Reproductive Genetics

Repro Genetics SG Cover:Layout 1

8/10/09

15:02

Page 1

Great progress has been made in the field of genetics within the past decade. This, combined with our growing knowledge, has impacted on this important area with interesting consequences. The ability to identify genetic defects before implantation, to diagnose fetal abnormalities and to introduce screening programmes means that genetic testing now has a major role in preventive medicine. These topics are discussed in detail in the book. In parallel with these advances, other aspects that cannot be ignored, such as education of the public and the potential ethical dilemmas that may arise by virtue of these new methodologies, are raised and discussed in this volume, which is based on the 57th RCOG Study Group and includes a set of consensus views from the expert participants.

Reproductive Genetics

This is a unique book, covering areas not available elsewhere. Within its pages, the authors discuss many diverse areas relating to reproduction and genetics.

12.5 mm Spine Width

This book provides topical and essential information for practising clinicians, researchers and other healthcare professionals interested in these fields of study.

Edited by Sean Kehoe, Lyn Chitty and Tessa Homfray

RCOG Press Royal College of Obstetricians and Gynaecologists 27 Sussex Place, Regent’s Park, London NW1 4RG

www.rcog.org.uk

12.5 mm Spine Width

Reproductive Genetics Edited by

Sean Kehoe Lyn Chitty and Tessa Homfray

Reproductive genetics

Since 1973 the Royal College of Obstetricians and Gynaecologists has regularly convened Study Groups to address important growth areas within obstetrics and gynaecology. An international group of eminent clinicians and scientists from various disciplines is invited to present the results of recent research and to take part in in-depth discussions. The resulting volume, containing enhanced versions of the papers presented, is published within a few months of the meeting and provides a summary of the subject that is both authoritative and up to date.

SOME PREVIOUS STUDY GROUP PUBLICATIONS AVAILABLE The Placenta: Basic Science and Implantation and Early Clinical Practice Development Edited by JCP Kingdom, ERM Jauniaux Edited by Hilary Critchley, Iain and PMS O’Brien Cameron and Stephen Smith Disorders of the Menstrual Cycle Edited by PMS O’Brien, IT Cameron and AB MacLean Infection and Pregnancy Edited by AB MacLean, L Regan and D Carrington

Contraception and Contraceptive Use Edited by Anna Glasier, Kaye Wellings and Hilary Critchley Multiple Pregnancy Edited by Mark Kilby, Phil Baker, Hilary Critchley and David Field

Pain in Obstetrics and Gynaecology Edited by A MacLean, R Stones and Heart Disease and Pregnancy S Thornton Edited by Philip J Steer, Michael A Gatzoulis and Philip Baker Incontinence in Women Edited by AB MacLean and L Cardozo Teenage Pregnancy and Reproductive Health Maternal Morbidity and Mortality Edited by Philip Baker, Kate Guthrie, Edited by AB MacLean and J Neilson Cindy Hutchinson, Roslyn Kane and Kaye Wellings Lower Genital Tract Neoplasia Edited by Allan B MacLean, Albert Obesity and Reproductive Health Singer and Hilary Critchley Edited by Philip Baker, Adam Balen, Lucilla Poston and Naveed Sattar Pre-eclampsia Edited by Hilary Critchley, Allan Renal Disease in Pregnancy MacLean, Lucilla Poston and James Edited by John M Davison, Catherine Walker Nelson-Piercy, Sean Kehoe and Philip Baker Preterm Birth Edited by Hilary Critchley, Phillip Cancer and Reproductive Health Bennett and Steven Thornton Edited by Sean Kehoe, Eric Jauniaux, Pierre Martin-Hirsch and Philip Savage Menopause and Hormone Replacement Reproductive Ageing Edited by Hilary Critchley, Ailsa Gebbie Edited by Susan Bewley, William and Valerie Beral Ledger and Dimitrios Nikolaou

Reproductive genetics Edited by

Sean Kehoe, Lyn Chitty and Tessa Homfray

Sean Kehoe MD FRCOG Convenor of Study Groups, Lead Consultant in Gynaecological Oncology, Oxford Gynaecological Cancer Centre, John Radcliffe Hospital, Headington, Oxford OX3 9DU Lyn Chitty PhD MRCOG Professor of Genetics and Fetal Medicine, Clinical Molecular Genetics Unit, Institute of Child Health, 30 Guilford Street, London WC1N 1EH and Consultant in Genetics and Fetal Medicine, University College London Hospitals NHS Foundation Trust, London Tessa Homfray FRCP Consultant, Medical Genetics, Department of Genetics, St George’s University of London, Cranmer Terrace, London SW17 0RE

Published by the RCOG Press at the Royal College of Obstetricians and Gynaecologists, 27 Sussex Place, Regent’s Park, London NW1 4RG www.rcog.org.uk Registered charity no. 213280 First published 2009 © 2009 The Royal College of Obstetricians and Gynaecologists No part of this publication may be reproduced, stored or transmitted in any form or by any means, without the prior written permission of the publisher or, in the case of reprographic reproduction, in accordance with the terms of licences issued by the Copyright Licensing Agency in the UK [www.cla.co.uk]. Enquiries concerning reproduction outside the terms stated here should be sent to the publisher at the UK address printed on this page. The use of registered names, trademarks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant laws and regulations and therefore for general use. While every effort has been made to ensure the accuracy of the information contained within this publication, the publisher can give no guarantee for information about drug dosage and application thereof contained in this book. In every individual case the respective user must check current indications and accuracy by consulting other pharmaceutical literature and following the guidelines laid down by the manufacturers of specific products and the relevant authorities in the country in which they are practising. The rights of Sean Kehoe, Lyn Chitty and Tessa Homfray to be identified as Editors of this work has been asserted by them in accordance with the Copyright, Designs and Patents Act 1988. ISBN 978-1-906985-16-5 A machine-readable catalogue record for this publication can be obtained from the British Library [www.bl.uk/catalogue/listings.html] Cover image: Control of new synthesised DNA helix © medicalpicture GmbH (image number 16874270) RCOG Editor: Andrew Welsh Original design by Karl Harrington, FiSH Books, London Typesetting by Andrew Welsh Index by Liza Furnival, Medical Indexing Ltd Printed by Henry Ling Ltd, The Dorset Press, Dorchester DT1 1HD

Contents

Participants

vii

Declarations of personal interest

x

Preface

xi

1

Genetic aetiology of infertility Anu Bashamboo, Celia Ravel and Ken McElreavey

2

Disorders of sex development Lin Lin and John C Achermann

15

3

Preimplantation genetic diagnosis: current practice and future possibilities Alison Lashwood and Tarek El-Toukhy

35

Ethical aspects of saviour siblings: procreative reasons and the treatment of children Mark Sheehan

59

4

1

5

Epigenetics, assisted reproductive technologies and growth restriction Jennifer M Frost, Sayeda Abu-Amero, Caroline Daelemans and Gudrun E Moore 71

6

Fetal stem cell therapy Jennifer Ryan, Michael Ting and Nicholas Fisk

7

Prenatal gene therapy Khalil Abi-Nader and Anna David

101

8

Ethical aspects of stem cell therapy and gene therapy Søren Holm

123

9

Fetal dysmorphology: the role of the geneticist in the fetal medicine unit in targeting diagnostic tests Tessa Homfray

131

10 Fetal karyotyping: what should we be offering and how? John Crolla 11

147

Non-invasive prenatal diagnosis: the future of prenatal genetic diagnosis? Lyn Chitty, Gail Norbury and Helen White 159

12 Non-invasive prenatal diagnosis for fetal blood group status Geoff Daniels, Kirstin Finning, Peter Martin and Edwin Massey 13

83

173

Selective termination of pregnancy and preimplantation genetic diagnosis: some ethical issues in the interpretation of the legal criteria Rosamund Scott 183

vi  |  CONTENTS

14 Implementation and auditing of new genetics and tests: translating genetic tests into practice in the NHS Rob Elles and Ian Frayling

193

15 New advances in prenatal genetic testing: the parent perspective Jane Fisher

199

16 Informed consent: what should we be doing? Jenny Hewison and Louise Bryant

205

17 Consensus views arising from the 57th Study Group: Reproductive Genetics

217

Index

221

Participants John C Achermann

Wellcome Trust Senior Research Fellow in Clinical Science, Developmental Endocrinology Research Group, Clinical and Molecular Genetics Unit, UCL Institute of Child Health, 30 Guilford Street, London WC1N 1EH, UK.

Lyn Chitty

Professor of Genetics and Fetal Medicine, Clinical Molecular Genetics Unit, Institute of Child Health, 30 Guilford Street, London WC1N 1EH, UK and Consultant in Genetics and Fetal Medicine, University College London Hospitals NHS Foundation Trust, London.

John Crolla

Consultant Clinical Scientist, Wessex Regional Genetics Laboratory, Salisbury District Hospital, Salisbury SP2 8BJ, UK.

Geoff Daniels

Head of Molecular Diagnostics, International Blood Group Reference Laboratory, NHS Blood and Transplant, 500 Northway, Filton, Bristol BS34 7QH, UK.

Anna David

Senior Lecturer and Honorary Consultant in Obstetrics and Maternal/Fetal Medicine, Institute for Women’s Health, University College London, 86–96 Chenies Mews, London WC1 6HX, UK.

Jane Fisher

Director, Antenatal Results and Choices (ARC), 73 Charlotte Street, London W1T 4PN, UK.

Nicholas Fisk

Director, University of Queensland Centre for Clinical Research, Royal Women’s and Brisbane Hospital campus, Building 71/918, Herston 4029, Brisbane, Queensland, Australia.

Ian Frayling

Laboratory Director, All-Wales Medical Genetics Service; Consultant in Genetic Pathology and Clinical Genetics; Honorary Senior Research Fellow, Cardiff University; Institute of Medical Genetics, University Hospital of Wales, Cardiff CF14 4XW, UK.

Jenny Hewison

Professor of the Psychology of Healthcare, Institute of Health Sciences, University of Leeds, Charles Thackrah Building, 101 Clarendon Road, Leeds LS2 9LJ, UK.

Søren Holm

Professor of Bioethics, CSEP, School of Law, University of Manchester, Oxford Road, Manchester M13 9PL, UK.

Tessa Homfray

Consultant, Medical Genetics, Department of Genetics, St George’s University of London, Cranmer Terrace, London SW17 0RE, UK.

Sean Kehoe

Convenor of Study Groups, Lead Consultant in Gynaecological Oncology, Oxford Gynaecological Cancer Centre, John Radcliffe Hospital, Headington, Oxford OX3 9DU, UK.

Alison Lashwood

Consultant Nurse in Genetics and PGD, Clinical Genetics, 7th Floor, Borough Wing, Guy’s Hospital, London SE1 9RT, UK.

viii  |  PARTICIPANTS

Ken McElreavey

Head, Human Developmental Genetics Unit, Institut Pasteur, 25 rue du Dr Roux, Paris 75724, France.

Gudrun E Moore

Professor of Clinical and Molecular Genetics, Institute of Child Health, University College London, 30 Guilford Street, London WC1N 1EH, UK.

Rosamund Scott

Professor of Medical Law and Ethics, Centre of Medical Law and Ethics and School of Law, King’s College London, Strand, London WC2R 2LS, UK.

Mark Sheehan

Oxford BRC Ethics Fellow and James Martin Research Fellow, Program on the Ethics of the New Biosciences, Suite 8, Littlegate House, 16/17 St Ebbe’s Street, Oxford OX1 1PT, UK.

Additional contributors Khalil Abi-Nader

Clinical Academic Research Fellow, Academic Department of Maternal Fetal Medicine, Institute for Women’s Health, University College London, 86–96 Chenies Mews, London WC1E 6HX, UK.

Sayeda Abu-Amero

Senior Research Fellow, Institute of Child Health, University College London, 30 Guilford Street, London WC1N 1EH, UK.

Anu Bashamboo

Chief Scientist, Human Developmental Genetics Unit, Institut Pasteur, 25 rue du Dr Roux, Paris 75724, France.

Louise Bryant

Lecturer in the Psychology of Healthcare, University of Leeds, Leeds Institute of Health Sciences, Room 1.12, Charles Thackrah Building, 101 Clarendon Road, Woodhouse, Leeds LS2 9LJ, UK.

Caroline Daelemans

Wellbeing of Women Research Fellow, Institute of Child Health, University College London, 30 Guilford Street, London WC1N 1EH, UK.

Rob Elles

Director of Molecular Genetics, Regional Molecular Genetics Service, Genetic Medicine (6th Floor), St Mary’s Hospital, Oxford Road, Manchester M13 9WL, UK.

Tarek El-Toukhy

Consultant Gynaecologist and Subspecialist in Reproductive Medicine, Assisted Conception Unit, 11th Floor, Tower Wing, Guy’s Hospital, London SE1 9RT, UK.

Kirstin Finning

Clinical Scientist, International Blood Group Reference Laboratory, NHS Blood and Transplant, 500 Northway, Filton, Bristol BS34 7QH, UK.

Jane Fisher

Director, ARC (Antenatal Results and Choices), 73 Charlotte Street, London W1T 4PN, UK.

Jennifer M Frost

MRC PhD student, Institute of Child Health, University College London, 30 Guilford Street, London WC1N 1EH, UK.

PARTICIPANTS  |  ix

Lin Lin

Postdoctoral Research Associate, Developmental Endocrinology Research Group, Clinical and Molecular Genetics Unit, UCL Institute of Child Health, 30 Guilford Street, London WC1N 1EH, UK.

Peter Martin

Clinical Scientist, International Blood Group Reference Laboratory, NHS Blood and Transplant, 500 Northway, Filton, Bristol BS34 7QH, UK.

Edwin Massey

Consultant Haematologist, NHS Blood and Transplant, 500 Northway, Filton, Bristol BS34 7QH, UK.

Gail Norbury

Director, North East Thames Regional Molecular Genetics Laboratory, Great Ormond Street Hospital, L6 York House, 37 Queen Square, London WC1N 3BH, UK.

Celia Ravel

University Clinical Lecturer, Human Developmental Genetics Unit, Institut Pasteur, 25 rue du Dr Roux, Paris 75724, France.

Jennifer Ryan

Postdoctoral Fellow, University of Queensland Centre for Clinical Research, Royal Women’s and Brisbane Hospital campus, Building 71/918, Herston 4029, Brisbane, Queensland, Australia.

Michael Ting

Postdoctoral Research Fellow, University of Queensland Centre for Clinical Research, Royal Women’s and Brisbane Hospital campus, Building 71/918, Herston 4029, Brisbane, Queensland, Australia.

Helen White

Senior Scientist, National Genetics Reference Laboratory (Wessex), Salisbury District Hospital, Salisbury, Wiltshire, SP2 8BJ, UK.

DECLARATIONS OF PERSONAL INTEREST All contributors to the Study Group were invited to make a specific Declaration of Interest in relation to the subject of the Study Group. This was undertaken and all contributors complied with this request. Khalil Abi-Nader’s department receives financial support from Ark Therapeutics Ltd, UK. John Achermann is a member of the annual meeting steering committee of the Endocrine Society. He has received reimbursement of approximately £300 each for chapters on sex development in books on internal medicine and on endocrinology. Lyn Chitty is a member of the National Screening Committee and the RCOG Ethics Committee. She is an editor of Prenatal Diagnosis and receives travel costs and honoraria. Geoff Daniels is President of the British Blood Transfusion Society. Rob Elles is a former chairman of the British Society for Human Genetics. He has received minimal editorial fees from Springer for the book Molecular Diagnosis of Genetic Diseases. Tarek El-Toukhy is a member of the Royal College of Obstetricians and Gynaecologists, the British Fertility Society and Infertility Network UK. Jane Fisher is a trustee of the Genetic Interest Group (GIG) and the Director of Antenatal Results and Choices (ARC). ARC is a member of the pro-choice coalition Voice for Choice. Nicholas Fisk acts as a medical legal consultant in the UK, USA and Australia. He has received travel expenses and accommodation from Ferring International. Ian Frayling is a member of Council and the Executive of the Royal College of Pathologists. He is also a member of the Clinical Molecular Genetics Society (CGMS) and thus also the British Society for Human Genetics (BSHG). Jenny Hewison is a member of the National Screening Committee's FAS Programme Steering Group. Gail Norbury is a member of the steering group for the NIHR-funded grant on Reliable and Accurate Non-Invasive Prenatal Diagnosis (RAPID).

P Preface

Reproduction and genetics without doubt encompass a very wide remit and, as is often the case in Study Groups, it is not possible to cover all the aspects relating to the topic that would fulfil everyone’s desires or aspirations. These constraints, however, do not prevent an attempt at addressing and discussing the relevant areas of the specialty with acknowledged experts from the UK and abroad. The progress and increased knowledge base in genetics within the past decade have impacted on this important area with interesting consequences. The ability to identify genetic defects prior to implantation, to diagnose fetal abnormalities and to introduce screening programmes means that genetic testing has a major role in preventive medicine. In parallel with these advances, there are other aspects that cannot be ignored, such as education of the public and the potential ethical dilemmas that may arise by virtue of these new methodologies. In this book, developed from the 57th RCOG Study Group, many of the above topics are discussed. It is hoped that the reader will find in a single book the diverse areas relating to reproduction and genetics, and it should be of interest to those involved in these specialist areas. As with all these expert groups, the RCOG thanks all the individuals who have selflessly given their time and expertise to ensure the publication of a unique book covering areas not available elsewhere. My thanks go to the co-editors for their patience and to all the team involved in making this book a reality. Sean Kehoe Convenor of Study Groups

1 Chapter 1

Genetic aetiology of infertility Anu Bashamboo, Celia Ravel and Ken McElreavey

Introduction In recent years there has been increasing concern about a possible decline in reproductive health.1,2 It is estimated that one in seven couples worldwide have problems conceiving and there is increasing demand for fertility treatments.1,2 These include intracytoplasmic sperm injection (ICSI) and in vitro fertilisation. In some European countries, such as Denmark, more than 6% of children are born after assisted reproduction.1,2 Sperm counts in many European countries are declining by around 2% per year.2 Although human infertility rates are high and increasing, our understanding of the genetic pathways and basic molecular mechanisms involved in gonadal development and function is limited. In this overview, we examine the various forms of infertility and the evidence that there is a genetic component, and discuss in some detail the known genetic causes of infertility.

