod. 1999;14(11):2762–9. 14. Luciano DE, Exacoustos C, Albrecht L, LaMonica R, Proffer A, Zupi E, et al. Three-dimensional ultrasound in diagnosis of adenomyosis: Histologic correlation with ultrasound targeted biopsies of the uterus. J Minim Invasive Gynecol. 2013; 20(6):803–10. 15. Saunders RD, Shwayder JM, Nakajima ST. Current methods of tubal patency assessment. Fertil Steril. 2011;95(7):2171–9.

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16. Almog B, Shehata F, Suissa S, Holzer H, ShalomPaz E, La Marca A, et al. Age-related normograms of serum antimüllerian hormone levels in a population of infertile women: A multicenter study. Fertil Steril. 2011;95(7):2359–64. 17. Campbell S. Ultrasound Evaluation in Female Infertility: Part 1, the Ovary and the Follicle. Obstet Gynecol Clin North Am. 2019;46(4): 683–96. 18. Dewailly D, Lujan ME, Carmina E, Cedars MI, Laven J, Norman RJ, et al. Definition and significance of polycystic ovarian morphology: A task force report from the androgen excess and polycystic ovary syndrome society. Hum Reprod Update. 2014;20(3):334–52. 19. Raine-Fenning N, Deb S, Jayaprakasan K CJ, Hopkisson J CB. Timing of oocyte maturation and egg collection during controlled ovarian stimulation: a randomized controlled trial evaluating manual and automated measurements of follicle diameter. Fertil Steril. 2010;94(1): 184–8. 20. Guerriero S, Ajossa S, Gerada M, D’Aquila M, Piras B, Melis GB. “Tenderness-guided” transvaginal ultrasonography: a new method for the detection of deep endometriosis in patients with chronic pelvic pain. Fertil Steril. 2007;88(5):1293–7. 21. Benacerraf BR, Abuhamad AZ, Bromley B, Goldstein SR, Groszmann Y, Shipp TD, et al. Consider ultrasound first for imaging the female pelvis. Am J Obstet Gynecol [Internet]. 2015;212 (4):450–5. Available from: http://dx.doi.org/ 10.1016/j.ajog.2015.02.015 22. Hudelist G, Fritzer N, Staettner S, Tammaa A, Tinelli A, Sparic R, et al. Uterine sliding sign: A simple sonographic predictor for presence of

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deep infiltrating endometriosis of the rectum. Ultrasound Obstet Gynecol. 2013;41(6):692–5. Caspi B, Weissman A, Zalel Y, Barash A, Tulandi T, Shoham Z. Ovarian Stimulation and In Vitro Fertilization in Women With Mature Cystic Teratomas. 1998;92(6):979–81. Jayaprakasan K, Jayaprakasan R, Al-Hasie HA, Clewes JS, Campbell BK, Johnson IR, et al. C a n quantitative three-dimensional power Doppler angiography be used to predict ovarian hyperstimulation syndrome? Ultrasound Obstet Gynecol. 2009;33(5):583–91. Mitchell P Rosen, Shehua Shen, Anthony T Dobson, Paolo F Rinaudo, Charles E McCulloch MIC. A quantitative assessment of follicle size on oocyte developmental competence. Fertil Steril. 2015;90(3):684–90. Rodriguez-Fuentes A, Hernandez J G-G, R, Chinea E, Iaconianni L PA. Prospective evaluation of automated follicle monitoring in 58 in vitro fertilization cycles/: follicular volume as a new indicator of oocyte maturity. Fertil Steril. 2010; 93(2):616–20. Nargund G. Time for an ultrasound revolution in reproductive medicine. Ultrasound Obstet Gynecol. 2002;20(2):107–11. Friedler S, Schenker JG, Herman A, Lewin A. The role of ultrasonography in the evaluation of endometrial receptivity following assisted reproductive treatments/: a critical review. Hum Reprod Update. 1996;2(4):323–35. Lenz S, Lauritsen JG. Ultrasonically guided percutaneous aspiration of human follicles under local anesthesia: A new method of collecting oocytes for in vitro fertilization. Fertil Steril. 1982;38(6):673–7.

14 Assessment of Endometrial Receptivity by Ultrasound Sunita Sharma, Sanghamitra Ghosh, Kamal Oswal and BN Chakravarty

Uterine receptivity is defined as self-limited temporary period when the endometrium acquires the ability to support embryonic implantation. Reproductive success depends on a healthy embryo, receptive endometrium and the positive molecular interaction through signaling cross-talk between them. Intricate but at the same time when coordinated while immunologic, molecular, cellular, structural, and vascular changes take place in the endometrium during this period. Ovarian hormones, estrogen and progesterone primarily coordinate these changes in the endometrium supporting embryo implantation.1 Most of the IVF cycles fail to result in a pregnancy due to implantation failure. Despite significant advancement in field of assisted reproductive technology (ART) research, implantation rate per embryo is considerably low. Although embryo quality is the key to IVF success, endometrial factors contribute 31–64%, there by also emphasizing the importance of endometrial receptivity.2,3 Thus, it is crucial to identify the time when the endometrium is most receptive and corresponds to the window of implantation resulting in an optimal outcome in ART. Receptivity markers pertaining to molecular (proteomic, and genetic) aspects of the endometrium are investigated. Though the reports are interesting, they are expensive, invasive and not necessarily applicable in defining a receptive endometrium.4 It is