Female infertility The main causes of female infertility are anovulation and anatomical causes such as obstruction in the genital tract (ovulatory infertility). About one-third of all cases of female infertility are due to obstruction in the genital tract.3 The obstruction can be in the fallopian tubes, uterus, cervix or vagina. Uterine abnormalities include congenitally absent (Mayer–Rokitansky–Küster–Hauser [MRKH] syndrome), bicornuate or double uterus, leiomyomas and Asherman syndrome.4 Endometriosis is an estrogen-dependent inflammatory disease that affects 5–10% of women of reproductive age in the USA.5 Its defining feature is the presence of endometrium-like tissue in sites outside the uterine cavity, primarily on the pelvic peritoneum and ovaries. Tubal and peritoneal blockades are due to tubal loss or impairment as a result of ectopic pregnancy, endometriosis or infections. Cervical factors (cervical stenosis, antisperm antibodies, non-receptive cervical mucus) and vaginal factors (vaginismus, vaginal obstruction) also contribute to ovulatory primary or secondary infertility. Primary ovarian insufficiency (POI) is characterised by primary or secondary amenorrhoea, hypergonadotrophinism and estrogen deficiency in women younger than 40 years.6,7 In the majority of cases, the aetiology remains unknown but known causes include chemotherapy, radiotherapy, surgery, associations with autoimmune © Anu Bashamboo, Celia Ravel and Ken McElreavey. Volume compilation © RCOG

2  |  ANU BASHAMBOO, CELIA RAVEL AND KEN McELREAVEY

diseases and infections. POI is the preferred term for the condition that was previously referred to as premature menopause or premature ovarian failure; other terms used for this condition include primary ovarian failure, hypergonadotrophic hypogonadism and gonadal dysgenesis.6,7 The condition differs from the menopause in that there is varying and unpredictable ovarian function in approximately 50% of cases, and about 5–10% of women conceive and give birth to a child after they have received the diagnosis.8–11 Women with Turner syndrome (monosomy  X) have a normal complement of oocytes until the third month of fetal life, after which apoptosis is accelerated, generally resulting in oocyte depletion by the end of the first decade of life. As a consequence, only 10% of women with Turner syndrome achieve menarche.12–14 In contrast, women with Turner syndrome and a chromosomal mosaicism (45,X/46,XX) are more likely (40% of such women) to menstruate and can do so for several years before developing overt POI. It is possible that undetected X chromosome mosaicism may account for some cases of unexplained POI.15 Anovulation is the cause of infertility in about one-third of couples attending fertility clinics, and polycystic ovary syndrome (PCOS) accounts for 90% of such cases.16 PCOS is one of the most common endocrine disorders in women of reproductive age and the most frequent cause of hyperandrogenism and anovulation/ oligo-ovulation.17,18 The anovulatory or oligo-ovulatory infertility could also be a result of endocrine dysfunction (androgen insensitivity and follicle-stimulating hormone [FSH] and luteinising hormone [LH] imbalance, thyroid disorders, adrenal dysfunction), hypothalamic–pituitary anomalies (Kallmann syndrome, Sheehan syndrome, hyperprolactinaemia, hypopituitarism), disorders of sex development (DSD), ovarian tumours and Turner syndrome. Evidence of a genetic contribution Many aspects of female reproductive function are strongly influenced by genetic factors and consequently there have been repeated attempts to identify genetic mutations associated with disorders of female reproductive function. Endometriosis can be inherited in a polygenic manner; its incidence in relatives of affected women is up to seven times the incidence in women without such a family history.19 Several twin studies also indicate a strong heritability.20–22 This has prompted a large number of genetic association studies including, more recently, genomewide linkage analysis on affected sib pairs and on extended families.23,24 Although these studies present evidence of linkage to chromosomes 7p13–15 and 10q26, the causal gene(s) have not been described.23,24 A meta-analysis of published association studies suggests that variants in the glutathione S-transferase gene may contribute to endometriosis but further studies are required.25 The prevalence of PCOS varies by ethnicity and has been reported at between 6.5% and 8% in women younger than 40 years.26–30 Both twin and familial studies suggest that there is a genetic component to PCOS with a polygenic pattern of inheritance31,32 but genetic studies have not yet identified robust associations between gene polymorphisms and PCOS.32 There are several possible reasons for the inconsistency between association studies on PCOS. These methodological problems are potential pitfalls that arise in all association studies. These include population stratification; that is, the difference in allelic frequency between case and control populations may be due to genetic differences between the case and control populations, such as population history, that are unrelated to the phenotype. Case and control selection biases may

GENETIC AETIOLOGY OF INFERTILITY  |  3

also influence results and sample sizes are often too small to have the power to detect anything other than major causal mutations. There is a wide variation in the age at which the normal menopause begins, varying from 40 years to just over 60 years. Several studies have indicated that the variation of the age of onset of normal menopause has a very strong genetic component,33,34 with several to many genes assumed to make an additive contribution to the variation. Estimates of heritability range up to 85%. The observation from these studies that a woman with one or more first-degree relatives with a history of early menopause is likely to experience early menopause herself suggest that genes, perhaps the same genes, are also involved in early reproductive failure. Indeed, POI shows both familial inheritance and varies by ethnicity, suggesting that genetic factors play a role in some cases. The incidence of familial idiopathic POI is reported to be between 4% and 31%.35–40 The wide range in occurrence reflects the genetic heterogeneity, the wide spectrum of pathologies included and the absence of clearly defined guidelines for the diagnosis of POI. The prevalence of POI is estimated to be 0.1–1.4% in women younger than 40 years and the occurrence varies considerably with ethnicity (1.4% of African American women, 1.4% of Hispanic women, 1.0% of European women, 0.5% of Chinese women and 0.1% of Japanese women), suggesting predisposing or protective genetic factors.41 Genetic causes Androgen insensitivity syndrome is an X-linked disorder characterised by variable defects in virilisation of 46,XY individuals. This is due to the loss of function of the androgen receptor gene (Xq11–12), resulting in peripheral androgen resistance with an estimated frequency of about one in 65 000 46,XY individuals.42 Several genetic causes are known to be associated with POI. These include monosomy  X and an expansion of the CGG trinucleotide repeat in the 5′ region of the ‘fragile X mental retardation 1’ (FMR1) gene within the premutation range.43 Premutation repeats range from 55 to 200 trinucleotides and increasing repeat length is correlated with decreasing age for ovarian failure.44 Other regions on the X  chromosome may also be associated with ovarian failure. These are the POF1, POF2 and POF3 loci: although they contain several candidates, the precise gene involved has not been identified.45–47 Mutations in genes that result in ovarian insufficiency often cause other somatic anomalies. These include autosomal recessive mutations in the AIRE, EIF2B and GALT genes.48–50 POI has also been observed to be associated with the blepharophimosis-ptosis-epicanthus inversus syndrome (BPES) caused by mutations in the FOXL2 gene.51,52 In non-syndromic forms of ovarian failure, rare recessive inactivating mutations of the FSH and LH receptors have been described.53–55 Mutations in the ovary-specific homeobox transcription factor NOBOX are also a rare cause of POI.56 Some reports have suggested that mutations in X-linked oocyte-secreted factor BMP15 cause non-syndromic ovarian failure57 but others have questioned these findings.58 As discussed below in the section on male infertility, a great deal of caution needs to be exercised before it can formally be established that a candidate gene is responsible for a given phenotype. Several uterine abnormalities, including MRKH syndrome and bicornuate or double uterus, are considered to have a genetic basis on evidence from a number of familial cases with these anomalies.59–61

4  |  ANU BASHAMBOO, CELIA RAVEL AND KEN McELREAVEY

Recurrent miscarriage is commonly defined as the loss of three or more consecutive pregnancies before 20–28 weeks of pregnancy and it affects up to 5% of couples.62,63 The examination of embryos from women with recurrent miscarriage indicates that most have a chromosomal anomaly.62 The majority of these chromosomal anomalies, including trisomies and structural rearrangements, are de novo but it has been suggested that as many as 10% are present in one of the parents.62

Male infertility A genetic contribution to spermatogenic failure is indicated by several families with multiple infertile or subfertile men.64–73 In some of these families an autosomal recessive mutation appears to be responsible while in others an autosomal dominant mutation with sex-limited expression is likely.64,68 In other families the genetic cause is known to involve either chromosomal anomalies or Y chromosome microdeletions.65 Familial clustering of male subfertility and impaired spermatogenesis has been well documented and considered as evidence of an important genetic contribution to the phenotype74,75 but other studies have questioned this interpretation, based on twin studies that suggest the shared environment may be more important.76 Although questions remain concerning the general genetic contribution to infertility and subfertility, several genetic causes have been documented that are responsible for a significant minority of cases. Chromosome disorders A chromosomal anomaly is carried by 5% of all infertile men.77,78 This is ten times higher than the frequency in the general population.77 In about 80% of these cases a sex chromosome is involved and in the remaining 20% the autosomes are involved.77,78 Klinefelter syndrome (47,XXY) is the most frequent cause of gonosomic anomalies, occurring in 0.1–0.2% of newborn males. The prevalence among infertile men is high, from 5% in those with severe oligozoospermia to 10% in those with azoospermia. The presence of an additional X chromosome is associated with testis hypotrophia and an increase in plasmatic gonadotrophins, thus representing the most frequent form of male hypogonadism. Before the advent of ICSI these men were regarded as being completely infertile. However, there are now more than 50 normal children born from fathers with Klinefelter syndrome, where rare spermatozoa were recovered with a testicular biopsy.79 In spite of the existence of a constitutional chromosomal anomaly, the risk for these men to transmit a chromosomal anomaly is small since fluorescence in situ hybridisation studies have shown that the rate of disomic XY or XX sperm rises only marginally in Klinefelter cases, suggesting these spermatozoa are produced from 46,XY spermatogonia.80 A 47,XYY karyotype is the second most frequent cause of gonosomic anomalies. Although the majority of these men are fertile, the frequency of this anomaly is four times higher in those who are infertile than in the general population.81 Almost 1% of men with azoospermia have a 46,XX karyotype.77,81,82 In most cases, the testis-determining gene SRY is present on the short arm of one of the X  chromosomes or on an autosome. In rare cases, SRY may be absent and the phenotype results then from a mutation in another gene involved in the formation of the testis. Although one case of a 46,XX male with a duplication of the SOX9 gene has been described,83 the genetic cause of most cases is unknown.

GENETIC AETIOLOGY OF INFERTILITY  |  5

Chromosomal translocations are found with a frequency 8–10 times higher in infertile men and may be acrocentric Robertsonian translocations (centric fusions) or reciprocal translocations.77,78,81,84 It also seems that the frequency of small supernumerary marker chromosomes, made up of the short arms of acrocentric chromosomes, is often increased in the infertile populations of men.77,78,81,84 Three Y chromosome microdeletions The male-specific portion of the Y  chromosome contains 78 genes or families of genes, many of which appear to be involved in spermatogenesis.85 These genes are located on both the short and long arms of the Y chromosome but most attention has focused on the long arm because it was deletions in this region that were first detected by conventional cytogenetic approaches in infertile men.86 Later molecular approaches defined three regions of the long arm associated with infertility: AZFa, AZFb and AZFc.87 In most studies, deletions are found at frequencies of around 10– 12% and are independent of the ethnic or geographic background.88 AZFc deletions and AZFc + AZFb deletions are the most frequent. AZFc deletions account for almost 60% of all Yq deletions, whereas AZFa and AZFb deletions are rare. Most infertile men carrying AZF deletions have either azoospermia or oligozoospermia. Men with complete AZFc deletions show a poor correlation with testicular histology, whereas complete AZFb deletions are usually associated with azoospermia and meiotic germ cell arrest on histology and men with complete AZFa deletions lack germ cells on histology.87,88 Rare partial deletions of AZFa and AZFb have been described associated with variable phenotypes.89,90 Thus, in cases of complete AZFa or AZFb deletion, a testicular biopsy is not recommended because the probability of finding viable sperm is almost zero. Apart from their infertility, men carrying AZF microdeletions do not present other known pathology. Since these deletions can be associated with some sperm production, cases of father-to-son transmission of AZF deletions has been described.91 The molecular mechanism of AZF microdeletions is due to the particular structure of the Y  chromosome consisting of complex repetitive regions.85 AZFc deletions occur as a consequence of intra-Y homologous recombination between repeat sequences of a complex palindromic structure.85,89 Partial deletions within the 3.5 Mb AZFc portion have been identified that result in the absence of some gene family members in the region.92 The deletions are structurally heterogeneous, with some of them being apparently polymorphic variants that have no obvious effect on fertility. One group of deletions, known as gr/gr deletions, are controversial since some studies have linked these deletions to infertility whereas other have not.89,92 Further studies are necessary to further characterise these deletions and define the Y chromosome background on which they occur. This is important because the class of Y chromosome (Y chromosome haplogroup or Y chromosome background) can have an effect on spermatogenesis89 and some gr/gr deletions that are fixed on Y chromosome haplogroups may in fact be acting as a surrogate marker for other Y variants that may affect spermatogenesis.89 The function of many of the Y  chromosome genes is now known or inferred from their X chromosome or autosomal homologue. Several of these genes may play a role in the epigenetic reprogramming that is known to occur in the specification and differentiation of the germline and during spermatogenesis. SMCY and UTY are known or suspected to have histone demethylase activity93,94 whereas CDY is a histone acetyltransferase.95 DAZ may be a meiotic competence factor.96 To date, only one

6  |  ANU BASHAMBOO, CELIA RAVEL AND KEN McELREAVEY

point mutation has been described in any of the Y chromosome genes associated with male infertility, suggesting that these mutations are rare.97 Autosomal genes Although many genes are known to be essential for gametogenesis and murine knockout studies have identified several hundred genes specifically associated with infertility, there are surprisingly few mutations in X chromosome or autosomal genes that have been conclusively demonstrated to cause spermatogenic failure. Homozygous mutations in the SPATA16 gene that may be involved in acrosome formation are associated with globozoospermia.98 The AURKC gene (aurora kinase C) expresses a cell cycle regulatory serine/threonine kinase mainly in the testis during meiosis. A homozygous, single base pair deletion that results in a premature stop codon was found associated with large-headed multiflagellar polyploid spermatozoa.99 This is a founder mutation with a carrier frequency of one in 50 in North African populations.99 The CFTR gene (cystic fibrosis transmembrane conductance regulator) encodes a chloride channel that regulates the transport of water and salt on both sides of the plasma membrane of epithelial cells. Infertile men with obstructive azoospermia due to congenital bilateral absence of the vas deferens (CBAVD) frequently harbour mutations of the CFTR gene. Around 80% of men with isolated CBAVD carry two CFTR mutations, usually in compound heterozygosity.100 In an isolated case of unilateral absence of the vas deferens, the association with CFTR mutations is controversial. Similarly, it has been reported that CFTR may also be involved in sperm capacitation and that mutations impairing CFTR function may lead to reduced sperm fertilising capacity and male infertility other than CBAVD.101 In both these situations, additional data are required. End-organ resistance to androgens is designated as androgen insensitivity syndrome and comprises complete and partial forms.102,103 Complete androgen insensitivity in a 46,XY individual is characterised by female external genitalia, a short blind-ending vagina, the absence of Müllerian ducts, the development of gynaecomastia and the absence of pubic and axillary hair. Partial androgen insensitivity syndrome covers a wide spectrum of undervirilised phenotypes ranging from clitoromegaly at birth to infertile men.102,103 More than 600 mutations have been described in the X-linked androgen receptor gene (AR) resulting in a 46,XY DSD (androgen insensitivity).104 The first exon of the AR gene contains a polymorphic sequence of CAG triplets. Spinal and bulbar muscular atrophy (SBMA; also called Kennedy disease) is a recessive neurodegenerative adult-onset disorder in men.105 Men with SBMA show slowly progressive spinal and bulbar muscular atrophy with fasciculations and generalised muscle weakness. Partial androgen insensitivity with gynaecomastia, impotence and reduced fertility is often seen. Affected men have about twice as many CAG repeats as unaffected men (38–72 repeats compared with 10–36 repeats, respectively).105 In contrast to SBMA, a reduction in the number of CAG repeats has been reported to be associated with an increased risk of cancer of the prostate in androgen-dependent tumours.106 Expansion of the CAG triplets associated with reduced transcriptional activity and leading to reduced sperm counts has been extensively studied and remains controversial. Although some studies find an association between length of CAG triplets and male infertility, many others do not.107 These discordant results may reflect a difference in ethnicity and geography in the selection of patients and controls. The need for adequate control populations is highlighted by studies on the X-linked testis-specific USP26 gene. Three variants that form a haplotype (371insACA, 494T>C and 1423C>T) were reported to be associated with azoospermia in two

GENETIC AETIOLOGY OF INFERTILITY  |  7

independent studies.108,109 Further analyses demonstrated that two of these changes are the ancestral sequence of the gene that is present in significant frequencies in subSaharan African and South and East Asian populations, including in individuals with known fertility.110 A large number of polymorphic variants have been reported to be associated with male infertility, including polymorphisms associated with the genes DAZL, MTHFR, BOULE, POLG, FSHR, ESR1, DNAI1, DNAH5, DNAH11, KIT, KITLG, ES and PRM1.111–114 However, the contribution of mutations in these genes remains to be established, since the association studies were often based on small sample sizes, and the population substructure, which could give spurious associations, was not taken into account in most of the studies.

Genetics and epigenetics Any consideration of genetic contributions to infertility needs to take into account the recent and sharp decline in human male reproductive health that has been widely reported.115 Prospective cross-sectional studies have indicated a general birth cohort decline in sperm quantity.116 Indeed, 20% of investigated young men from Denmark and Norway had a sperm concentration of below the World Health Organization reference values for oligozoospermia.116 Testicular germ cell cancer is now the most common malignancy in males aged between 20 and 45 years and may have increased in incidence over the past 50 years.117 These phenotypes, together with undescended testes and anomalies of the male external genitalia, may have a common aetiology resulting from disruption of the gonadal environment during fetal life, and the unifying term testicular dysgenesis syndrome (TDS) has been used describe them.118 The rapid rise in the incidence of TDS suggest an environmental aetiology perhaps in genetically susceptible individuals. The influence of genetic and environmental factors is the subject of a continuing debate but reports of declining semen quality in industrialised countries, particularly in Western Europe, over the past 60  years suggests a major environmental influence.118 A considerable body of data suggests that exposure of a developing male fetus in vivo to a number of environmental factors can negatively influence sexual development and testicular function. Much of these data come from well-documented studies of wildlife and experimental laboratory animals exposed at critical periods in their life stage to synthetic chemicals that alter hormone activity in the body (endocrine disruptors). Human exposure is variable but widespread, and sources include consumer products at doses predicted to have a negative impact on reproductive health.118,119 A correlation has been demonstrated between primary metabolites of phthalates in the urine of expectant mothers and boys born with reduced anogenital distance (a sensitive measure of testosterone activity).120 A correlation with phthalate metabolites in breast milk and reduced testosterone levels in infants has also been noted.121 These suggest a link between phthalates and anomalies of the male reproductive system but, as with all association studies, other factors could be involved and the interpretation of statistical trends remains controversial. Associations can be fortuitous and other lifestyle factors could be involved. A move away from trend analyses can be seen in the increasing number of studies that directly evaluate the effects of these chemicals on the synthesis or activity of proteins known to be involved in testicular descent or spermatogenesis.122 In the longer term, data from functional studies may provide a more persuasive argument demonstrating the negative impact of these chemicals on male reproductive development and function. A number of studies have suggested that the detrimental influence of environmental agents on male germ cells may be an epigenetic phenomenon that alters DNA

8  |  ANU BASHAMBOO, CELIA RAVEL AND KEN McELREAVEY

methylation. In utero exposure of fetal male rodents to fungicide at the moment of testis determination promotes heritable male germ cell defects in multiple generations.123,124 These defects are associated with an acquired and apparently permanent hypermethylation inherited through the paternal allele.123,124 In the human, several studies have examined the methylation profiles in sperm from infertile men, with differing results. Loss of hypermethylation at the paternally imprinted H19 locus was originally described in the sperm of men with unexplained reduced sperm counts.125 A further study of seven loci subjected to either maternal or paternal imprinting revealed anomalies of both types of locus in 14–20% of men with moderate or severe oligozoospermia.126 A genome-wide analysis has suggested a global hypermethylation of DNA from poor-quality sperm and improper erasure of DNA methylation during germ cell development.127 These data suggest that the epigenetic landscape may be altered in some men but, from such a limited number of studies, the relationship between these changes and infertility remains obscure.

Conclusion There is evidence for a major genetic contribution to human infertility but the genetic causes themselves remain to be identified. There is a limited basic understanding of the molecular mechanisms involved in gonad development and in gametogenesis. For example, there is considerable interest in deriving gametes from embryonic stem cells but the underlying genetic and epigenetic mechanisms are unknown. There is accumulating evidence of modifications of the epigenetic landscape in sperm from men with reduced sperm counts. These modifications may be related to infertility. Genetic association studies aimed at identifying genetic causes of infertility have also been largely unsuccessful because of limitations that are common in most genetic association studies – variable definition of patient/control phenotypes, small sample size, variable inclusion/exclusion criteria and ascertainment bias. These limitations can be overcome, at least in part, by the creation of national and international biobanks of biological material from well-characterised patient and control groups. Understanding of the genetic basis of infertility is likely to increase dramatically in the future. New technologies are available that permit high-throughput detailed genetic analysis. This includes advances in sequencing technologies such as sequencecapture methods that enable defined regions of the genome (such as the exome) to be enriched by hybridisation, then sequenced using new high-throughput technologies. A significant number of patients with infertility problems carry chromosomal rearrangements. It is likely that other chromosomal anomalies are present that are not detected by the current limits of resolution of conventional cytogenetic approaches. High-resolution comparative genome hybridisation using oligoarrays can identify changes at resolutions of up to 5–10 kb depending on the platform. Some individuals with infertility may be carrying these submicroscopic rearrangements that are causing infertility, whereas copy number variants may confer a genetic susceptibility to infertility. A recent example of such a copy number variant has been described in the ESR1 gene where an intragenic 2.2 kb deletion is present at a frequency of 10% in the Spanish population and may confer a susceptibility to infertility.128 A combination of these approaches together with strict diagnostic criteria will increase the likelihood of success in understanding the genetic basis of infertility.

GENETIC AETIOLOGY OF INFERTILITY  |  9

References 1.

2. 3. 4. 5. 6.

7. 8. 9.

10.

11.

12. 13. 14. 15. 16. 17.

18. 19. 20. 21. 22. 23. 24.

Thonneau P, Marchand S, Tallec A, Ferila ML, Ducot B, Lansac J, et al. Incidence and main causes of infertility in a resident population (1 850 000) of three French regions (1988–1989). Hum Reprod 1991;6:811–16. Skakkebaek NE, Jørgensen N, Main KM, Rajpert-De Meyts E, Leffers H, Andersson AM, et al. Is human fecundity declining ? Int J Androl 2006;29:2–11. Brassard M, AinMelk Y, Baillargeon JP. Basic infertility including polycystic ovary syndrome. Med Clin North Am 2008;92:1163–92. Acién P, Acién M, Sánchez-Ferrer M. Complex malformations of the female genital tract. New types and revision of classification. Hum Reprod 2004;19:2377–84. Giudice LC, Kao LC. Endometriosis. Lancet 2004;364:1789–99. Albright F, Smith PH, Fraser R. A syndrome characterized by primary ovarian insufficiency and decreased stature: report of 11 cases with a digression on hormonal control of axillary and pubic hair. Am J Med Sci 1942;204:625–48. Welt CK. Primary ovarian insufficiency: a more accurate term for premature ovarian failure. Clin Endocrinol (Oxf) 2008;68:499–509. Rebar RW, Connolly HV. Clinical features of young women with hypergonadotropic amenorrhea. Fertil Steril 1990;53:804–10. Nelson LM, Anasti JN, Kimzey LM, Defensor RA, Lipetz KJ, White BJ, et al. Development of luteinized graafian follicles in patients with karyotypically normal spontaneous premature ovarian failure. J Clin Endocrinol Metab 1994;79:1470–5. van Kasteren YM, Schoemaker J. Premature ovarian failure: a systematic review on therapeutic interventions to restore ovarian function and achieve pregnancy. Hum Reprod Update 1999;5:483–92. Bakalov VK, Anasti JN, Calis KA, Vanderhoof VH, Premkumar A, Chen S, et al. Autoimmune oophoritis as a mechanism of follicular dysfunction in women with 46,XX spontaneous premature ovarian failure. Fertil Steril 2005;84:958–65. Singh RP, Carr DH. The anatomy and histology of XO human embryos and fetuses. Anat Rec 1966;155:369–83. Cunniff C, Jones KL, Benirschke K. Ovarian dysgenesis in individuals with chromosomal abnormalities. Hum Genet 1991;86:552–6. Modi DN, Sane S, Bhartiya D. Accelerated germ cell apoptosis in sex chromosome aneuploid fetal human gonads. Mol Hum Reprod 2003;9:219–25. Devi AS, Metzger DA, Luciano AA, Benn PA. 45,X/46,XX mosaicism in patients with idiopathic premature ovarian failure. Fertil Steril 1998;70:89–93. Balen AH, Michelmore K. What is polycystic ovary syndrome? Are national views important? Hum Reprod 2002;17:2219–27. Carmina E, Rosato F, Jannì A, Rizzo M, Longo RA. Extensive clinical experience: relative prevalence of different androgen excess disorders in 950 women referred because of clinical hyperandrogenism. J Clin Endocrinol Metab 2006;91:2–6. Ehrmann DA. Polycystic ovary syndrome, N Engl J Med 2005;352:1223–36. Simpson JL, Elias S, Malinak LR, Buttram VC Jr. Heritable aspects of endometriosis. I. Genetic studies. Am J Obstet Gynecol 1980;137:327–31. Vigano P, Somigliana E, Vignali M, Busacca M, Blasio AM. Genetics of endometriosis: current status and prospects. Front Biosci 2007;12:3247–55. Treloar SA, O’Connor DT, O’Connor VM, Martin NG. Genetic influences on endometriosis in an Australian twin sample. Fertil Steril 1999;71:701–10. Bischoff F, Simpson JL. Genetic basis of endometriosis. Ann N Y Acad Sci 2004;1034:284–99. Montgomery GW, Nyholt DR, Zhao ZZ, Treloar SA, Painter JN, Missmer SA, et al. The search for genes contributing to endometriosis risk. Hum Reprod Update 2008;14:447–57. Treloar SA, Wicks J, Nyholt DR, Montgomery GW, Bahlo M, Smith V, et al. Genomewide linkage study in 1,176 affected sister pair families identifies a significant susceptibility locus for endometriosis on chromosome 10q26. Am J Hum Genet 2005;77:365–76.

10  |  ANU BASHAMBOO, CELIA RAVEL AND KEN McELREAVEY 25. 26. 27. 28.

30.

31. 32.

33. 34. 35.

36. 37. 38. 39.

40. 41. 42. 43. 44. 45.

46. 47.

48. 49. 50.

Guo SW. Glutathione S-transferases M1/T1 gene polymorphisms and endometriosis: a metaanalysis of genetic association studies. Mol Hum Reprod 2005;11:729–43. Allahbadia GN, Merchant R. Polycystic ovary syndrome in the Indian Subcontinent. Semin Reprod Med 2008;26:22–34. Yu Ng EH, Ho PC. Polycystic ovary syndrome in asian women. Semin Reprod Med 2008;26:14–21. Goodarzi MO, Quiñones MJ, Azziz R, Rotter JI, Hsueh WA, Yang H. Polycystic ovary syndrome in Mexican-Americans: prevalence and association with the severity of insulin resistance. Fertil Steril 2005;84:766–9. Davis SR, Knight S, White V, Claridge C, Davis BJ, Bell R. Preliminary indication of a high prevalence of polycystic ovary syndrome in indigenous Australian women. Gynecol Endocrinol 2002;16:443–6. Jahanfar S, Eden JA. Genetic and non-genetic theories on the etiology of polycystic ovary syndrome. Gynecol Endocrinol 1996;10:357–64. Simoni M, Tempfer CB, Destenaves B, Fauser BC. Functional genetic polymorphisms and female reproductive disorders: Part I: Polycystic ovary syndrome and ovarian response. Hum Reprod Update 2008;14:459–84. de Bruin JP, Bovenhuis H, van Noord PA, Pearson PL, van Arendonk JA, te Velde ER, et al. The role of genetic factors in age at natural menopause. Hum Reprod 2001;16:2014–18. Kok HS, van Asselt KM, van der Schouw YT, Peeters PH, Wijmenga C. Genetic studies to identify genes underlying menopausal age. Hum Reprod Update 2005;11:483–93. van Kasteren YM, Hundscheid RD, Smits AP, Cremers FP, van Zonneveld P, Braat DD. Familial idiopathic premature ovarian failure: an overrated and underestimated genetic disease? Hum Reprod 1999;14:2455–9. Starup J, Sele V. Premature ovarian failure. Acta Obstet Gynecol Scand 1973;52:259–68. Conway GS, Kaltsas G, Patel A, Davies MC, Jacobs HS. Characterization of idiopathic premature ovarian failure. Fertil Steril 1996;65:337–41. Cramer DW, Xu H, Harlow BL. Family history as a predictor of early menopause. Fertil Steril 1995;64:740–5. Vegetti W, Grazia Tibiletti M, Testa G, de Lauretis Yankowski, Alagna F, Castoldi E, et al. Inheritance in idiopathic premature ovarian failure: analysis of 71 cases. Hum Reprod 1998;13:1796–1800. Torgerson DJ, Thomas RE, Reid DM. Mothers and daughters menopausal ages: is there a link? Eur J Obstet Gynecol Reprod Biol 1997;74;63–6. Luborsky JL, Meyer P, Sowers MF, Gold EB, Santoro N. Premature menopause in a multi-ethnic population study of the menopause transition. Hum Reprod 2003;18:199–206. McPhaul MJ. Androgen receptor mutations and androgen insensitivity Mol Cell Endocrinol 2002;198:61–7. Sherman SL. Premature ovarian failure in the fragile X syndrome. Am J Med Genet 2000;97:189–94. Van Esch H. The Fragile X premutation: new insights and clinical consequences. Eur J Med Genet 2006;49:1–8. Lacombe A, Lee H, Zahed L, Choucair M, Muller JM, Nelson SF, et al. Disruption of POF1B binding to nonmuscle actin filaments is associated with premature ovarian failure. Am J Hum Genet 2006;79:113–19. Davison RM, Fox M, Conway GS. Mapping of the POF1 locus and identification of putative genes for premature ovarian failure. Mol Hum Reprod 2000;6:314–18. Marozzi A, Manfredini E, Tibiletti MG, Furlan D, Villa N, Vegetti W, et al. Molecular definition of Xq common-deleted region in patients affected by premature ovarian failure. Hum Genet 2000;107:304–11. Finnish-German APECED Consortium. An autoimmune disease, APECED, caused by mutations in a novel gene featuring two PHD-type zinc-finger domains. Nat Genet 1997;7:399–403. Leslie ND. Insights into the pathogenesis of galactosemia. Annu Rev Nutr 2003;23:59–80. Fogli A, Gauthier-Barichard F, Schiffmann R, Vanderhoof VH, Bakalov VK, Nelson LM, et al. Screening for known mutations in EIF2B genes in a large panel of patients with premature ovarian failure. BMC Womens Health 2004;4:8.

GENETIC AETIOLOGY OF INFERTILITY  |  11 51. 52.

53.

54.

55.

56. 57. 58. 59. 60. 61. 62. 63. 64.

65. 66. 67. 68. 69. 70. 71. 72. 73.

74.

De Baere E, Lemercier B, Christin-Maitre S, et al. FOXL2 mutation screening in a large panel of POF patients and XX males. J Med Genet 2002;39:e43. Chatterjee S, Modi D, Maitra A, Kadam S, Patel Z, Gokrall J, et al. Screening for FOXL2 gene mutations in women with premature ovarian failure: an Indian experience. Reprod Biomed Online 2007;15:554–60. Aittomäki K, Lucena JL, Pakarinen P, Sistonen P, Tapanainen J, Gromoll J, et al. Mutation in the follicle-stimulating hormone receptor gene causes hereditary hypergonadotropic ovarian failure. Cell 1995;82:959–68. Latronico AC, Anasti J, Arnhold IJ, Rapaport R, Mendonca BB, Bloise W, et al. Brief report: testicular and ovarian resistance to luteinizing hormone caused by inactivating mutations of the luteinizing hormone-receptor gene. N Engl J Med 1996;334:507–12. Toledo SP, Brunner HG, Kraaij R, Post M, Dahia PL, Hayashida CY, et al. An inactivating mutation of the luteinizing hormone receptor causes amenorrhea in a 46,XX female. J Clin Endocrinol Metab 1996;81:3850–4. Qin Y, Choi Y, Zhao H, Simpson JL, Chen ZJ, Rajkovic A. NOBOX homeobox mutation causes premature ovarian failure. Am J Hum Genet 2007;81:576–81. Dixit H, Rao LK, Padmalatha VV, Kanakavalli M, Deenadayal M, Gupta N, et al. Missense mutations in the BMP15 gene are associated with ovarian failure. Hum Genet 2006;119:408–15. Ledig S, Röpke A, Haeusler G, Hinney B, Wieacker P. BMP15 mutations in XX gonadal dysgenesis and premature ovarian failure. Am J Obstet Gynecol 2008;198:84.e1–5. Hammoud AO, Gibson M, Peterson CM, Kerber RA, Mineau GP, Hatasaka H. Quantification of the familial contribution to Mullerian anomalies. Obstet Gynecol 2008;111:378–84. Verp MS, Simpson JL, Elias S, Carson SA, Sarto GA, Feingold M. Heritable aspects of uterine anomalies I. Three familial aggregrates with Müllerian fusion anomalies. Fertil Steril 1983;40:80–5. Carson SA, Simpson JL, Malinak LR, Elias S, Gerbie AB, Buttram VC Jr, et al. Heritable aspects of uterine anomalies II. Genetic analysis of Müllerian aplasia. Fertil Steril 1983;40:86–90. Carp H, Toder V, Aviram A, Daniely M, Mashiach S, Barkai G. Karyotype of the abortus in recurrent miscarriage. Fert Steril 2001;75:678–82. Pandey MK, Rani R, Agrawal S. An update in recurrent spontaneous abortion. Arch Gynecol Obstet 2005;272:95–108. Tuerlings JH, van Golde RJ, Oudakker AR, Yntema HG, Kremer JA. Familial oligoasthenoteratozoospermia: evidence of autosomal dominant inheritance with sex-limited expression. Fertil Steril 2002;77:415–18. Chaganti RS, German J. Human male infertility, probably genetically determined, due to defective meiosis and spermatogenic arrest. Am J Hum Genet 1979;31:634–41. Léonard C, Bisson JP, David G. Male sterility associated with familial translocation heterozygosity: t(8;15) (q22;p11). Arch Androl 1979;2:269–75. Cantú JM, Rivas F, Hernández-Jáuregui P, Díaz M, Cortés-Gallegos V, Vaca G, et al. Meiotic arrest at first spermatocyte level: a new inherited infertility disorder. Hum Genet 1981;59:380–5. Chang PL, Sauer MV, Brown S. Y chromosome microdeletion in a father and his four infertile sons. Hum Reprod 1999;14:2689–94. Saut N, Terriou P, Navarro A, Lévy N, Mitchell MJ. The human Y chromosome genes BPY2, CDY1 and DAZ are not essential for sustained fertility. Mol Hum Reprod 2000;6:789–93. Shabtai F, Bichacho S, Halbrecht I. The fragile site on chromosome 16 (q21q22). Data on four new families. Hum Genet 1980;55:19–22. Rivera H, Alvarez-Arratia MC, Moller M, Díaz M, Cantú JM. Familial inv(1) (p3500q21.3) associated with azoospermia. Hum Genet 1984;66:165–7. Rolf C, Gromoll J, Simoni M, Nieschlag E. Natural transmission of a partial AZFb deletion of the Y chromosome over three generations: case report. Hum Reprod 2002;17:2267–71. Meschede D, Froster UG, Bergmann M, Nieschlag E. Familial pericentric inversion of chromosome 1 (p34q23) and male infertility with stage specific spermatogenic arrest. J Med Genet 1994;31:573–5. Meschede D, Lemcke B, Behre HM, De Geyter C, Nieschlag E, Horst J. Clustering of male infertility in the families of couples treated with intracytoplasmic sperm injection. Hum Reprod 2000;15:1604–8.

12  |  ANU BASHAMBOO, CELIA RAVEL AND KEN McELREAVEY 75.

76. 77.

78. 79. 80.

81. 82. 83. 84. 85.

86. 87.

88. 89. 90. 91.

92.

93.

94. 95.

96. 97.

Gianotten J, Westerveld GH, Leschot NJ, Tanck MW, Lilford RJ, Lombardi MP,. Familial clustering of impaired spermatogenesis: no evidence for a common genetic inheritance pattern. Hum Reprod 2004;19:71–6. Cloonan YK, Holt VL, Goldberg J. Male factor infertility: a twin study. Paediatr Perinat Epidemiol 2007;21:229–34. Ravel C, Berthaut I, Bresson JL, Siffroi JP; Genetics Commission of the French Federation of CECOS. Prevalence of chromosomal abnormalities in phenotypically normal and fertile adult males: large-scale survey of over 10,000 sperm donor karyotypes. Hum Reprod 2006;21:1484–9. Chandley AC. Genetic contribution to male infertility. Hum Reprod 1998;13 Suppl 3:76–83. Ferlin A, Garolla A, Foresta C. Chromosome abnormalities in sperm of individuals with constitutional sex chromosomal abnormalities. Cytogenet Genome Res 2005;111:310–16. Yamamoto Y, Sofikitis N, Mio Y, Loutradis D, Kaponis A, Miyagawa I. Morphometric and cytogenetic characteristics of testicular germ cells and Sertoli cell secretory function in men with non-mosaic Klinefelter’s syndrome. Hum Reprod 2002;17:886–96. Yoshida A, Miura K, Shirai M. Cytogenetic survey of 1,007 infertile males. Urol Int 1997;58:166–76. Mau-Holzmann UA. Somatic chromosomal abnormalities in infertile men and women. Cytogenet Genome Res 2005;111:317–36. Huang B, Wang S, Ning Y, Lamb AN, Bartley J. Autosomal XX sex reversal caused by duplication of SOX9. Am J Med Genet 1999;87:349–53. Martin RH. Cytogenetic determinants of male fertility. Hum Reprod Update 2008;14:379–90. Skaletsky H, Kuroda-Kawaguchi T, Minx PJ, Cordum HS, Hillier L, Brown LG, et al. The male-specific region of the human Y chromosome is a mosaic of discrete sequence classes. Nature 2003;423:825–37. Tiepolo L, Zuffardi O. Localization of factors controlling spermatogenesis in the nonfluorescent portion of the human Y chromosome long arm. Hum Genet 1976;34:119–24. Vogt PH, Edelmann A, Kirsch S, Henegariu O, Hirschmann P, Kiesewetter F, et al. Human Y chromosome azoospermia factors (AZF) mapped to different subregions in Yq11. Hum Mol Genet 1996;5:933–43. Sadeghi-Nejad H, Oates RD. The Y chromosome and male infertility. Curr Opin Urol 2008;18:628–32. McElreavey K, Ravel C, Chantot-Bastaraud S, Siffroi JP. Y chromosome variants and male reproductive function. Int J Androl 2006;29:298–303. Vinci G, Raicu F, Popa L, Popa O, Cocos R, McElreavey K. A deletion of a novel heat shock gene on the Y chromosome associated with azoospermia. Mol Hum Reprod 2005;11:295–8. Kühnert B, Gromoll J, Kostova E, Tschanter P, Luetjens CM, Simoni M, et al. Case report: natural transmission of an AZFc Y-chromosomal microdeletion from father to his sons. Hum Reprod 2004;19:886–8. Repping S, Skaletsky H, Brown L, van Daalen SK, Korver CM, Pyntikova T, et al. Polymorphism for a 1.6-Mb deletion of the human Y chromosome persists through balance between recurrent mutation and haploid selection. Nat Genet 2003;35:247–51. Iwase S, Lan F, Bayliss P, de la Torre-Ubieta L, Huarte M, Qi HH, et al. The X-linked mental retardation gene SMCX/JARID1C defines a family of histone H3 lysine 4 demethylases. Cell 2007;128:1077–88. Lan F, Bayliss PE, Rinn JL, Whetstine JR, Wang JK, Chen S, et al. A histone H3 lysine 27 demethylase regulates animal posterior development. Nature 2007 ;449:689–94. Lahn BT, Tang ZL, Zhou J, Barndt RJ, Parvinen M, Allis CD, et al. Previously uncharacterized histone acetyltransferases implicated in mammalian spermatogenesis. Proc Natl Acad Sci U S A 2002;99:8707–12. Lin Y, Gill ME, Koubova J, Page DC. Germ cell-intrinsic and -extrinsic factors govern meiotic initiation in mouse embryos. Science 2008;322:1685–7. Sun C, Skaletsky H, Birren B, Devon K, Tang Z, Silber S, et al. An azoospermic man with a de novo point mutation in the Y-chromosomal gene USP9Y. Nat Genet 1999;23:429–32.

GENETIC AETIOLOGY OF INFERTILITY  |  13 98.

99.

100. 101.

102. 103. 104. 105. 106.

107. 108. 109. 110.

111. 112.

113.

114. 115.

116.

117. 118. 119.

120.