therefore imperative to find a less expensive, non-invasivealternative diagnostic method of assessing endometrial receptivity with reproducibility. Ultrasound has become an essential tool for the assessment and management of infertile women undergoing ART. Transvaginal ultrasonography (TVS) may representan ideal non-invasive technique to detect receptive endometrium, which includes endometrial thickness (ET), pattern, uterine perfusion, endometrial contraction and volume. This chapter highlight show ultrasound imaging can assess the markers of endometrial receptivity to improve ART outcome. BASIC ASPECT OF EMBRYO IMPLANTATION

Embryo implantation is a well-organized and orchestrated process where the following events take place: • Apposition of the blastocyst on the most receptive site of the endometrium • Adhesion of the embryonic trophectoderm directly with the endometrial epithelium • Penetration and invasion by embryonic trophoblast while displacing endometrial epithelium, destroying the basal membrane and getting access to the stroma and blood vessels • Secretion of growth factors and substrates locally • Stromal response to the proteins and hormones from ovary and embryo

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Ultrasound Aspects of Endometrial Receptivity Endometrium forms the innermost glandular layer of the uterus and is the location for implantation. It consists of superficial functional layer and deep basal layer. It undergoes morphological changes during different phases of menstrual cycle under influence of increasing estrogen from the developing follicles. In the menstrual phase and very early proliferative phase, endometrium is usually less than 4 mm, homogenous and brightly echogenic. In the mid and late proliferative phase, the functional layer thickens and appears comparatively hypoechoic. A pronounced triple-line pattern is noted due to higher estradiol levels during the peri-ovulatory period. Following ovulation, when progesterone is released, the triple-line pattern disappears and a fully secretory heterogeneous endometrial pattern is observed; representing luteinised endometrial tissues. Secretory phase endometrium is echogenic due to thickened soft edematous glycogen rich functional endometrium under the influence of progesterone. This process is also associated with elongation and tortousity of spiral arteries which is as increased vascularity on Doppler imaging. The use of high-resolution TVS probes has made it possible for a follow up of endometrial changes throughout the cycle. Ultrasound monitoring of the endometrium usually involves measuring the ET and studying its morphological changes till ovulation. ET is measured in the midsagittal plane of

the uterus as the maximum distance between the two interfaces of the endometrialmyometrial junction. For optimal ART success we need to know the best timing for embryo transfer. High-resolution TVS probes with 3D advancement, is suitable to follow up uterine features like endometrial thickness, morphology and uterine vascularity throughout the stimulated cycle. Various parameters that are observed on ultrasound to assess the endometrial receptivity are covered in detail in this chapter. Endometrial Thickness There are conflicting reports regarding ET and ART outcome. Some studies observed thicker endometrium in conception cycles than in non-conception cycles5 while others did not confirm it.6 Though there is evidence that thin endometrium is associated with lower pregnancy rate, an ET as low as 4 mm was reported to have successful pregnancy. Dickey, et al. in 1993 reported that endometrial thickness 14 mm.8 Therefore, one needs to be cautious to administer trigger, if ET is 7 mm.10 Casper RF explained that a thin endometrium is not suitable for implantation, where the functional layer is very thin or absent and thereby exposing the transferred embryo in direct contact with basal layer. Increased vascularity of spiral arteries in basal layer exposes the embryos to high oxygen tension which may be detrimental to embryo implantation.11

a homogenous luteal pattern (Type C) at the time of hCG trigger. It reflects a coordinated follicular and endometrial development.12 Early secretory changes in endometrium are suggestive of early progesterone rise and hence may lower pregnancy rates in an ART cycle.14,15 However, others pose contradictory views, 16,17 suggesting that the rise in progesterone was not early enough or high enough to affect embryo implantation. Thus, ultrasound also helps in interpretation of premature luteinization (Fig. 14.2).