Dam AH, Koscinski I, Kremer JA, Moutou C, Jaeger AS, Oudakker AR, et al. Homozygous mutation in SPATA16 is associated with male infertility in human globozoospermia. Am J Hum Genet 2007;81:813–20. Dieterich K, Soto Rifo R, Faure AK, Hennebicq S, Ben Amar B, et al. Homozygous mutation of AURKC yields large-headed polyploid spermatozoa and causes male infertility. Nat Genet 2007 39:661–5. Chillón M, Casals T, Mercier B, Bassas L, Lissens W, Silber S, et al. Mutations in the cystic fibrosis gene in patients with congenital absence of the vas deferens. N Engl J Med 1995;332:1475–80. Xu WM, Shi QX, Chen WY, Zhou CX, Ni Y, Rowlands DK, et al. Cystic fibrosis transmembrane conductance regulator is vital to sperm fertilizing capacity and male fertility. Proc Natl Acad Sci U S A 2007;104:9816–21. Brinkmann AO. Molecular basis of androgen insensitivity. Mol Cell Endocrinol 2001;179:105–9. Sultan C, Paris F, Terouanne B, Balaguer P, Georget V, Poujol N, et al. Disorders linked to insufficient androgen action in male children. Hum Reprod Update 2001;7:314–22. Gottlieb B, Beitel LK, Wu JH, Trifiro M. The androgen receptor gene mutations database (ARDB): 2004 update. Hum Mutat 2004;23:527–33. La Spada AR, Wilson EM, Lubahn DB, Harding AE, Fischbeck KH. Androgen receptor gene mutations in X-linked spinal and bulbar muscular atrophy. Nature 1991;352:77–9. Giovannucci E, Stampfer MJ, Krithivas K, Brown M, Dahl D, Brufsky A, et al. The CAG repeat within the androgen receptor gene and its relationship to prostate cancer. Proc Natl Acad Sci U S A 1997;94:3320–3. Erratum in: Proc Natl Acad Sci U S A 1997;94:8272. Tüttelmann F, Rajpert-De Meyts E, Nieschlag E, Simoni M. Gene polymorphisms and male infertility – a meta-analysis and literature review. Reprod Biomed Online 2007;15:643–58. Stouffs K, Lissens W, Tournaye H, Van Steirteghem A, Liebaers I. Possible role of USP26 in patients with severely impaired spermatogenesis. Eur J Hum Genet 2005;13:336–40. Paduch DA, Mielnik A, Schlegel PN. Novel mutations in testis-specific ubiquitin protease 26 gene may cause male infertility and hypogonadism. Reprod Biomed Online 2005;10:747–54. Ravel C, El Houate B, Chantot S, Lourenço D, Dumaine A, Rouba H, et al. Haplotypes, mutations and male fertility: the story of the testis-specific ubiquitin protease USP26. Mol Hum Reprod 2006;12:643–6. Teng YN, Lin YM, Sun HF, Hsu PY, Chung CL, Kuo PL. Association of DAZL haplotypes with spermatogenic failure in infertile men. Fertil Steril 2006;86:129–35. Thangaraj K, Deepa SR, Pavani K, Gupta NJ, Reddy P, Reddy AG, et al. A to G transitions at 260, 386 and 437 in DAZL gene are not associated with spermatogenic failure in Indian population. Int J Androl 2006;29:510–14. Kukuvitis A, Georgiou I, Bouba I, Tsirka A, Giannouli CH, Yapijakis C, et al. Association of oestrogen receptor alpha polymorphisms and androgen receptor CAG trinucleotide repeats with male infertility: a study in 109 Greek infertile men. Int J Androl 2002;25:149–52. Oliva R. Protamines and male infertility. Hum Reprod Update 2006;12:417–35. Jørgensen N, Asklund C, Carlsen E, Skakkebaek NE. Coordinated European investigations of semen quality: results from studies of Scandinavian young men is a matter of concern. Int J Androl. 2006:29:54–61. Jørgensen N, Carlsen E, Nermoen I, Punab M, Suominen J, Andersen AG, et al. East–West gradient in semen quality in the Nordic-Baltic area: a study of men from the general population in Denmark, Norway, Estonia and Finland. Hum Reprod 2002;17:2199–208. Møller H. Clues to the aetiology of testicular germ cell tumours from descriptive epidemiology. Eur Urol 1993;23:8–13. Skakkebaek NE, Rajpert-De Meyts E, Main KM. Testicular dysgenesis syndrome: an increasingly common developmental disorder with environmental aspects. Hum Reprod 2001;16:972–8. vom Saal FS, Akingbemi BT, Belcher SM, Birnbaum LS, Crain DA, Eriksen M, et al, Chapel Hill bisphenol A expert panel consensus statement: integration of mechanisms, effects in animals and potential to impact human health at current levels of exposure. Reprod Toxicol 2007;24:131–8. Swan SH, Main KM, Liu F, Stewart SL, Kruse RL, Calafat AM, et al. Study for Future Families Research Team. Decrease in anogenital distance among male infants with prenatal phthalate exposure. Environ Health Perspect 2005;113:1056–61.

14  |  ANU BASHAMBOO, CELIA RAVEL AND KEN McELREAVEY 121. Main KM, Mortensen GK, Kaleva MM, Boisen KA, Damgaard IN, Chellakooty M, et al. Human breast milk contamination with phthalates and alterations of endogenous reproductive hormones in infants three months of age. Environ Health Perspect 2006;114:270–6. 122. Laguë E, Tremblay JJ. Antagonistic effects of testosterone and the endocrine disruptor mono-(2ethylhexyl) phthalate on INSL3 transcription in Leydig cells. Endocrinol 2008;149:4688–94. 123. Anway MD, Cupp AS, Uzumcu M, Skinner MK. Epigenetic transgenerational actions of endocrine disruptors and male fertility. Science 2005;308:1466–9. 124. Chang HS, Anway MD, Rekow SS, Skinner MK. Transgenerational epigenetic imprinting of the male germline by endocrine disruptor exposure during gonadal sex determination. Endocrinology 2006;147:5524–41. 125. Marques CJ, Carvalho F, Sousa M, Barros A. Genomic imprinting in disruptive spermatogenesis. Lancet 2004;363:1700–2. 126. Kobayashi H, Sato A, Otsu E, Hiura H, Tomatsu C, Utsunomiya T, et al. Aberrant DNA methylation of imprinted loci in sperm from oligospermic patients. Hum Mol Genet 2007;16:2542–51. 127. Houshdaran S, Cortessis VK, Siegmund K, Yang A, Laird PW, Sokol RZ. Widespread epigenetic abnormalities suggest a broad DNA methylation erasure defect in abnormal human sperm. PLoS One 2007;2:e1289. 128. Galan JJ, Buch B, Pedrinaci S, Jimenez-Gamiz P, Gonzalez A, Serrano-Rios M, et al. Identification of a 2244 base pair interstitial deletion within the human ESR1 gene in the Spanish population J Med Genet 2008;45:420–4.

2 Chapter 2

Disorders of sex development Lin Lin and John C Achermann

Introduction Disorders of sex development (DSD) are defined as ‘congenital conditions in which development of chromosomal, gonadal or anatomical sex is atypical’.1,2 DSD therefore represent a diverse range of conditions that can present at different ages and to a range of different healthcare professionals. Typically, a baby born with ambiguous genitalia will be referred to a paediatric endocrinologist or urologist but DSD can also present in adolescence to gynaecologists because of primary amenorrhoea in a girl, or even in adulthood to fertility services because of difficulty having children. Furthermore, an increasing number of individuals with DSD are being diagnosed in fetal medicine units following a discordance between prenatal karyotyping (performed for another reason) and genital appearance on ultrasound or at birth. An awareness of this range of conditions is thus important for practitioners in many different fields. Given the diverse and complex nature of DSD, a multidisciplinary team (MDT) approach involving various healthcare professionals with experience and interest in this area is essential (for example, endocrinologist, urologist, gynaecologist, psychologist, geneticist, cytogeneticist, biochemist and ethicist).1 While this team need not necessarily be involved in every case of apparently ‘simple’ hypospadias if managed by an experienced urologist, an MDT approach is necessary for more complex cases of DSD where there are diagnostic, management and sex assignment issues. Our knowledge of the causes of DSD, especially at the molecular level, has increased significantly in the past 20 years.3 In the past, the two most frequent diagnoses made were 21-hydroxylase deficiency (congenital adrenal hyperplasia) in a 46,XX baby with ambiguous genitalia, and complete androgen insensitivity syndrome (CAIS) in a 46,XY girl with pubertal development but primary amenorrhoea. Although these two conditions remain the most frequent DSD diagnoses, it is also emerging that many other specific conditions can present as DSD and that the pathways underlying gonad development, gonad function and steroidogenesis are more complex than the simple reductionist models we tended to use. Indeed, in a study of adults with a 46,XY karyotype and DSD, the diagnosis, on review of the data available, was found to be incomplete, absent or even incorrect in a substantial proportion of cases.4 This is not entirely surprising as: n many different conditions might result in a similar phenotype (such as ambiguous genitalia) © Lin Lin and John C Achermann. Volume compilation © RCOG

16  |  LIN LIN AND JOHN C ACHERMANN n each condition probably has a spectrum of severity ranging from a complete

form to milder or partial forms n biochemical and endocrine markers have limited specificity in some cases n some of the biological events resulting in the phenotype may only be manifest during critical developmental windows. Therefore, a genetic-based approach to diagnosis may be useful in some situations. Reaching a specific diagnosis based on molecular genetics also improves our understanding of the biological basis of these conditions. It might also help in making the best decisions for sex assignment and subsequent management based on likely gender identity, urological and sexual function, likely need for endocrine replacement, fertility options, tumour risk and the possibility of associated features developing (for example, adrenal dysfunction). A genetic diagnosis can also help in appropriate counselling of individuals and their families, in stratifying long-term outcome studies based on specific diagnoses, and can in some cases be associated with a sense of resolution. However, genetic analysis in itself generates a number of scientific and ethical challenges, which are important to get right if improved knowledge in this area is to have a significant positive impact in the long term. In this chapter, we review some recent advances in our understanding of human sex development, discuss a suggested new nomenclature and classification system for DSD, provide a brief overview of some known genetic causes of DSD, and outline some challenges for research and clinical practice in this field related to the theme of ‘reproductive genetics’.

Human sex development Human sex development can be broken down into three major components: n chromosomal sex (the presence or absence of X or Y chromosomes) n gonadal sex (development of the gonad into either testis or ovary) n phenotypic (anatomical) sex (the appearance of the internal and external genitalia). Sometimes ‘brain sex’ (gender identity, sex role behaviour, sexual orientation) is also considered as a distinct entity. It is important to note that no single component dictates an individual’s sex. However, considering these separate aspects can help in focusing on a specific diagnosis, which might in some cases be confirmed by molecular analysis. Chromosomal sex The importance of the Y chromosome in testis development has been known for many years and the quest to identify a ‘testis-determining factor’ began in the 1950s. An increasingly narrow region of the Y  chromosome was identified that could contain this factor and, following a series of seminal studies published between 1989 and 1991, the gene SRY (sex-determining region, Y) was shown to be the main testis-determining factor in mice and humans.5 Even to this day, SRY seems to be most important molecular switch involved in testis development. This finding underscores the importance of an intact Y  chromosome for the testis to form, which occurs even in the presence of multiple copies of the X chromosome (for example, 47,XXY).

DISORDERS OF SEX DEVELOPMENT  |  17

Gonadal and phenotypic sex In humans, the primitive gonad develops from a condensation of mesoderm in the medioventral region of the urogenital ridge at around 4–5 weeks of gestation. At this time, primordial germ cells (PGCs) – the precursors of gametes – migrate into the gonad from their origin at the dorsal endoderm of the yolk sac. The presence of intact germ cells appears to be necessary for the ovary to develop but is not needed for testis development to occur. This primitive gonad remains ‘bipotential’ and undifferentiated until around 42 days of gestation, at which point a ‘wave’ of SRY expression occurs in the developing testis, which results in a series of downstream events and commitment to testis formation. At around 7 weeks of gestation, primitive Sertoli cells form and release Müllerian inhibiting substance (MIS) (also known as antimüllerian hormone, AMH), which results in the regression of the Müllerian structures that are destined to form the fallopian tubes, uterus and upper two-thirds of the vagina. At around 8 weeks of gestation, early fetal Leydig cells start to produce testosterone. Testosterone acts through the androgen receptor to stabilise the Wolffian structures (epididymes, vasa deferentia and seminal vesicles) (Figure  2.1). Testosterone also reaches the perineum where it is converted in target tissues by the enzyme 5α-reductase type 2 to its more active metabolite dihydrotestosterone (DHT). DHT also activates the androgen receptor resulting in elongation of the developing phallus and fusion of urethral folds (Figure 2.2).6,7 Thus, the 8- to 12-week period of fetal development is a crucial time for the phenotype to become established. During these early stages of sex development, Leydig cell steroidogenesis is largely under the control of placental human chorionic gonadotrophin (hCG) signalling through the shared luteinising hormone/hCG receptor (LHCGR). However, the hypothalamic– pituitary (gonadotrope) axis becomes active by 20 weeks of gestation, at which point pituitary-derived LH also has a role in stimulating Leydig cell steroidogenesis and testis descent (see below). Although relatively little is known about these specific processes in humans, detailed studies in mice are starting to reveal a cascade of genetic, structural and paracrine events that occur during gonad development.8 For example, following expression of SRY, and subsequent expression of SOX9, a series of discrete events occurs in the developing testis such as: n cellular proliferation n migration of cells into the testis from the neighbouring mesonephros n organisation into primitive cords n development of a coelomic vessel that runs lengthwise along the gonad n Leydig cell differentiation. During this period, testis development seems to be a much more active process morphologically than ovarian development, which in some respects has been considered a constitutive (or ‘default’) process. However, detailed studies of differential gene expression patterns are starting to show a distinct set of genes expressed in the developing ovary, so it seems that ovarian differentiation is an active process too.9,10 While some of these genes may be necessary to maintain ovarian integrity, it is emerging that certain genes expressed in the ovary (DAX1, WNT4/RSPO1) may be needed to oppose testis development – the so called ‘anti-testis’ genes.11 In contrast to the case with the testis, most data suggest that the ovary is steroidogenically quiescent during fetal life.

18  |  LIN LIN AND JOHN C ACHERMANN

Other sexually dimorphic processes: germ cell fate and gonad position Several other important differences occur between the developing testis and ovary in the fetus, such as variations in germ cell fate and gonad position. In the first few months of gestation, PGCs undergo multiple cycles of mitotic division. In the testis, a ‘self-renewable’ population of undifferentiated germ cells

Figure 2.1

Simplified overview of steroidogenesis in the testis and adrenal gland. Testis-specific pathways are shown in grey boxes, adrenal-specific pathways in white boxes, and shared testis/adrenal pathways in black boxes. During male sex development, placental human chorionic gonadotrophin (hCG) or pituitary luteinising hormone (LH) stimulates the LHCG receptor on the fetal Leydig cell to increase cholesterol update into the mitochondria by steroidogenic acute regulatory protein (StAR). Following conversion to pregnenolone by P450 side-chain cleavage (P450scc, CYP11A1), a series of enzymatic modifications occur that ultimately result in the synthesis of the androgen testosterone by 17β-hydroxysteroid dehydrogenase 3 (17β-HSD 3). Testosterone is released from the testis and converted to the more potent androgen dihydrotestosterone (DHT), where it acts on the androgen receptor to stimulate external genital development. Defects anywhere along this pathway can result in impaired androgen synthesis and action. Defects in StAR, P450ssc and 3β-hydroxysteroid dehydrogenase type 2 (3β-HSD 2) cause adrenal and gonadal failure, whereas defects on 17α-hydroxylase (which also has 17,20-lyase activity) cause hypertension due to adrenal steroid excess. In 46,XX fetuses with congenital adrenal hyperplasia, blocks in 21-hydroxylase or 11β-hydroxylase result in reduced cortisol feedback and elevated adrenocorticotrophin (ACTH) drive, shunting steroid precursors into the androgenic pathway and causing androgenisation of the female fetus. Milder effects are seen with blocks in 3β-HSD 2. All these conditions can be associated with adrenal dysfunction. In addition to the pathways shown, disruption of P450 oxidoreductase (POR) – a co-enzyme for 17α-hydroxylase and 21-hydroxylase – can cause under-androgenisation (46,XY) or over-androgenisation (46,XX) and a steroid pattern reflecting defects in both these enzyme systems; DHEA = dehydroepiandrosterone; DOC = deoxycorticosterone

DISORDERS OF SEX DEVELOPMENT  |  19

Figure 2.2 Differentiation of male external genitalia in humans between 8 and 10 weeks post conception (wpc): (A) undifferentiated human external genitalia at 8 wpc; (B) differentiation of scrotal folds and fusion of the urethral folds (asterisks mark patent regions, either side) at 10 wpc; gs = genital swelling; gt = genital tubercle; sf = scrotal folds; uf = urethral folds; scale bars = 500 µm; reproduced with permission from Goto et al.6 (© 2006 American Society for Clinical Investigation)

exists, but most PGCs commit to differentiation and, after several cycles of mitotic division, enter mitotic arrest. Subsequent testicular development can occur in the absence of this germ cell population. Meiosis only occurs during the progress of spermatogenesis after puberty. In the developing ovary, primordial ova (oogonia) undergo mitotic expansion in the first few months of gestation (5–24  weeks), followed by meiotic division (8– 36 weeks) and a process of meiotic arrest (oocytes). Retinoic acid signalling from the mesonephros may stimulate this process.12,13 The presence of the meiotic oocytes is critical for differentiation of pre-follicular cells into follicular cells and for maintaining ovarian development. More than six million oogonia and prophase oocytes exist in the developing ovary at around 16 weeks of gestation. Formation of oogonia from PGCs stops by 7 months of gestation. At this stage, some oocytes are found to be associated with somatic pregranulosa cells forming primitive or primordial follicles. However, most oogonia do not form follicles and undergo apoptosis, so that approximately one million germ cells are present in the ovary at the time of birth. These ‘resting’ primodial follicles can remain in this stage of development throughout the woman’s reproductive life, and meiosis only progresses in response to ovulation of the Graafian follicle (approximately 400 in a woman’s reproductive lifetime). Traditional doctrine has been that the population of germ cells is ‘fixed’ at birth but more recent data have raised the possibility of a potential self-renewable population of germline stem cells in the mouse ovary that are active into adult life or of potential repopulation of the ovary by germ cells from other sites.14 It is highly controversial whether such mechanisms exist in primates.15 Whereas the ovary remains in an intra-abdominal position, the developing testis undergoes a two-stage process of descent that starts at around 12 weeks of gestation and is usually complete by the middle of the third trimester. The initial transabdominal stage of testicular descent involves contraction and thickening of the gubernacular ligament. This stage is mediated by the testis itself, following secretion of factors such

20  |  LIN LIN AND JOHN C ACHERMANN

as insulin-like 3 (INSL3, relaxin-like factor) which acts through a G-protein-coupled receptor, LGR8 (GREAT). The subsequent trans-inguinal phase of testicular descent involves stimulation by androgens and LH. Thus, dysgenetic testes are usually intraabdominal whereas testes are inguinal in disorders of androgen synthesis and action. Early postnatal changes The hypothalamic–pituitary (gonadotrope)–gonadal (HPG) axis develops in midgestation and actively secretes gonadotrophins (LH and follicle-stimulating hormone [FSH]) from around 20  weeks of gestation. Around the time of birth, boys have relatively high levels of testosterone, which fall in the first week of life. After a period of postnatal quiescence, the HPG axis becomes reactivated and a so-called ‘minipuberty’ occurs from 6 weeks to 6 months with a peak of testosterone in the low adult range at around 3 months of age. Thereafter, the HPG axis remains quiescent again until puberty. The role of this HPG activation in early infancy is not known although it is associated with a small acceleration in penile growth and may occur before certain further developments in germ cell maturation.16 It is likely that postnatal HPG axis activation occurs in girls but the significance of this is not known. Two relatively new hormones are emerging as useful markers of postnatal testis function. Inhibin  B is secreted predominantly by Leydig cells. It is detectable at birth, falls during infancy and rises again at puberty, thus following the pattern of testosterone secretion to some degree. MIS/AMH is secreted predominantly by Sertoli cells and is high at birth, rises in the first few weeks of life and remains high throughout childhood before falling to adult levels during puberty. Both markers will be low if testicular dysgenesis or regression has occurred.17 Inhibin A and AMH may reflect ovarian integrity but levels are usually low in infancy and childhood. In both sexes, reactivation of the HPG axis in late childhood results in onset of puberty in adolescence. In boys, LH stimulates Leydig cells to produce androgens (testosterone) necessary for penile enlargement, hair growth (pubic, axillary and facial) and voice changes. FSH stimulates Sertoli cells to activate spermatogenesis, although intra-testicular testosterone also has a facilitative role in this regard. In girls, LH and FSH stimulate the production of estrogens by the steroidogenic pathways of the ovarian theca and granulosa cells. Estrogens promote breast development and uterine growth. In addition, gonadotrophins regulate certain aspects of follicular development so that regular ovulatory cycles develop by the end of puberty. More detailed descriptions of spermatogenesis and folliculogenesis are beyond the scope of this chapter.

Disorders of sex development The past 20 years have seen significant advances in our understanding of the underlying aetiology of many forms of DSD. Coupled with this has been the need to develop a classification system based on a more precise diagnosis wherever possible and awareness that many of the terms used to describe DSD are out of date, scientifically inaccurate and/or perceived as pejorative by many individuals with these conditions. In 2005, a consensus meeting to discuss these issues was convened in Chicago.1,2 Although this was driven by the major paediatric endocrinology societies, participants included surgeons, psychologists, geneticists and representatives of patient advocacy groups. The outcome of this meeting was published as a series of guidelines and statements in 2006. This process generated many more questions than answers, for

DISORDERS OF SEX DEVELOPMENT  |  21

example regarding the controversies of surgical intervention and lack of long-term outcome data. However, two important elements to emerge from this consensus meeting were a proposed new nomenclature system and a classification system that can be used to incorporate a specific molecular diagnosis where available. Nomenclature The need for a revised nomenclature system has been apparent for some time.18,19 Many of the traditional terms used were viewed negatively by individuals with these conditions and were not helpful diagnostically. For example, a woman with CAIS, who would typically present in late adolescence with primary amenorrhoea but who has a female gender identity and sex role behaviour, would subsequently be called ‘male pseudohermaphrodite’. The consensus meeting proposed that a more generic term – ‘disorders of sex development’ – be used instead of intersex, and that ‘46,XY DSD’ and ‘46,XX DSD’ could replace male and female pseudohermaphroditism, respectively (Table 2.1).1,2 Wherever possible, a more specific diagnosis (for example, ‘gonadal dysgenesis’ or ‘androgen insensitivity’) could be used and an exact molecular diagnosis incorporated into this if known. Classification and diagnosis Together with a revised nomenclature system, an updated classification system for DSD was proposed. This system divides the conditions into: n sex chromosome DSD n 46,XY DSD n 46,XX DSD. An overview of a potential classification system is shown in Table  2.2. It is very important to realise that the karyotype in itself is not necessarily the most important factor in determining outcome and the karyotype does not define an individual’s ‘sex’. However, rapid diagnostic tests such as fluorescence in situ hybridisation (FISH) (using probes for SRY and the X chromosome) or quantitative polymerase chain reaction (PCR) mean that a preliminary karyotype result can be available within a matter of hours or days, which can be very useful for guiding initial investigation, management and counselling, especially in a newborn child presenting with ambiguous genitalia. Thus, this form of genetic/cytogenetic analysis is very important in the initial approach to investigation of DSD. Such a wide-reaching definition and classification system can be criticised to some degree. For example, ‘sex chromosome DSD’ (sex chromosome aneuploidy) includes monosomy X (45,X; Turner syndrome) and 47,XXY (Klinefelter sydrome), which

Table 2.1

An updated nomenclature for disorders of sex development

Terminology used previously Intersex Male pseudohermaphrodite Female pseudohermaphrodite True hermaphrodite XX male

Proposed new terminology Disorders of sex development (DSD) 46,XY DSD 46,XX DSD Ovotesticular DSD Testicular DSD

C: Other 1. Syndromic associations of male genital development (≥ 50) (e.g. cloacal anomalies, Robinow syndrome, Aarskog syndrome, hand-foot-genital syndrome, popliteal pterygium syndrome) 2. Persistent Müllerian duct syndrome 3. Vanishing testis syndrome 4. Isolated hypospadias 5. Congenital hypogonadotrophic hypogonadism 6. Cryptorchidism 7. Environmental influences

B: Disorders of androgen synthesis or action 1. Disorders of androgen synthesis:  – LH receptor mutations  C: 45,X/46,XY mosaicism – Smith-Lemli-Opitz syndrome  (mixed gonadal dysgenesis) – Steroidogenic acute regulatory protein  – Cholesterol side-chain cleavage  D: 46,XX/46,XY – 3β-hydroxysteroid dehydrogenase type 2 (chimerism) – 17α-hydroxylase/17,20-lyase – P450 oxidoreductase  – 17β-hydroxysteroid dehydrogenase type 3 – 5α-reductase 2 2. Disorders of androgen action:  – androgen insensitivity syndrome  – drugs and environmental modulators

B: 45,X (Turner syndrome and variants)

46,XY DSD

A: Disorders of gonadal (testis) development 1. Complete or partial gonadal dysgenesis (e.g. SRY, SOX9, SF1, WT1, DHH) 2. Ovotesticular DSD 3. Testis regression

A: 47,XXY (Klinefelter syndrome and variants)

Syndromic associations (e.g. cloacal anomalies) Müllerian agenesis/hypoplasia (e.g. MURCS) Uterine abnormalities (e.g. MODY5) Vaginal atresia (e.g. McKusick–Kaufman syndrome) 5. Labial adhesions

1. 2. 3. 4.

C: Other

B: Androgen excess 1. Fetal:  – 3β-hydroxysteroid dehydrogenase type 2 – 21-hydroxylase  – P450 oxidoreductase  – 11β-hydroxylase – glucocorticoid receptor mutations 2. Fetoplacental:  – aromatase deficiency  – oxidoreductase deficiency 3. Maternal:  – maternal virilising tumours (e.g. luteomas)  – androgenic drugs

A: Disorders of gonadal (ovary) development 1. Gonadal dysgenesis 2. Ovotesticular DSD 3. Testicular DSD (e.g. SRY+, dup SOX9, RSPO1)

46,XX DSD

A classification system for disorders of sex development; modified with permission from Hughes et al.1 (© 2006 BMJ Publishing Group Ltd and the Royal College of Paediatrics and Child Health)

Sex chromosome DSD

Table 2.2

22  |  LIN LIN AND JOHN C ACHERMANN

DISORDERS OF SEX DEVELOPMENT  |  23

were not previously considered ‘intersex’ conditions. In addition, the androgenisation of 46,XX individuals with congenital adrenal hyperplasia (CAH) reflects an adrenal disease with reproductive consequences, rather than a true DSD. Finally, more frequently diagnosed conditions such as hypospadias or undescended testes may not represent a classic DSD and including such individuals in this global diagnosis may not be appropriate. Nevertheless, the group of conditions outlined in this classification do share some diagnostic, surgical or reproductive common ground and a subset of boys with hypospadias/undescended testes may have conditions at the milder end of the DSD spectrum. It is also worth noting that a large number (more than 100) of genetic syndromes have genital or reproductive anomalies listed as part of the phenotype. In most cases, this reflects a more frequent than expected occurrence of undescended testes, micropenis or hypospadias in boys. However, complete or partial testicular dysgenesis, or genital ambiguity can occur in some cases and several well-defined syndromic associations of DSD are described (for example, SOX9/campomelic dysplasia and ARX/X-linked lissencephaly; see Table 2.3). Furthermore, several syndromes affect female development, causing apparent clitoromegaly or anomalies in ovarian, uterine or vaginal development (Table 2.2). Using the classification system, the initial determination of karyotype together with clinical features can lead into a series of biochemical, endocrine, radiological and surgical investigations that can help determine the likely diagnostic category for the child’s or adult’s condition. Further detailed genetic analysis can then be used to reach a specific diagnosis at the molecular level. In certain well-established conditions, where the diagnosis is likely from the biochemical data (for example, disorders of steroidogenesis),20 genetic analysis can be useful to confirm the diagnosis at the molecular level and can help to counsel family members or potentially for guiding prenatal treatment. The likelihood of finding a specific genetic defect in such cases is relatively high (above 80%). In other forms of DSD, such as testicular dysgenesis without associated features or partial androgen insensitivity syndrome (PAIS), there may be few clues as to the exact cause of the condition and a more systematic approach involving analysis of several known candidate genes may be needed. Even in such cases, the current rate of identifying a specific disease-causing change may be of the order of only 20–30%. The remainder of this section briefly discusses some key issues from the three main types of DSD as shown in Table  2.2, with a focus on reproductive genetics. More detailed information of these conditions and their management can be found elsewhere.3 Sex chromosome DSD Sex chromosome DSD includes all cases of sex chromosome aneuploidy. As discussed above, monosomy  X and its variants (Turner syndrome) and 46,XXY and its variants (Klinefelter syndrome) were not classed as ‘intersex’ conditions previously, although the presence of a Y chromosome fragment in Turner syndrome can cause androgenisation and an increased gonadal tumour risk,21 and new data suggest that the prevalence of hypospadias is higher in Klinefelter syndrome than in the general population, especially when multiple copies of the X chromosome are present (for example, 48,XXXY and 49,XXXXY).22 Furthermore, fertility issues related to DSD may be relevant to some of these cases of sex chromosome aneuploidy, so the MDT can provide valuable input in some cases.

Gene

Uterus Adrenal Associated features present defect

↑ pregnenolone, progesterone, 11-deoxycorticosterone, ↓ 17-hydroxylated steroids (except in isolated 17,20-lyase deficiency) and adrenal/gonadal androgens, ↑ LH

↑ Δ5 : Δ4 ratio +/− mineralocorticoid insufficiency

Impaired production of all steroids

↑ LH, poor androgen response to hCG Impaired production of all steroids

↑ 7-dehydrocholesterol

− Androgen biosynthetic defect too − − − − − − − − −

Proteinuria

−

Diagnostic biochemical features

Some recognised single-gene or chromosomal defects found in disorders of sex development, with associated phenotypic and biochemical features; modified with permission from Achermann et al.46 (© 2002 The Endocrine Society)

45,X/46,XY mosaicism Mixed gonadal dysgenesis − +/− − +/− Turner syndrome-like features 46,XY DSD – disorders of testis development (gonadal dysgenesis) WAGR, Denys–Drash and WT1 +/− − Wilms’ tumour, renal abnormalities, gonadal tumours Frasier syndromes CBX2 CBX2 + − − Steroidogenic factor 1 NR5A1 +/− +/− +/− partial hypogonadotrophic hypogonadism SRY SRY +/− − − Campomelic dysplasia SOX9 SOX9 +/− − Desert hedgehog DHH + − +/− Minifascicular neuropathy X-linked lissencephaly ARX − − Lissencephaly, epilepsy SIDDT syndrome TSPYL1 − − Sudden infant death 9p24.3 deletion DMRT1 +/− − Developmental delay Xq13.3 deletion ATRX − − α-thalassaemia, developmental delay Xp21 duplication DAX1 +/− − − 1q35 duplication WNT4a +/− − − 46,XY DSD – disorders of androgen synthesis and action Smith–Lemli–Opitz DHCR7 − +/− Coarse facies, second-third toe syndactyly, developmental delay, syndrome cardiac and visceral abnormalities LH resistance LHGCR − − Leydig cell hypoplasia Congenital lipoid adrenal Lipid-filled adrenals, pubertal failure (46,XY), anovulation (46,XX) StAR − + hyperplasia Cholesterol side-chain CYP11A1 − + Pubertal failure cleavage deficiency + Pubertal failure 3β-hydroxysteroid dehydro- HSD3B2 − genase deficiency type II CYP17 − + Hypertension due to ↑ 11-deoxycorticosterone (except in isolated 17α-hydroxylase/17,2017,20-lyase deficiency), pubertal failure lyase deficiency

Condition

Table 2.3

24  |  LIN LIN AND JOHN C ACHERMANN

Gene

+

+

+

POR

CYP19

GRα

P450 oxidoreductase deficiency Aromatase insufficiency

Glucocorticoid resistance

Mild, partial androgenisation due to ↑ conversion of DHEA

↑ ACTH, ↑ 17-hydroxyprogesterone, +/− mineralocorticoid insufficiency Hypertension due to ↑ 11-deoxycorticosterone but often normotensive ↑ ACTH, ↑ 11-deoxycorticosterone, ↑ 11-deoxycortisol or even mild salt loss in early life, premature virilisation +/− Antley–Bixler craniosynostosis Mixed features of 21-hydroxylase deficiency, 17α-hydroxylase/17,20-lyase deficiency, salt loss rare Maternal androgenisation during pregnancy, absent breast ↑ A4, testosterone, ↓ estrogens, ↑ FSH/LH development at puberty except in partial cases, polycystic ovaries, delayed bone age Hypertension ↑ ACTH, 17-hydroxyprogesterone, cortisol, mineralocorticoids and androgens, failure of dexamethasone suppression (NB a patient with ambiguous genitalia was heterozygous for a mutation in CYP21 too)

+

+

−

−

+

+

− − Palmarplantar hyperkeratosis, squamous cell carcinoma

− − −

+ = present; − = absent; ↑ = increasing levels of; ↓ = decreasing levels of; ACTH = adrenocorticotrophin; DHEA = dehydroepiandrosterone; DHT = dihydrotestosterone; FSH = follicle-stimulating hormone; hCG = human chorionic gonadotrophin; LH = luteinising hormone; SIDDT = sudden infant death with dysgenesis of the testes a This region also contains RSPO1

CYP11B1 +

11β-hydroxylase deficiency

↑ ACTH, ↑ ∆5 : ∆4 ratio, +/− mineralocorticoid insufficiency

−

−

Premature virilisation

hCG-responsive ↑ androgens hCG-responsive ↑ androgens hCG-responsive ↑ androgens

Partial androgenisation at puberty

−

↑ testosterone : DHT ratio (often > 20, may need hCG stimulation) Variable ↑ testosterone and LH/FSH, ↑ AMH

Partial androgenisation at puberty

Mixed features of 21-hydroxylase deficiency, 17α-hydroxylase/17,20-lyase deficiency, salt loss rare ↓ testosterone : androstenedione ratio (C SNP in exon 5 of RHCE encoding Ala226Pro in the fourth extracellular loop of the protein.38 Typing for  E, therefore, involves detection of C676. The k/K polymorphism results from a 698C>T SNP in exon  6 of KEL encoding Thr193Met.40 There are only a few published reports of non-invasive fetal genotyping for  C, c and E.34,41–43 All involve RQPCR with allele-specific primers or probes. The genotyping results, compared with serological determinations following birth, show 100% accuracy. K typing presents more of a problem. Finning et al.42 were unable to obtain a satisfactory level of specificity by conventional RQPCR methods owing to mispriming of the K (KEL*1) allele-specific primer on the k (KEL*2) allele. This was overcome, with a sacrifice of reduced sensitivity, by employing locked nucleic acids (LNAs) and the introduction of a mismatch into the allele-specific primer. LNAs are nucleic acid analogues that lock the structure into a rigid bicyclic formation.44 Oligonucleotides that contain LNAs have exceptionally high affinity for complementary DNA strands and excellent mismatch discrimination. The only false negative result was obtained in a sample taken at 17 weeks of gestation that had a particularly low yield of total DNA. Consequently, it is a recommendation in England that a K-negative result obtained before 28 weeks of gestation should be followed up with a repeat test at or after 28 weeks.

180  |  GEOFF DANIELS, KIRSTIN FINNING, PETER MARTIN AND EDWIN MASSEY

Li et al.45 obtained 94% accuracy in fetal K  detection by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS)-based single allele-based extension reaction (SABER). Most K antibodies are stimulated by blood transfusion rather than pregnancy.46 Consequently, the partners of many women with anti-K are K-negative, indicating a K-negative fetus. It is valuable, therefore, to test the father for K before making the decision to carry out fetal testing. Development of a non-invasive genotyping method for determining fetal HPA-1a platelet antigen would be beneficial because anti-HPA-1a is the most common cause of neonatal alloimmune thrombocytopenia.

Quality assurance As an increasing number of laboratories internationally are introducing non-invasive fetal blood group testing, it is important that their performances are monitored through external quality assurance schemes. The International Society of Blood Transfusion (ISBT) organised international workshops in molecular blood group genotyping in 2004,17 200618 and 2008.19 In the most recent workshop, two plasma samples from pregnant D-negative women, one with a D-positive and one with a D-negative fetus, were distributed to 17  laboratories (14 from Europe and one each from Australia, Brazil and China). From a total of 31 results submitted, 27 were correct, two were inconclusive and two reported the D-positive fetus as D-negative (Table  12.1). All laboratories employed RQPCR.19 A European network of excellence on special non-invasive advances in fetal and neonatal evaluation (SAFE), funded by the European Union, has provided money for research, meetings and workshops, increasing communication between workers in the field.47 A SAFE workshop on extraction of fetal DNA from maternal plasma demonstrated that the highest yield was obtained by the QIAamp DSP virus kit.48

Conclusion To date, the enormous promise of the application of free fetal DNA in maternal plasma to prenatal diagnostics has only been realised in fetal blood grouping and fetal sexing. Fetal blood grouping often plays an important role in the avoidance of unnecessary procedures and is the standard of care in England for pregnant women with significant levels of anti-D. Since a reliable non-invasive test is available, it could be considered unethical to perform amniocenteses purely for fetal RhD typing. In the future, high-throughput fetal D typing will reduce wastage of anti-D immunoglobulin and avoid unnecessary treatment of pregnant women with blood products. As intellectual property rights have been granted for free fetal DNA testing worldwide, let us hope that progress in this field is not hampered by financial and legal issues.

Table 12.1 Fetal D type D-negative D-positive

Plasma fetal RHD typing results from the International Society of Blood Transfusion (ISBT) workshop in 2008; 17 laboratories received samples19 Correct 14 13

Incorrect 0 2

Inconclusive 1 1

No results reported 2 1

NON-INVASIVE PRENATAL DIAGNOSIS FOR FETAL BLOOD GROUP STATUS  |  181

References 1. 2.

3. 4. 5. 6. 7. 8. 9.

10. 11.

12. 13. 14. 15. 16. 17. 18. 19. 20.

21.

22.

23.

24. 25.

Lo YM, Corbetta N, Chamberlain PF, Rai V, Sargent IL, Redman CW, et al. Presence of fetal DNA in maternal plasma and serum. Lancet 1997;350:485–7. Lo YM, Tein MS, Lau TK, Haines CJ, Leung TN, Poon PM, et al. Quantitative analysis of fetal DNA in maternal plasma and serum: implications for noninvasive prenatal diagnosis. Am J Hum Genet 1998;62:768–75. Chan KC, Zhang J, Hui AB, Wong N, Lau TK, Leung TN, et al. Size distributions of maternal and fetal DNA in maternal plasma. Clin Chem 2004;50:88–92. National Institute for Clinical Excellence. Guidance on the Use of Routine Antenatal Anti-D Prophylaxis for RhD-negative Women. Technology appraisal guidance 41. London: NICE; 2002. Kumar S, Regan F. Management of pregnancies with RhD alloimmunisation. BMJ 2005;330:1255–8. Wilson RD. Amniocentesis and chorionic villus sampling. Curr Opin Obstet Gynecol 2000;12: 81–6. Tabor A, Bang J, Nørgaard-Pedersen B. Feto-maternal haemorrhage associated with genetic amniocentesis: results of a randomized trial. Br J Obstet Gynecol 1987;94:528–34. Wagner FF, Flegel WA. RHD gene deletion occurred in the Rhesus box. Blood 2000;95:3662–8. Singleton BK, Green CA, Avent ND, Martin PG, Smart E, Daka A, et al. The presence of an RHD pseudogene containing a 37 base pair duplication and a nonsense mutation in most Africans with the Rh D-negative blood group phenotype. Blood 2000;95:12–18. Faas BH, Beckers EA, Wildoer P, Ligthart PC, Overbeeke MA, Zondervan HA, et al. Molecular background of VS and weak C expression in blacks. Transfusion 1997;37:38–44. Daniels GL, Faas BH, Green CA, Smart E, Maaskant-van Wijk PA, Avent ND, et al. The Rh VS and V blood group polymorphisms in Africans: a serological and molecular analysis. Transfusion 1998;38:951–8. Daniels G. Human Blood Groups. 2nd ed. Oxford: Blackwell Science; 2002. Wagner FF, Gassner C, Müller TH, Schönitzer D, Schunter F, Flegel WA. Molecular basis of weak D phenotypes. Blood 1999;93:385–93. Westhoff CM. Rh complexities: serology and DNA genotyping. Transfusion 2007;47(1 Suppl):17S–22S. Mouro I, Le Van Kim C, Rouillac C, van Rhenen DJ, Le Pennec PY, et al. Rearrangements of the blood group RhD gene associated with the DVI category phenotype. Blood 1994;83:1129–35. Chiu RW, Poon LL, Lau TK, Leung TN, Wong EM, Lo YM. Effects of blood-processing protocols on fetal and total DNA quantification in maternal plasma. Clin Chem 2001;9:1607–13. Daniels G, van der Schoot CE, Olsson ML. Report of the first international workshop on molecular blood group genotyping. Vox Sang 2005;88:136–42. Daniels G, van der Schoot E, Olsson ML. Report of the Second International Workshop on molecular blood group genotyping. Vox Sang 2007;93:83–8. Daniels G, van der Schoot CE, Gassner C, Olsson ML. Report of the third international workshop on molecular blood group genotyping. Vox Sang 2009;96:337–43. Finning KM, Martin PG, Soothill PW, Avent ND. Prediction of fetal D status from maternal plasma: introduction of a new noninvasive fetal RHD genotyping service. Transfusion 2002;42:1079–85. Aubin JT, Le Van Kim C, Mouro I, Colin Y, Bignozzi C, Brossard Y, et al. Specificity and sensitivity of RHD genotyping methods by PCR-based DNA amplification. Br J Haematol 1997;98:356–64. Rouillac-Le Sciellour C, Puillandre P, Gillot R, Baulard C, Métral S, Le Van Kim C, et al. Largescale pre-diagnosis study of fetal RHD genotyping by PCR on plasma DNA from RhD-negative pregnant women. Mol Diagn 2004;8:23–31. Rouillac-Le Sciellour C, Sérazin V, Brossard Y, Oudin O, Le Van Kim C, Colin Y, et al. Noninvasive fetal RHD genotyping from maternal plasma. Use of a new developed Free DNA Fetal Kit RhD. Transfus Clin Biol 2007;14:572–7. van der Schoot CE, Hahn S, Chitty LS. Non-invasive pre-natal diagnosis and determination of fetal Rh status. Semin Fetal Neonatal Med 2008;13:63–8. Sequenom [www.sequenom.com].

182  |  GEOFF DANIELS, KIRSTIN FINNING, PETER MARTIN AND EDWIN MASSEY 26. 27. 28. 29.

30.

31. 32.

33.

34.

35. 36.

37.

38. 39. 40. 41.

42. 43. 44.

45.

46. 47. 48.