Endometrial Pattern (Echogenicity) Synchronized endometrial maturation and embryo development is the key to successful implantation. An association has been observed between ultrasound findings of endometrial morphology and serum hormonal levels.12 After estrogen priming in the proliferative phase, progesterone causes secretory changes in the endometrium which is observed as loss of trilaminar pattern on ultrasound. Smith, et. al. were the first to stress on timing of hCG trigger depending on thickness and morphology of the endometrium. Endometrial morphology can be classified as Type A (triple layer characterized by a hypoechoic endometrium with a central line (Fig. 14.2A), Type B (isoechoic endometrium with less defined outer walls of myometrium and central echogenic line) and Type C, an entirely homogeneous endometrium.13 The best pattern suggested is the three-layered pattern (Type A) as most favorablepattern for implantation than

Endometrial Vascularity Uterine perfusion and neoangiogenesis play an important role in proliferation of endometrium and helps in normal implantation. Doppler ultrasound estimates blood flow in uterine arteries by assessing pulsatility index (PI) and resistive index (RI) (Fig. 14.3). Lower resistance

Fig. 14.3: Uterine artery perfusion measured by 2D Doppler ultrasound

Fig. 14.2: Endometrial morphology classified as (A) triple layer characterized by a hypoechoic endometrium with a central line (Type A), (B) isoechoic endometrium with less defined outer walls of myometrium and central echogenic line (Type B) and (C) an entirely homogeneous endometrium (Type C)

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of uterine blood flow is observed during early and mid-luteal phase.18 Introduction of power Doppler images have facilitated assessment of smaller endometrial vessels. Furthermore, it is rational to assess endometrial function in terms of direct assessment of endometrial vascularization. Grading of endometrial vascularization is done based on penetration of blood vessels in endometrial and periendometrial area adopting Applebaum criteria (Table 14.1 and Fig. 14.4).19 Table 14.1: Grading of endometrium based on vascularization Applebaum criteria Zone 1 A 2-mm thick area surrounding the hyperechoic outer layer of the endometrium Zone 2 Hyperechoic outer layer of the endometrium Zone 3 Hypoechoic inner layer of the endometrium Zone 4 Endometrial cavity

However, certain precautionary measures should be taken during evaluation of endometrial perfusion. While scanning, the endometrium should preferably occupy at least 50% of the screen color gate (increasing the frame rate), and waiting for few second to allow endometrial contractions to go off and flow signals to appear. In efforts to maximize ART outcome, the role of uterine and endometrial blood flow with Doppler and 3D imaging has been emphasized. Few studies have suggested that sub-fertile women have decreased early luteal phase blood flow in comparison to healthy subjects20–22 but others did not confirm it.23–25 Uterine arterial blood flow does not correlate with actual blood flow in the endometrium which was earlier used as a predictor of endometrial receptivity. It has been reported that increased PI in uterine arteries is associated with poor implantation and lower

Fig. 14.4A to D: Endometrial and sub-endometrial blood flow classified as (A) Zone 1, (B) Zone 2, (C) Zone 3, (D) Zone 419

Assessment of Endometrial Receptivity by Ultrasound

pregnancy rates in one study, but others did not corroborate with such claim.26 In view of the fact, evaluation of endometrial and subendometrial blood flow with 3D power Doppler is more meaningful to assess the implantation potential. Absent endometrial and sub-endometrial blood flow is reported to be associated with poor pregnancy and implantation rates. In contrast, higher clinical pregnancy rates were observed with good blood flow in the endometrium and subendometrium. 27,28 An explanation of this observation may be attributed to better perfusion of endometrium leading to a better placentation, lower risk of miscarriage and a higher live birth rate after ART.29 However, few studies hold contrary opinion.30 Endometrial Motion/Contractility Endomyometrial junction also known as junctional zone, is recognized as a separate structure and is crucial in regulating normal uterine function including fertility. Subtle contractile activity of endomyometrial junction appears as wave like movement of the endometrium on real time TVS. 31 Since these waves are responsive to estrogen and progesterone, they exhibit cycle dependent frequency and amplitude. 32 During late follicular phase, waves propagate from cervix to fundus and its intensity increases around the time of ovulation. However, movement of such waves are in reverse during early follicular phase. This motion of the junctional zone is considered to help in sperm transport. In contrast, during luteal phase a relative uterine quiescence is observed which is supposed to facilitate embryo implantation and supply nutrition to decidua.33 Although origin of these waves is unclear, role of local nitric oxide is attributed to this phenomenon.34 As observed by Lensy and Killick, frequency of uterine waves >5/min during embryo transfer were associated with lower implantation rate when compared with 1–3/min contractions. Endomyometrial contractions are usually not perceived. However, due to intense contractions during menstruation, it may be felt and can be painful. 35 Stimulated ART

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cycles involve supraphysiological estrogen and are demonstrated to be associated with higher frequency of myometrial contractions. These vigorous waves if seen during embryo transfer, may expel the embryo and affect ART outcome. 36 Moreover, direction of these endometrial waves may change under influence of prostaglandins during difficult embryo transfer (when cervix is held by tenaculum) which may lead to embryo expulsion. Patients may also complain of uterine cramps at the time of embryo transfer. Postponing embryo transfer can be beneficial when increased myometrial contractions are observed during IVF cycles. Although endometrial contractions are not monitored r o u t i n e l y , t h e y m a y b e considered in patients with history of implantation failure. Endometrial Volume Introduction of 3D ultrasonography has provided the opportunity to precisely measure the volume of endometrium (Fig. 14.5).37 Some studies show that endometrial volume is a better predictor of implantation than endometrial thickness. 38 Low endometrial volume of