Geifman-Holtzman O, Grotegut CA, Gaughan JP. Diagnostic accuracy of noninvasive fetal genotyping from maternal blood – a meta analysis. Am J Obstet Gynecol 2006;195:1163–73. Lenetix [www.lenetix.com]. Minon JM, Semterre JM, Schaaps JP, Foidart JP. An unusual false positive fetal RHD typing result using DNA derived from maternal plasma from a solid organ recipient. Transfusion 2006;46:1454. Page-Christiaens GC, Bossers B, van der Schoot CE, de Haas M. Use of bi-allelic insertion/ deletion polymorphisms as a positive control in maternal blood. First clinical experience. Ann N Y Acad Sci 2006;1075:123–9. Pertl B, Pieber D, Panzitt T, Haeusler MC, Winter R, Tului L, et al. RhD genotyping by quantitative fluorescent polymerase chain reaction: a new approach. Br J Obstet Gynaecol 2000;107:1498–502. Liu FM, Wang XY, Feng X, Wang W, Ye YX, Hong C. Feasibility study of using fetal DNA in maternal plasma for non-invasive prenatal diagnosis. Acta Obstet Gynecol Scand 2007;86:535–41. Chan KC, Ding C, Gerovassili A, Yeung SW, Chiu RW, Leung TN, et al. Hypermethylated RASSF1A in maternal plasma: a universal fetal DNA marker that improves the reliability of noninvasive prenatal diagnosis. Clin Chem 2006;52:2211–18. Costa JM, Giovangrandi Y, Ernault P, Lohmann L, Nataf V, El Halali N, et al. Fetal RHD genotyping in maternal serum during the first trimester of pregnancy. Br J Haematol 2002;119:255–60. Legler TJ, Lynen R, Maas JH, Pindur G, Kulenkampff D, Suren A, et al. Prediction of fetal Rh D and Rh CcEe phenotype from maternal plasma with real-time polymerase chain reaction. Transfus Apher Sci 2002;27:217–223. Van der Schoot CE, Soussan AA, Koelewijn J, Bonsel G, Paget-Christiaens LG, de Haas M. Noninvasive antenatal RHD typing. Transfus Clin Biol 2006;13:53–7. Finning K, Martin P, Summers J, Massey E, Poole G, Daniels G. Effect of high throughput RHD typing of fetal DNA in maternal plasma on use of anti-RhD immunoglobulin in RhD negative pregnant women: prospective feasibility study. BMJ 2008;336:816–18. Müller SP, Bartels I, Stein W, Emons G, Gutensohn K, Köhler M, et al. The determination of the fetal D status from maternal plasma for decision making on Rh prophylaxis is feasible. Transfusion 2008;48:2292–301. Mouro I, Colin Y, Chérif-Zahar B, Cartron JP, Le Van Kim C. Molecular genetic basis of the human Rhesus blood group system. Nature Genet 1993;5:62–5. Poulter M, Kemp TJ, Carritt B. DNA-based Rhesus typing: simultaneous determination of RHC and RHD status using the polymerase chain reaction. Vox Sang 1996;70:164–8. Lee S, Wu X, Reid M, Zelinski T, Redman C. Molecular basis of the Kell (K1) phenotype. Blood 1995;85:912–16. Hromadnikova I, Vechetova L, Vesela K, Benesova B, Doucha J, Vlk R. Non-invasive fetal RHD and RHCE genotyping using real-time PCR testing of maternal plasma in RhD-negative pregnancies. J Histochem Cytochem 2005;53:301–5. Finning K, Martin P, Summers J, Daniels G. Fetal genotyping for the K (Kell) and Rh C, c, and E blood groups on cell-free fetal DNA from maternal plasma. Transfusion 2007;47:2126–33. Orzińska A, Guz K, Brojer E, Zupańska B. Preliminary results with fetal Rhc examination in plasma of pregnant women with anti-c. Prenat Diagn 2008;28:335–7. Petersen K, Vogel U, Rockenbauer E, Nielsen KV, Kølvraa S, Bolund L, et al. Short PNA molecular beacons for real-time PCR allelic discrimination of single nucleotide polymorphisms. Mol Cell Probes 2004;18:117–22. Li Y, Finning K, Daniels G, Hahn S, Zhong X, Holzgreve W. Noninvasive genotyping fetal Kell blood group (KEL1) using cell-free fetal DNA in maternal plasma by MALDI-TOF mass spectometry. Prenat Diagn 2008;28:203–8. Klein HG, Anstee DJ. Blood Transfusion in Clinical Medicine. 11th ed. Oxford: Blackwell; 2005. Chitty LS, van der Schoot CE, Hahn S, Avent ND. SAFE – The Special Non-invasive Advances in Fetal and Neonatal Evaluation Network: aims and achievements. Prenat Diagn 2008;28:83–8. Legler TJ, Liu Z, Mavrou A, Finning K, Hromadnikova I, Galbiati S, et al. Workshop report on the extraction of foetal DNA from maternal plasma. Prenat Diagn 2007;27:824–9.

13 Chapter 13

Selective termination of pregnancy and preimplantation genetic diagnosis: some ethical issues in the interpretation of the legal criteria Rosamund Scott

Introduction This chapter considers some of the ethical issues at stake in the legal interpretation of the grounds for selective termination of pregnancy on the one hand and preimplantation genetic diagnosis (PGD) on the other.*1 Termination of pregnancy is legal under section 1(1)(d) of the Abortion Act 19672 (as amended by the Human Fertilisation and Embryology [HFE] Act 19903) if two doctors have formed an opinion in good faith that ‘there is a substantial risk that if the child were born it would suffer from such physical or mental abnormalities as to be seriously handicapped’.† These terms leave considerable scope for interpretation, particularly about what is meant by ‘seriously’, and to date there has been no direct judicial interpretation of this section. In a similar but not identical vein, PGD is legal if there is a significant risk ‘that a person … will have or develop a serious physical or mental disability, a serious illness or any other serious medical condition’.‡3,4 In both cases, then, great reliance is placed on the idea of seriousness. A certain degree of risk is also essential in both cases – a substantial risk in the context of termination of pregnancy and a significant risk in the case of PGD. In this chapter, I focus on some of the difficulties in interpreting seriousness in either context. My analysis will draw on a key and widely endorsed distinction in the bioethics literature between a life that may not be worth living and one that is worth living. Attention to this distinction shows that it is only where a person would have a condition that is so serious that he or she may not think his or her life worth living that selective termination or PGD can truly be said to be done for the sake of the person who would otherwise be born. This is likely to be the case in relation to surprisingly * Some parts of this chapter have been adapted from my work in Scott.1 † The relevant part of section 1(1) of the Abortion Act 19672 as amended by the HFE Act 19903 reads: ‘Subject to the provisions of this section, a person shall not be guilty of an offence under the law relating to abortion when a pregnancy is terminated by a registered medical practitioner if two registered medical practitioners are of the opinion, formed in good faith… (d) that there is a substantial risk that if the child were born it would suffer from such physical or mental abnormalities as to be seriously handicapped’. ‡ HFE Act 19903 as amended by the HFE Act 2008,4 section 1(Z)(A)(2). See also section 1(Z)(A)(1) and (3) for similar uses of the term ‘serious’.

© Rosamund Scott. Volume compilation © RCOG

184  |  ROSAMUND SCOTT

few conditions. In all other cases, since it is not against the interests of a person with a life worth living to be born, we need to consider in whose potential interests prenatal diagnosis (PND), selective termination and PGD are. I shall argue that it is legitimate, to a certain extent at least, for parental (and wider family) interests to be taken into account in the interpretation of the notion of ‘serious’ in both contexts. I shall first briefly discuss the distinction between a life that someone may not think is worth living and one that someone will think is worth living and consider its implications for the question of whose interests may be at stake in PND and PGD. I then turn to consider the interpretation of the grounds for selective termination of pregnancy in English law. I conclude that parental interests have a legitimate place in the interpretation of ‘serious’ under the Act but that it is not clear that parental interests could justify termination up until birth. Rather, it may be that after 24 weeks of gestation, fetal interests – and so the protection of a future child from a so-called ‘wrongful life’ – provide the most legitimate grounds for termination. In the latter part of the chapter I briefly touch on the recommendations that laid the groundwork for the current criteria for PGD, which have been accepted by Parliament in the HFE Act 2008.4 I also raise some questions about the interpretation of the relevant provisions of the new Act and two aspects of the draft guidance in the forthcoming Human Fertilisation and Embryology Authority (HFEA) Eighth Code of Practice.

The severity of a condition: whose interests? When born, a child may be or become so severely impaired that we might ask whether his or her life is overall good or bad for him or her, in terms of the wellbeing it contains. Part of this question entails reflection on the severity of the condition that a child would have. Tay–Sachs disease appears to be one example of a very serious condition.5 Another might be Lesch–Nyhan syndrome.6 Judgements by third parties about the quality of life of possible others are obviously difficult and sensitive. As Jonathan Glover7 has suggested, it may be best to restrict ourselves to the question of whether there is a ‘serious risk’ that someone may have a life he or she does not think worth living, a life of very low quality. It may be that this could fairly be said about life with the two conditions noted here. When we move away from these extreme and rare conditions, judging the seriousness of a condition becomes more difficult and thus controversial. This has been acknowledged by the Human Genetics Commission (HGC):8 It has proved impossible to define what ‘serious’ should mean in this context. We have listed some factors that should be taken into account when considering seriousness, but perhaps the most important is that this technique should not be used for the purposes of trait selection or in a manner which could give rise to eugenic outcomes.

The HFEA and Advisory Committee on Genetic Testing (AGCT), in their Consultation Document on Preimplantation Genetic Diagnosis,9 observe that: … individual judgments on seriousness will vary depending on personal and family circumstances and on the nature and severity of the condition and the likelihood of transmission.

Parents, people with relevant conditions of some kind and the medical profession may well have diverging views about this issue. Although it is likely that we have some hold on these issues at the extremes – for instance Tay–Sachs versus the webbing of two toes – difficulties attach to the large range of ‘mid-spectrum’ conditions in

SELECTIVE TERMINATION AND PGD: ETHICAL ISSUES IN THE INTERPRETATION OF LEGAL CRITERIA  |  185

between.*10 I am thinking here of conditions such as cystic fibrosis, spina bifida and Down syndrome, the seriousness of which (depending on the prognosis, especially in the first two cases) is very much a matter of debate. Despite the significant difficulties that will be experienced in the lives of people with Down syndrome or cystic fibrosis, on balance it may be fair to say that a person with one of these conditions will probably have a life he or she thinks is worth living, one that particularly he or she may also think is of reasonable quality. Indeed, there is evidence from positive psychological research on disability and disease that seems to suggest that in very many cases our subjective wellbeing is largely genetic, so that whether we have a condition of some kind matters a lot less than people may think.11 So, although the severity of these conditions varies, even at their worst it seems that it would not have been against the interests of someone with one of them to have been born.†12 This means that PND or PGD for conditions such as cystic fibrosis or Down syndrome could not be said to be conducted for the sake of the future person that a given fetus or embryo will become. Nevertheless, a child’s condition may still implicate his parents’ interests in reproduction in serious ways: he or she might have significant mental impairment or serious health problems requiring repeated hospitalisation, with an uncertain future. So, although people may reasonably disagree about this, and although the spectrum of severity of a given condition varies, it is arguable that some children born with a condition such as Down syndrome or cystic fibrosis may fairly be judged to have a serious condition, even though it was not against their own interests to be born, so that they do not have a ‘wrongful life’. In this light, it appears that testing and selection decisions can in fact be made for either of two principal reasons: first, where the child’s quality of life would be one of extremely low (sometimes called ‘subzero’) quality, as in the case of Tay–Sachs, primarily because of what that life would be like for the resulting child (here the parents’ interests will also be implicated); second, where the child would in fact have a reasonable quality of life but their condition may nevertheless have the potential adversely to impact on their parents, because of their interests in the kind of child who will be born. Selection against Down syndrome or cystic fibrosis (in some cases) may put special weight on the parents’ interests rather than those of the child. In such cases, prospective parents arguably do have a legitimate moral interest (at least in the earlier rather than later stages of pregnancy) in deciding whether to have a child with a serious condition that would nevertheless mean that a child had a life he or she thought worth living. Furthermore, this interest is one aspect of their broader interest in deciding whether to have any child. There are a number of writers who have noted the possible distinction between the interests of the future child on the one hand and those of the parents on the other in such decisions, including Jonathan Glover, Sally Sheldon, Stephen Wilkinson and Tom Shakespeare.13 It is not clear whether or to what extent the distinction between those rare cases in which birth might not be in the interests of the future child, given the severity of its impairment or condition on the one hand, and cases in which selection is really, potentially, in the parents’ interests or those of their wider family on the other, is explicitly acknowledged in the conduct of selection practices. In some ways, the idea that parents may prefer to avoid the birth of an impaired child for their own sake or that of their family is somewhat taboo. In this regard, one obstetrician observed * Page 263 onwards in Wertz10 discusses the difference of views among practitioners as to what is serious. † For interesting work on the views of people with conditions such as cystic fibrosis, Down syndrome and spina bifida, both on their lives and on screening practices, see Alderson.12

186  |  ROSAMUND SCOTT

that she considers that clinicians tend always to cite and use the disability ground of the Abortion Act in relation to termination for fetal disability because this is easier for parents.14 That those working in the field do reflect on this issue is evidenced by a research project in which I was involved that looked at the views and experience of those working in the field of PGD. We interviewed healthcare professionals and scientists at two London sites and conducted ethics discussion groups with them. In one discussion group, a scientist observed [my emphasis]:* I was at a conference … Parent Project UK, which is a charity which is aimed at … therapy for Duchenne muscular dystrophy people, and they were all parents. So one presented a talk actually which I found very interesting, and they looked at the quality of life for families with boys with Duchenne muscular dystrophy, which is a severe disease. The average lifespan now is about 19. And the quality of life of – the perception of the quality of life of the affected boy was rated differently by parents, by the clinicians looking after them and by the boys themselves. And the boys themselves … gave their rating of quality of life the same as any healthy controlled sample. And the parents gave them the lowest quality and the clinicians gave them somewhere in between the two, which was interesting, I thought. … So that implies we’re doing this for the parents and not for the child in some respects.

This person also said: Obviously people want to have children and when they have children with disability or handicap, to some extent that makes their life a bit more miserable compared to what they’re hoping for.

The potential impact of a child’s disability on parents was observed by a number of participants in this project. For instance, another scientist† referred to the ‘huge burden’ that parents may experience. More particularly, he or she also alluded to the idea of undoubtedly serious conditions on the one hand and conditions about which people may disagree on the other: I mean I think there are conditions which are under all circumstances, horrendous. And can be put very firmly on that list. But I completely agree with [participant], I mean I think there are lots of conditions which aren’t clearcut and which for some families might be considered serious and others not.

Can the requirement of seriousness in the statutory criteria for termination of pregnancy and PGD legitimately be interpreted in such a way as to mean that prospective parents’ interests can be taken into account in those cases which appear to form the majority of selection decisions? These are the cases in which birth would not in fact be against the interests of the child who would be born. I turn first to selective termination of pregnancy.

Interpreting the law on selective termination of pregnancy and PGD Termination of pregnancy A few years ago, Reverend Joanna Jepson brought the case of Jepson versus The Chief Constable of West Mercia Police Constabulary,15 which concerned an application * Ethics discussion group 2, Scientist 21, Wellcome Trust Biomedical Ethics Programme, grant no. 074935. The project was entitled ‘Facilitating Choice, Framing Choice: the Experience of Staff Working in PGD’, January 2005 to June 2007, and was conducted by Clare Williams (principal researcher), Kathryn Ehrich, Bobbie Farsides, Clare Sandall, Rosamund Scott and Peter Braude. † Scientist 8.

SELECTIVE TERMINATION AND PGD: ETHICAL ISSUES IN THE INTERPRETATION OF LEGAL CRITERIA  |  187

for judicial review of the police’s decision not to prosecute doctors in relation to a termination of pregnancy for cleft lip and palate at 28 weeks of gestation. (I do not further discuss the facts of this case here.) The case did not progress beyond the initial hearing but at that hearing she contended that a key error of law in that case was ‘that the medical practitioners who signed the certificate … took into account the views of the parents involved’ and ‘that … in relation to the decision in question, the parents’ views, as a matter of law, could have no weight’. Certainly the Act states that two doctors must be ‘of the opinion, formed in good faith’ that the impairment is serious. Whether two doctors can decide in good faith that the condition is serious and mean (or also mean) in so doing ‘serious for this woman’ (their patient for the purposes of a legal termination) is a crucial question. Doctors could decide that a woman’s views as to seriousness are relevant to their opinion of whether the criterion of seriousness is satisfied if they consider that at least one purpose of the disability section of the Act is to give women (and couples) choice about the birth of a seriously disabled child and recognise that, in making this choice, women (together with their husbands or partners, family and wider social network) will have views about the degree of impairment in a child they feel able to take on. Some scholars have indeed suggested that, where the fetus could have a reasonable quality of life as a born child, it is in fact the woman’s or parents’ interests that underlie this section of the Abortion Act. Glanville Williams16 laid the ground for this argument some time ago and the basis of this section has been reconsidered by Sally Sheldon and Stephen Wilkinson.17 They argue that, because very few fetuses aborted under this ground would have lives of little or no quality if born, most terminations of pregnancy under this section cannot be seen as protecting the fetus from such a life. Rather, in most cases in which this section is invoked, the real concern is with the woman’s or parents’ interests. This is a moral argument as to the legal interpretation of this section of the Act and one I support. Whether or not parents’ interests could be relevant to the interpretation of the disability ground of the Act was set to be a key issue in the Jepson case. When the police investigated the matter, it contacted the Royal College of Obstetricians and Gynaecologists (RCOG). The original advice given to them by its Vice President was (in part) as follows:15 The decision to abort under Clause E would always ultimately be with the mother having taken into consideration the perception of the parents to the serious nature of the handicap following counselling and information from experts with special knowledge of the condition.

On one reading, this means the woman is to take into account her own views, but this cannot be what was intended. Another reading is that legally the woman is required to take account of her partner’s views, but this is legally incorrect. A further interpretation is that although discussions about the seriousness of the impairment will take place between the woman (perhaps her partner), practitioners and relevant specialists, at the end of the day, assuming two doctors think that the legal criteria for a termination of pregnancy are made out, it is the woman who decides whether to continue the pregnancy. This makes the most sense. In contrast, since the Act requires that two doctors consider the condition to be serious, the sentence cannot mean that the woman decides whether the criterion of seriousness is satisfied. Despite this, whether two doctors can certify in good faith that the condition is serious – and mean in so doing (at least to some degree) ‘serious for this woman or couple’ – is the critical issue. The claimant in the Jepson case was seeking to steer the law away from any such interpretation. A further interpretation of this important sentence could be

188  |  ROSAMUND SCOTT

that it contained a ‘typo’ and that it should have read [my emphasis]:14 ‘The decision to abort under Clause E would always ultimately be with the doctor having taken into consideration the perception of the parents to the serious nature of the handicap following counselling and information from experts with special knowledge of the condition.’ (Alternatively, the ‘typo’ may have entered the scene in the case report.) Indeed, this would be consistent with an earlier sentence in the Vice President’s letter that read: ‘There is no precise definition of “serious handicap” and the decision is therefore one for the practitioner to make in consultation with the parents and other interested parties.’ On this point, one clinician commented that ‘it would be normal good medical practice for a doctor to take account of parents’ views’.15 Given that the Vice President alluded to discussions between a woman and her doctor, her statement may be describing the practice that typically occurs and has built up over the years. This practice may partly be a response to the discomfort some practitioners evidently feel at being the gatekeepers to termination of pregnancy and the administrators of a system in which a pregnant woman’s autonomy is deeply at stake but lacks legal protection in the form of a right to terminate.*18 The question, however, is whether such a practice is lawful. One way to reflect on this would be to try to reconcile use of section 1(1)(a) of the Act in relation to fetal anomaly with section 1(1)(d), the disability ground. Section 1(1)(a) permits termination of pregnancy where two doctors are of a goodfaith opinion ‘that the pregnancy has not exceeded twenty-four weeks and that the continuance of the pregnancy would involve risk, greater than if the pregnancy were terminated, of injury to the physical or mental health of the pregnant woman or any existing children of her family’. The possible use of two different sections of the Abortion Act in relation to fetal impairment is not apparent from the terms of the Act itself. However, it seems right that section 1(1)(a) can be interpreted in this way given its concern with the mental (or physical) health of the woman and the RCOG has itself endorsed such use in some cases.19 Women vary in their reaction to being told that their fetus is, or may be, abnormal. Occasionally a woman feels strongly that she is unable to accept a probability of risk or a degree of handicap that her medical practitioners consider less than substantial or serious. Under such circumstances, and only when the gestation is less than 24  weeks, the practitioners may decide that abortion has become necessary to protect her mental health.

What can we glean from the use of section 1(1)(a) for our interpretation of the disability ground of the Act? When section 1(1)(a) is used in this way, essentially the thought is that, although doctors do not think either that the risk is substantial or the condition serious, a woman may be seriously concerned about what she is able to take on, to the extent that there are risks to her physical or mental health greater than those inherent in termination. Yet, since raising any child, but perhaps particularly a disabled one, has at least the potential to have considerable implications for the life of a woman (and her partner), any termination for serious fetal anomaly is likely seriously to implicate a woman’s (and her partner’s) interests, including where the termination is legally justified on the basis of section 1(1)(d). * In discussions between healthcare professionals, a philosopher and sociologists, one (unnamed) obstetrician said: ‘I think things are probably made more difficult because most obstetricians and gynaecologists know that terminations for social reasons, or whatever, are done effectively on demand, and one of the main reasons for that is because of anxiety generated about the pregnancy continuing … our sort of baseline has shifted because of shifts in how the Abortion Act has been applied over the past 20 years or so.’22

SELECTIVE TERMINATION AND PGD: ETHICAL ISSUES IN THE INTERPRETATION OF LEGAL CRITERIA  |  189

On the one hand, then, section 1(1)(a) of the Act does not explicitly indicate that it might, in effect, be used in relation to fetal impairment, but arguably can be interpreted in this way to some degree, as evidenced in the RCOG guidelines; on the other hand, the disability ground of the Act does not state that prospective parents’ views about impairment can be a legitimate factor in the interpretation of this section but arguably can be interpreted in this way. Notably, this may particularly be where a ‘serious’ condition may nevertheless result in a life that a child would think worth living, so that termination cannot be claimed, morally speaking, to be in the interests of the fetus or the child it would become. Indeed, assuming section 1(1)(a) can be used in relation to fetal impairment (which must be right), it would be hard to assert that prospective parents’ views, particularly about the seriousness of an impairment, are legally irrelevant under section 1(1)(d), the disability ground, even though that section is not based on risks to the woman’s mental or physical health. Rather, the use of section 1(1)(a) in relation to fetal anomaly confirms that the question of the degree of risk or, particularly, the seriousness of an impairment cannot be a purely objective matter. As noted earlier, this has been acknowledged by others, including the HGC. Importantly, we therefore have good reason to think that doctors can, to some extent at least, take the views and interests of a woman (and her partner) into account when deciding whether the criteria of the disability ground of the Act are satisfied. If so, then the practice to which the Vice President alludes in her letter, which was also referred to by a clinician, is arguably defensible at law. Importantly, if parents’ views about raising a disabled child and their thoughts about the seriousness of its disability were irrelevant to the legality of termination on the basis of the disability ground of the Act, then it is likely that the legality of many terminations that are currently performed should at least be questioned, as should the purpose of certain kinds of screening and testing. Indeed, this would probably be in line with Reverend Jepson’s desires and beliefs in relation to selective termination of pregnancy. For instance, given that it is most unlikely to be against the interests of someone with Down syndrome to be born, screening, testing and termination for Down syndrome could rarely, perhaps never, be said to be based on the seriousness of the condition from the child’s point of view (although one in ten children do not survive the first year of life).*20,21 As regards the third-trimester fetus, the pressure for terminations of pregnancy after 24  weeks to be for particularly serious reasons is rightly strong. The RCOG guidelines on termination of pregnancy after this time assert that terminations must be for particularly serious reasons:22 As the protection due increases with embryonic development and fetal growth, reasons for termination, at no stage trivial, must be more pressing the longer pregnancy has progressed.

The guidelines thus endorse a gradualist approach to fetal moral status, which holds that the longer a pregnancy has been allowed to develop, the greater must be the justification for in any way compromising it. It is a position that I suggest is intuitively appealing and one I have explored and defended in detail elsewhere.23 Can the disability ground of the Act be invoked on behalf of the pregnant woman (and her partner) after 24 weeks? While I have argued that parental views about the seriousness of the disability that a child would have should be taken into account in the interpretation of the disability ground of the Act, at least where disabilities are the subject of reasonable disagreement, it does not follow that this means that their possible * There is also a high rate of stillbirth between screening and birth.

190  |  ROSAMUND SCOTT

wish not to have a disabled child can carry weight up until birth. This is owing to the increasing moral claims of the fetus. Since the law has to have a clear line, legally speaking the shift in the strength of those claims occurs at the 24th week, after which the reasons for termination of pregnancy must be that much more stringent. Looking at the other grounds of the Act, we see this in the continuing relevance only of the risk to a woman’s life and the need to avoid grave permanent damage to her physical or mental health (in sections 1(1)(c) and (b), respectively).* It is therefore arguable that the disability ground of the Act should also be more stringently interpreted after this time. In this light, if termination on the grounds of fetal anomaly after 24 weeks is permissible in the interests of the pregnant woman, it may be that this can only be on the basis of section 1(1)(b). Although it seems unlikely that termination on the grounds of disability could be required, and therefore justified, to prevent grave permanent damage to her mental health under section 1(1)(b), a meritorious case cannot be categorically ruled out, particularly where there are other complicating factors. Moving now to consider the fetus, at any stage of pregnancy a termination could only be said to be in its interests when there is a real risk that a subsequently born child would not think his or her life worth living. As the RCOG report on third-trimester terminations notes, termination in the fetus’s interests will in effect be appropriate where as a neonate it would not be in its best interests to continue to receive lifesustaining medical treatment.22 It is beyond the scope of this chapter to say more about late-term terminations of pregnancy. PGD I now turn briefly to PGD. Elsewhere I have reviewed in detail the HFEA and HGC recommendations and discussions that led to the original formulation of the PGD criteria of ‘a significant risk of a serious genetic condition’.1 In essence, these are the criteria that have now been put on a statutory footing. How should they be interpreted? The original HFEA and HGC recommendations sought to recognise, at least to some extent, the personal nature of the issues at stake in PGD but also to observe limits to the legitimacy of prospective parents’ views. Essentially, this was to be achieved by requiring discussion between prospective parents and healthcare professionals and scientists about the degree of risk and the seriousness of any given condition. The HFEA and HGC also put these professionals in the position of being something of a ‘check’ on what these bodies saw as the potentially excessively wide views of those seeking treatment.† Under the new HFE Act, the HFEA must itself be satisfied as to the seriousness of the condition. In this regard, early in 2009 the HFEA sought views as to whether it should make two particular changes to its guidance to clinics. In the first case, the previous guidance stated:24 The use of PGD should be considered only where there is a significant risk of a serious genetic condition being present in the embryo. The perception of the level of risk by those seeking treatment is an important factor in the decision making process. The seriousness of the condition should be a matter for discussion between the people seeking treatment and the clinical team.

The HFEA proposed that the last sentence of the above should be dropped. In addition, among the list of factors that clinics are to consider in deciding whether * Section 1(1)(c) permits abortion where ‘the continuance of the pregnancy would involve risk to the life of the pregnant woman, greater than if the pregnancy were terminated’; section 1(1)(b) permits abortion where ‘necessary to prevent grave permanent injury to the physical or mental health of the pregnant woman’. † HFEA licence committees are of course a further possible check but, at least until now, it has been likely that if a clinic supports an application for PGD, the licence committee will usually agree.

SELECTIVE TERMINATION AND PGD: ETHICAL ISSUES IN THE INTERPRETATION OF LEGAL CRITERIA  |  191

PGD is appropriate, the first item used to be ‘the view of the people seeking treatment of the condition’.* The HFEA consulted about whether the word ‘view’ should be changed to ‘experience’, with the thought that this might decrease any subjectivity in the decision-making process. In the documentation relating to these possible changes, the HFEA observed:25 Of particular relevance is the intent behind the updated legislation to ensure that the licensing process for PGD is set out within clear boundaries. This is a response to concerns that over time, PGD could be used to avoid less serious conditions. With this in mind, the law will now explicitly require that the HFEA, rather than the clinic in consultation with the people seeking treatment, be satisfied of the seriousness of that condition.

My research into the HFEA’s and HGC’s working groups that originally formulated the PGD criteria and accompanying guidance suggests that this would be contrary to the original intentions in the formulation of the guidance, which in fact allowed for at least some regard to be given to parental views.1 The proposed changes also appear unnecessary in that it is not clear that Parliament, in broadly confirming the original criteria in the HFE Act 2008, was seeking to make those grounds any more stringent.26 Moreover, in any event, we saw that the HGC, which worked with the HFEA in establishing the criteria for PGD, suggested that ‘objectivity’ in relation to serious disability is elusive. This thought is echoed in much of the literature.†27 At the time of writing, it is understood that the HFEA is likely to modify these proposed changes in the light of feedback from its consultation exercise.

Conclusion Looking to the future of PGD and PND, with regard to PGD there may be a tendency to assume that there are lower costs in choosing between embryos (at least once several embryos exist) than in terminating an existing pregnancy. Theoretically at least, there is greater scope to focus on less serious considerations, including ones that would be unlikely to influence prospective parents in deciding to terminate. However, the physical and emotional burdens of undergoing in vitro fertilisation will always be considerable. In this light, the concerns of people engaging in PGD are likely to be very serious indeed given their past reproductive experience and possible reproductive future. They will have in mind both the interests of their possible child and, particularly when a child’s impaired existence is nevertheless compatible with a reasonable quality of life but they remain concerned about the possible impact of such a birth on them, their own interests in reproduction. Turning to PND, despite the advances in testing, including in non-invasive testing, and the concerns that are sometimes expressed that a pregnancy will be terminated for trivial reasons, it seems unlikely that parents would seek a termination of pregnancy for a condition that they did not see as in some way seriously implicating their interests. Overall, both in PGD and PND, where a given child would have a life he or she thought worth living, parental interests may be the ones that are most often potentially invoked. In my view, it would be helpful to recognise and accept this * The original list in full, as formulated by the HGC and HFEA and then adopted by the HFEA in its Code of Practice at paragraph 12.3.3 stated: ‘In any particular situation the following factors should be considered when deciding the appropriateness of PGD: the view of the people seeking treatment of the condition to be avoided; their previous reproductive experience; the likely degree of suffering associated with the condition; the availability of effective therapy, now and in the future; the speed of degeneration in progressive disorders; the extent of any intellectual impairment; the extent of social support available; and the family circumstances of the people seeking treatment.’ † See, for example, comments to this effect in the Introduction by Parens and Asch to their Prenatal Testing and Disability Rights,27 which resulted from a Hastings Center Working Party.

192  |  ROSAMUND SCOTT

more clearly in our interpretation of the legal criteria in both contexts. This does not mean we have to accept parental views and reasons at face value. What it does mean is recognising that parents may be seriously affected by the condition of the child that is born and that ‘serious’ can therefore also mean serious for the parents and their family. This would more clearly enable their interests to be taken into account in the interpretation of the criterion of seriousness.

References 1. 2. 3. 4. 5. 6. 7. 8.

9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27.

Scott R. Choosing Between Possible Lives: Law and Ethics of Prenatal and Preimplantation Genetic Diagnosis. Oxford: Hart Publishing; 2007. Abortion Act 1967 [www.statutelaw.gov.uk/content.aspx?activeTextDocId=1181037]. Human Fertilisation and Embryology Act 1990 [www.opsi.gov.uk/acts/acts1990/ Ukpga_19900037_en_1.htm]. Human Fertilisation and Embryology Act 2008 [www.opsi.gov.uk/acts/acts2008/ ukpga_20080022_en_1]. National Tay-Sachs and Allied Diseases Association [www.ntsad.org]. National Institute of Neurological Disorders and Stroke [www.ninds.nih.gov/disorders/lesch_ nyhan/lesch_nyhan.htm]. Glover J. Choosing Children: Genes, Disability and Design. Oxford: Oxford University Press; 2006. Human Genetics Commission. Draft Response to the HFEA on the Outcome of the HFEA/ACGT Consultation on Preimplantation Genetic Diagnosis. Annex D in: Human Genetics Commission. Preimplantation Genetic Diagnosis. London: HGC; 2001 [www.hgc.gov.uk/UploadDocs/DocPub/ Document/hgc01-p2.pdf]. Human Fertilisation and Embryology Authority and Advisory Committee on Genetic Testing. Consultation Document on Preimplantation Genetic Diagnosis. November 1999 [www.hfea.gov.uk/cps/ rde/xbcr/hfea/PGD_document.pdf]. Wertz D. Drawing lines: notes for policymakers. In: Parens E, Asch A, editors. Prenatal Testing and Disability Rights. Washington, DC: Georgetown University Press; 2000. p. 261–87. Fave A, Massimini F. The relevance of subjective well-being to social policies: optimal experience and tailored intervention. In: Huppert F, Baylis N, Kaverne B. The Science of Wellbeing. Oxford: Oxford University Press; 2005. p. 379. Alderson P. Prenatal counselling and images of disability. In: Dickenson D, editor. Ethical Issues in Maternal–Fetal Medicine. Cambridge: Cambridge University Press; 2002. p. 195–212. Shakespeare T. Disability Rights and Wrongs. London: Routledge; 2006. S Bewley, personal communication. High Court of England and Wales [2003] E.W.H.C. 3318. Williams G. Textbook of Criminal Law. 1st ed. London: Stevens; 1978. Sheldon S, Wilkinson S. Termination of pregnancy for reason of foetal disability: are there grounds for a special exception in law? Med Law Rev 2001;9:85–109. Williams C, Alderson P, Farsides B. ‘Drawing the line’ in prenatal screening and testing: health practitioners’ discussions. Health Risk Soc 2002;4:61–75. Royal College of Obstetricians and Gynaecologists. Termination of Pregnancy for Fetal Abnormality in England, Wales and Scotland. London: RCOG Press; 1996. Julian-Reynier C, Aurran Y, Dumaret A, Maron A, Chabal F, Giraud F, et al. Attitudes towards Down’s syndrome: follow up of a cohort of 280 cases. J Med Genet 1995;32:597–9. Won RH, Currier RJ, Lorey F, Towner DR. The timing of demise in fetuses with trisomy 21 and trisomy 18. Prenat Diagn 2005;25:608–11. Royal College of Obstetricians and Gynaecologists Ethics Committee. A Consideration of the Law and Ethics in Relation to Late Termination of Pregnancy for Fetal Abnormality. London: RCOG Press; 1998. Scott R. Rights, Duties and the Body: Law and Ethics of the Maternal–Fetal Conflict. Oxford: Hart Publishing; 2002. Human Fertilisation and Embryology Authority. Code of Practice. 7th ed. London: HFEA; 2007. Human Fertilisation and Embryology Authority. Regulating Preimplantation Genetic Diagnosis for the Future: Listening to Your Views. London: HFEA; 2009. E Harris, MP, personal communication. Parens E, Asch A. Introduction. In: Parens E, Asch A, editors. Prenatal Testing and Disability Rights. Washington, DC: Georgetown University Press; 2000. p. 3–43

14 Chapter 14

Implementation and auditing of new genetics and tests: translating genetic tests into practice in the NHS Rob Elles and Ian Frayling

Background Genetic testing for single-gene disorders has been available in the NHS since the mid-1980s and for chromosomal disorders since the early 1970s. A mature network of multidisciplinary regional genetics centres has evolved combining clinical genetics and counselling, laboratory diagnostics and research.1 The link between research and diagnostics has proved to be an effective mechanism for rapid translation of tests and technologies into practice and the close involvement of clinical geneticists has meant that the introduction of new tests and gatekeeping of referrals to ensure appropriate use has been relatively effective. The professionally led network approach to test provision that avoided much duplication of service provision for single-gene disorders was formalised and supported by the Government through the formation of the UK Genetic Testing Network (UKGTN).2 A key part of the remit of the UKGTN is to evaluate new tests proposed for service and recommend them to specialist service commissioners for NHS funding. Our discussions will be limited to potentially heritable, germline genetic disorders.

The UK approach to genetic test evaluation To fulfil its evaluation role, UKGTN adopted the ACCE framework (‘analytic validity; clinical validity; clinical utility; and ethical, legal, and social implications’) developed by the US Centers for Disease Control and Prevention3 and adapted by Kroese et al.4 into a practical system for individual laboratories to submit a data set for peer group evaluation. This ‘gene dossier’ consists of a number of headings designed to define the test precisely in a clinical context and set out its characteristics referenced against the research literature and the results of an adequate series of in-house validation tests. This concise description of the test is generally sufficient for a small expert panel to make a judgement on whether a test should be recommended to the specialist genetic commissioners who meet as the Genetics Commissioning Advisory Group (GenCAG)5 for adoption on the UKGTN menu of tests (now over 400 in number). © Rob Elles and Ian Frayling. Volume compilation © RCOG

194  |  ROB ELLES AND IAN FRAYLING

International frameworks The UK experience has helped lead and influence international guidelines in genetic testing. NHS practice is largely compliant with international recommendations such as those adopted by the Organisation for Economic Co-operation and Development in 2007 for quality assurance in molecular genetic testing.6 The OECD guidelines make some important framework statements that are increasingly relevant as some UK-based private companies have become active in the ‘lifestyle genetics’ and ‘nutrigenomics’ areas and internet-ordered direct-to-consumer tests have become available. An example is a test for early fetal sexing from a maternal blood sample.7 The OECD recommendations relevant to the evaluation of new genetic tests and the context in which they are offered for service include:6 n ‘Molecular genetic testing should be delivered within the framework of health care.’ n ‘Pre and post test counselling should be available. It should be proportionate and appropriate to the characteristics of the test, the test limitations, the potential for harm, and the relevance of test results to individuals and their relatives.’ n ‘Laboratories should make available to service users current evidence concerning the clinical validity and utility of the tests they offer.’

The ACCE framework translated to a gene dossier Test definition The UKGTN gene dossier asks the service provider to define their proposed test in the context of the disease and associated gene(s) targeted, the mutation spectrum detectable by the set of assays used, the patient group targeted and, most importantly, those clinicians from whom referrals are acceptable. This may be defined by a set of symptoms (for example, the analysis of the FMR gene in the context of women with primary ovarian insufficiency (previously known as premature ovarian failure) or its application in boys with learning difficulty to exclude or establish a diagnosis of fragile X syndrome) or a risk (for example, the prior risk of carrying a mutation in a breast cancer predisposition gene calculated from the family history of the index case). Analytical validity The analytical validity of the technical assay (which is usually formulated by the service proposers) is documented by in-house trials. A trial programme is specifically designed to calculate the analytical sensitivity and specificity of the assay; in other words, its ability to accurately detect genetic variants in the sequence targeted. Assays may be complex, for example in a condition exhibiting genetic heterogeneity, a scan for genetic variants may employ a cascade strategy examining a series of genes, any one of which may be responsible for the condition. In this case, the overall yield of genetic variants detected (compared with reference sequences) is the measure required. This is established by using the assay in a baseline set of index cases (where the phenotype is ascertained by independent methods. An example might be congenital adrenal hyperplasia, where the presence of disease is defined by biochemical parameters. A second example would be a panel of patients in full compliance with formal diagnostic criteria (for example, in adult polycystic kidney disease the presence of bilateral cysts with more than one cyst in one kidney). Alternatively, the variant assay is referenced against a genotype established by statistically significant genetic linkage score using an independent set of markers.

IMPLEMENTATION AND AUDITING OF NEW GENETIC TESTS IN THE NHS  |  195

Clinical validity Establishing the clinical validity of the test requires a judgement of the strength of the evidence that a variant or set of variants in the gene(s) assayed are associated with the condition. These data are usually documented from research literature and include a discussion of the penetrance of the variants described (that is, the likelihood that a variant will manifest as a phenotype). The data are often incomplete as the research effort is unlikely to have surveilled all possible variants in a gene for a rare condition. The in-house validation trial may supplement this evidence and include a consideration of the data drawn from the local and clinically ascertained population (as opposed to patients ascertained for research purposes). As such, it is often a more accurate (and frequently lower than published) estimate of both the clinical sensitivity and the specificity of the test and its positive and negative predictive value. Clinical utility Clinical utility is the measure on which the evaluation of a test by UKGTN may stand or fall. Laboratories are asked to indicate the endpoint of the test in terms of its power to: n exclude or establish a diagnosis where the test indication is a sign or symptom; the ability of a test to provide a definitive diagnosis and end a diagnostic odyssey for a family is considered to be a significant utility n modify a carrier risk in an asymptomatic individual and in an autosomal recessive condition such as cystic fibrosis to open reproductive decision-making options n predict the onset of a condition and to improve prognostic information and the management and/or treatment of individuals. The gene dossier includes a discussion of the economic costs and benefits of the proposed test and laboratories are asked to use a standardised formula to calculate the cost of the test, including staff and materials costs and institutional overheads. In addition, the gene dossier asks for indicative details of savings to the NHS and to the time, effort and psychosocial costs to the patient and their family in terms of alternative investigations avoided by a genetic test outcome. The potential of a genetic test to end regular tumour surveillance under anaesthetic in children at risk of retinoblastoma is a good example of this type of argument. In contrast, laboratories are also asked to indicate alternative tests to a molecular genetic analysis that may be a cheaper, effective and more straightforward alternative. Adult polycystic kidney disease is an example of a condition where a simple and cheap abdominal ultrasound scan has been adopted in preference to the complex, difficult and expensive molecular genetic test for this condition. Ethical legal and social issues The gene dossier asks for any indication of significant ethical, legal and social issues associated with the test. Genetic testing within the UKGTN lies within the governance framework of the NHS and pre- and post-test counselling is generally considered to be accessible, adequate and appropriate to the clinical situation. Examples of infrequent requests, for example for prenatal diagnosis for variants indicative of adult-onset conditions such as familial cancers, or sensory conditions such as inherited forms of adult-onset blindness, are considered within the context of this counselling framework.

196  |  ROB ELLES AND IAN FRAYLING

Gatekeeping Service proposers are asked to outline their experience of the test in the validation phase and indicate the strength of connection to research and local clinical expertise. In addition, they are asked to indicate how gatekeeping is to be organised in terms of the categories of specialist clinicians from whom referrals would normally be accepted and, if necessary, how potentially inappropriate referrals are to be managed. Information for commissioning The gene dossier provides information on the potential national impact of the proposed test by detailing the incidence and prevalence of the condition, as well as any downstream costs that the NHS will incur. An example of this would be the cost of implantable defibrillators in at-risk family members ascertained by predictive genetic testing, consequent upon finding a familial mutation causing an inherited form of cardiac dysrhythmia. Service proposers are asked to estimate any backlog of cases, the national steady-state demand and what proportion of this caseload they will service on an annual basis. This informs local commissioners of the expected yearon-year funding level for UKGTN services in their region. In turn, commissioners can be satisfied that test requests will be made by clinicians, and likewise accepted by laboratories consistent with the referral criteria specified in the gene dossiers. The annual audit of activity carried out by UKGTN also enables the demand for and availability of testing across the UK to be assessed. Auditing effectiveness UKGTN’s aim is to provide equity of access to genetic tests and to ensure that they are performed to service quality standards. It audits reporting turnaround times against the targets specified in the genetics White Paper of 20038 and has begun to measure the uptake and estimate the costs of new tests evaluated and approved for commissioning by GenCAG. In addition, it has returned to consider long-established tests ‘grandfathered’ onto the UKGTN list when first assembled. Examples include tests for cystic fibrosis and fragile X syndrome. As a result, the normal clinical criteria for requesting these tests have been defined. Are the UKGTN systems future-proof? The UKGTN gene dossier-based system of genetic test evaluation has served providers, commissioners and end users of tests (clinicians and their patients) adequately over the past 5 years. The number of tests available through UKGTN has increased each year. This is despite complaints from service providers concerning the apparent gap in the National Institute for Health Research remit, which does not extend to genetic test evaluation studies. Resources to translate research findings to clinical services are limited and patient advocates, frustrated by the pace of translation, have frequently stepped in to support the assay design, test development and validation studies required to present a gene dossier for consideration for NHS commissioning. Despite these limitations, the UKGTN system has proved to be a practical solution in which individual service laboratories can present a limited data set for evaluation. The system works best where the arguments for utility are clear and qualitative arguments can be advanced based on the obvious effects of high-penetrance variants on phenotype. As more genes causing rare conditions are identified, this system will continue to be used

IMPLEMENTATION AND AUDITING OF NEW GENETIC TESTS IN THE NHS  |  197

and to be effective. The services considered by UKGTN to date are those in which the impacts on individuals and their families are explicit in decision making or influencing clinical management (even if treatment improvements are rarely demonstrable). The case for clinical utility will change as previously intractable genetic conditions become open to clinical trials, often using safe and well-characterised drugs (for example, prescription of losartan to moderate the effects of Marfan syndrome9). However, the gene dossier was designed to evaluate tests for rare single-gene disorders. It has become increasingly obvious that this framework will need to be adapted and evaluation mechanisms strengthened as genetic testing moves towards mainstream medicine. Genetic tests for common complex disorders have been proposed. These envisage tests for genetic variants representing low-penetrance biomarkers that raise the risk of late-onset conditions such as type  2 diabetes, coronary disease and age-related macular degeneration. The genetic test result may interact with less defined but important genetic factors (family history) and environmental factors (body mass index, diet, smoking, etc). Evaluation of this type of test will be essential to avoid their adoption in an ad hoc way and a consequent waste of resources, risking tests being discredited as expensive and ineffective as tools in NHS clinical care. Other major challenges to the evaluation system include the potential impact of the next-generation technologies that allow a parallel scan for sequence and copy number variants in many genes and chromosomal regions relevant to a phenotype or disease pathway. In addition, technologies allowing non-invasive prenatal diagnosis to screen for common aneuploidies may require as careful a consideration of their wide social impact and impact on the NHS as a service delivery system as their technical features if they are to be accepted by the public.

What changes or new systems should be considered? The ACCE framework is a useful starting point and Burke and Zimmern10 have used it in considering the issue of evaluating genetic testing for common complex disorders. They emphasise the difference between the technical ‘assay’ for one or more genetic variants and the ‘test’ that is designed to identify a set of genotypes for a particular condition in a defined clinical population and, importantly, for a specific purpose. They propose expanded dimensions of the ACCE headings, in particular considering the clinical validity of the test and distinguishing between closed assays (for a specific set of variants) and open assays (for any variant associated with a phenotypic indicator). Burke and Zimmern disaggregate the concept of clinical utility into a consideration of the defined purpose of the test. They differentiate between high- and low-penetrance variants contributing to a disease phenotype or risk. They consider measures of health quality including legitimacy (for example, questioning whether a prediction contributes to individual wellbeing in the absence of treatment) and how utility is considered and regulated differently when preimplantation genetic diagnosis or prenatal diagnosis are the proposed applications of the genetic test. Genetic test proposals with significant public health impact and implications for NHS policy, resources and service configuration require commensurately more and better-quality data. In-depth evaluation will involve multidisciplinary expertise (epidemiologists, health economists and public engagement) and policy decisions involving commissioners and service providers at all levels of NHS care. The Public Health Genetics Foundation workshop report and National Institute for Health Research-funded RAPID study to facilitate the translation into service of non-invasive

198  |  ROB ELLES AND IAN FRAYLING

prenatal diagnosis using cell-free fetal DNA in maternal circulation is an example of such a multidisciplinary evaluation study. More generally, Burke and Zimmern10 propose prioritising genetic test evaluations according to their likely scale of impact and removing the responsibility for data generation from commercial interests and service providers that inevitably have a vested interest in the outcome. These issues were considered more broadly at a diagnostic summit organised by the Royal College of Pathologists in January 2008, which recommended that a new body (that has been called a NICE [National Institute for Health and Clinical Excellence] for diagnostic tests) be established for laboratory-based diagnostic tests taking responsibility for evaluation and making data on the validity and utility of all tests, new or historical, publicly available. The evaluation of new genetic tests cannot be separated from the enabling technologies that will allow them to be realised. Next-generation genomic technologies (such as microarrays and parallel DNA sequencing) will require full health technology and economic assessment. These studies should pool the valid requirements of the genetic service network, laboratory medicine and mainstream medical disciplines. They should consider the rising numbers of clinically useful tests against falling unit costs. They will recognise that the implementation of new technology will incur an increased global cost for genomic diagnostics and ask whether this spending will be offset by the health benefits to patients and savings in diagnosis and treatment elsewhere in the NHS.

References 1. 2. 3. 4.

5.

6. 7. 8.

9. 10.

Donnai D, Elles R. Integrated regional genetic services: current and future provision. BMJ 2001;322:1048–52. UK Genetic Testing Network [www.ukgtn.nhs.uk]. US Centers for Disease Control and Prevention. Genomic Translation: ACCE Model Process for Evaluating Genetic Tests [www.cdc.gov/genomics/gtesting/ACCE/index.htm]. Kroese M, Zimmern RL, Farndon P, Stewart F, Whittaker J. How can genetic tests be evaluated for clinical use? Experience of the UK Genetic Testing Network. Eur J Hum Genet 2007;15:917–21. Department of Health. Genetics Commissioning Advisory Group (GenCAG) [www. dh.gov.uk/en/Publichealth/Scientificdevelopmentgeneticsandbioethics/Genetics/ Geneticsgeneralinformation/DH_4117687]. Organisation for Economic Co-operation and Development. OECD Guidelines for Quality Assurance in Molecular Genetic Testing [www.oecd.org/dataoecd/43/6/38839788.pdf]. Baby Gender Mentor [babygendermentor.com/information.php?information_id=2]. Department of Health. Our Inheritance, Our Future: Realising The Potential of Genetics in the NHS. London: Department of Health; 2003 [www.dh.gov.uk/en/Publicationsandstatistics/Publications/ PublicationsPolicyAndGuidance/DH_4006538]. Marfan syndrome trial example [clinicaltrials.gov/ct2/show/NCT00593710]. Burke W, Zimmern R. Moving Beyond ACCE: an Expanded Framework for Genetic Test Evaluation. UKGTN; 2007 [www.phgfoundation.org/pages/work7.htm#acce].

15 Chapter 15

New advances in prenatal genetic testing: the parent perspective Jane Fisher

Introduction Antenatal Results and Choices (ARC), which was established in 1988, is the only UK charity providing non-directive information and specialised support to parents before, during and after antenatal testing and when an abnormality is diagnosed in an unborn baby. Continuing support is offered for as long as required, whatever decision is made about the future of the pregnancy. ARC also runs a well-established training programme for healthcare professionals in communication skills, breaking bad news and supporting parent decision making, all in the context of antenatal testing For more than 20 years, ARC’s core support service has been its national helpline. We take calls from across the UK on all aspects of antenatal testing and its aftermath. This chapter will use ARC’s extensive experience with expectant parents to explore the potential implications for them of new techniques in prenatal genetic testing, particularly advances in non-invasive prenatal diagnosis (NIPD). Throughout the chapter I will refer to women and their partners as parents and the fetus as a baby, as this is how the vast majority of callers to ARC conceptualise themselves and their fetus from the earliest stages of the pregnancy.

ARC’s contact with parents making decisions about invasive diagnostic tests By far the most common call on the ARC helpline is from parents after a screening result or ultrasound finding, when they are deciding whether to have a diagnostic procedure, either chorionic villus sampling (CVS) or amniocentesis. Approximately half of all helpline contacts by phone or email are of this kind. Most hospitals quote a procedure-associated miscarriage risk of 1% and so a large number of contacts are from parents who have been given a Down syndrome screening result that means they have the chance of having an affected pregnancy of between one in 100 and one in 250. In other words, they are told that they have a higher than average risk of their baby having Down syndrome and then offered a procedure that carries a higher or at least comparable risk of miscarriage. This can make the decision-making process particularly onerous. © Jane Fisher. Volume compilation © RCOG

200  |  JANE FISHER

ARC’s specialised helpline team helps parents work out how to proceed in a way that is right for them in their individual circumstances. We have no agenda other than to enable them to make the decision they can best live with. It is often a very distressing process for parents as they weigh up the importance to them of having a confirmed diagnosis (or gaining reassurance) against the small but significant chance of losing a wanted pregnancy as a result of the procedure. We are fortunate at ARC to be able to spend as long as individual parents need to explore potential outcomes. Different parents bring different factors to their decision making, including how they feel about the condition being tested for and whether they would consider termination of pregnancy if their baby were affected. For many, a major concern is whether they will be able to manage their anxieties for what is usually over half the pregnancy without having a definitive answer. Conversations will almost always include discussion of how the procedure is performed and what, if anything, can minimise the risk of miscarriage. While some will consider CVS because it can provide an earlier diagnosis, others perceive it as a riskier procedure and prefer to wait to undergo an amniocentesis. Many parents are desperate to know whether there is a risk-free alternative. Some want to know what more information can be gained from ultrasound scans, and the media coverage of research into NIPD using cell-free fetal DNA (cffDNA) has led some to ask us whether they can access the test privately or as part of a clinical trial.

Implications for parents of the future implementation of NIPD From our experience, we can confidently predict that a risk-free genetic diagnostic test will be welcomed by parents; with the proviso that the test is robust and reliable. There will be a wait of some years before the sensitivity and specificity of cffDNA testing or competing non-invasive techniques are such that it can be routinely used in practice as a diagnostic tool for aneuploidy but research is currently making strides, with the added spur of huge potential demand. The fact that NIPD is likely to be offered well within the first trimester will also be welcome to many parents as potentially providing earlier reassurance. However, we must avoid making assumptions that an earlier diagnosis will necessarily be easier for parents to cope with or that decisions about the future of the pregnancy will be any less emotionally painful. The diagnosis of a genetic condition in their baby will have a significant emotional impact on parents whatever the gestation. A choice sounded better than not having a choice. Yet, the idea of choosing was overwhelming and confusing. We were devastated at the thought of either course of action. It wasn’t until much later that I realised the real tragedy was the loss of the healthy child and that I had not had a choice about. The loss had to be accepted and mourned; and before that could take place we had to make an irreversible decision.1

Making difficult decisions about the future of what is most often a wanted pregnancy is distressing regardless of gestation. Furthermore, there is no evidence that earlier terminations for fetal abnormality have substantially less emotional impact on women and couples than those carried out later in the pregnancy.2 I went in there twelve and a half weeks pregnant, and woke after a brief sleep, no longer carrying a baby. I don’t know what I dreamed of while asleep, but I woke crying silently. I know it must have been so much easier than a later induced termination – but the loss is less tangible. We never even saw our baby, except kicking around on that fateful scan.3

NEW ADVANCES IN PRENATAL GENETIC TESTING: THE PARENT PERSPECTIVE  |  201

In England and Wales, approximately 400 000 women undergo Down syndrome screening annually and around 25 000 diagnostic procedures are carried out.4 Much of this screening is currently done in the second trimester. In 2006, 1132 prenatal diagnoses of Down syndrome were made. In approximately 92% of cases, parents decided to terminate.5 If a comparable number of women who currently have screening should opt for first-trimester non-invasive diagnostic testing for Down syndrome and the proportion ending an affected pregnancy remains consistent, we can predict that there will be a rise in the diagnoses of affected pregnancies, as many cases will be diagnosed that would have otherwise been lost spontaneously before the second trimester. Indeed, there may even be a higher uptake, as the procedure itself does not put the pregnancy at risk. This will have implications for the provision of first-trimester termination of pregnancy services. At present within the NHS, women who decide to have a termination after a diagnosis of fetal abnormality before 13  weeks of gestation are offered a surgical procedure to end the pregnancy and medical induction for diagnoses after 13 weeks. If, as expected, the roll-out of NIPD gives rise to an increase in early diagnoses of aneuploidy, this in turn could result in more surgical procedures. It is likely that most will take place on gynaecological wards or be provided by independent service providers such as the British Pregnancy Advisory Service or Marie Stopes International. This will require staff training in providing individualised care for women having terminations for abnormality as within these settings most women are ending unwanted pregnancies for nonmedical reasons. Even if it remains the case that most women choose to have a termination, it will also be essential that carefully coordinated multidisciplinary care pathways are in place for women who want to continue after a diagnosis of aneuploidy.

NIPD and the challenge of informed consent Even though there has been a national programme in place for over 5 years, there is evidence that pre-Down syndrome screening discussions are limited and that many women perceive the tests as routine rather than requiring them to make an informed choice to opt in.6 I wasn’t aware I could have a choice not to have the test. I was just invited and turned up. I was invited by letter with an appointment saying please come for test. Nothing on [the] letter said it was optional. If I’d known I’d have still had the test but they should make it clear it’s optional. Other people would choose not to. I needed to know everything was okay.*

With the current roll-out throughout England of the combined Down syndrome screening programme and haemoglobinapathy screening, which are both offered in the first trimester, there is pressure on women to book ever earlier and pressure on staff to accommodate both early booking and time for screening discussions. If diagnostic genetic tests are to be offered earlier, there will need to be careful consideration of how best to ensure women have time to explore the implications of taking up the offer. It will no longer be a two-stage process with the opportunity for parents to reflect on whether they want diagnostic procedures after the screening stage and to discuss this in consultation with their healthcare professionals.

* Quote taken from a questionnaire distributed for ARC’s evaluation of combined screening for Down syndrome in four units in South West London, March 2009 (qualitative data Q11–07).

202  |  JANE FISHER

A further concern from the parental perspective is the form NIPD testing takes, being a simple blood test. Women are very accustomed to having blood taken in pregnancy and, while holding out an arm for a needle to be inserted is not always pleasant, it is something with which every pregnant woman is familiar. The ‘routine’ nature of the procedure could mean that some women embark on it without considering the possible outcomes and so will be particularly distressed if test results bring unexpected news about the pregnancy. Healthcare professionals may also perceive it as being more ‘routine’, which may impair pre-test counselling.7 As is currently the case with antenatal screening programmes, by far the majority of women who will undergo future NIPD testing will be reassured by a negative result. It is a continuing challenge to maintain a careful balance in order that informed consent is achieved without raising anxieties in all pregnant women to unmanageable levels.

Lessons to be learned from antenatal ultrasound scanning There is a precedent for a non-invasive diagnostic tool in routine use in antenatal care, namely ultrasound scanning. Every time an ultrasound probe is placed on the abdomen of a pregnant woman, there is the possibility that an abnormality will be detected. Although information provision to women about the purpose of antenatal ultrasound is improving, we cannot overestimate the profound impact when the scan shows that there may be something wrong with an unborn baby. However wellinformed a woman may be, such news will always come as a shock and will generate considerable anxiety and distress. This will also be the case for a diagnosis made from cffDNA testing, even if it comes earlier in pregnancy. Just as we do now in the context of ultrasound, ARC would advocate the implementation of planned care pathways in the instance of the diagnosis of an affected pregnancy. This will help ensure that parents have the information and support they need to decide how to proceed.

New techniques in preimplantation genetic screening and diagnosis Parents undergoing in vitro fertilisation (IVF) treatment are desperate for a successful outcome. It is more than 30 years since the birth of Louise Brown and great strides have been made: in 2006 the success rate per treatment rose to 23%, with a total of 12 956 babies born.8 However, the process remains physically and emotionally demanding for women. Therefore tests that can be carried out before implantation with the goal of improving success rates will often be attractive to parents undergoing IVF. They will also often be particularly anxious to avoid invasive testing because of its associated miscarriage risk. Many clinics offer preimplantation genetic screening (PGS) of embryos for chromosomal abnormalities and media interest in these techniques is high. In one of the most recent advances, a fertility clinic in Nottingham announced their first pregnancy after using comparative genomic hybridisation on an unfertilised egg, which was covered on BBC News.9 At the same time, it is worth noting that the British Fertility Society (BFS) updated its guidelines on PGS in July 2008, stating the following:10 It remains possible that PGS may be of benefit under certain circumstances, but the BFS currently recommends that PGS should therefore preferably be offered within the context of well-designed randomised trials performed in suitably experienced centres. Patients should be made aware that there is no robust evidence that PGS for advanced maternal age improves live birth rate; indeed from the evidence currently available the live birth rate may be significantly reduced following PGS.

NEW ADVANCES IN PRENATAL GENETIC TESTING: THE PARENT PERSPECTIVE  |  203

Here is an example of how the excitement and hype surrounding a new technology can mask its actual success in practice. Another topic that regularly attracts media interest is preimplantation genetic diagnosis (PGD). The reality is that very small numbers of parents undergo PGD. The latest figures from the Human Fertilisation and Embryology Authority state that there were 183 cycles that included PGD in 2006 (0.41% of all IVF cycles). These cycles led to 39 live births and a total of 46 babies (0.38% of the total of IVF-conceived babies).11 PGD is no easy option and is part of an IVF process that is still more likely to fail than not. It is undertaken by a small number of parents for whom it represents at least a chance of having a baby without a debilitating medical condition.

Ethical concerns: ‘eugenics’ and ‘designer babies’ Media interest in advances in prenatal genetic testing is high and news stories almost always spark debates about ethical issues. There are those who feel strongly that there is a state-endorsed eugenic purpose behind any form of prenatal test that leads to the offer of terminating an affected pregnancy.12 Media coverage has included claims that clinicians put pressure on parents to terminate affected pregnancies,13 which adds further fuel to the eugenic argument. Our two decades of experience of parental and professional attitudes to prenatal genetic diagnosis bears out a different reality. Parents never take ending a pregnancy lightly after a diagnosis; it is an agonising process as they work out whether to end their wanted unborn baby’s life before it has begun. They are very conscious of the ethical dimension; a major contributor to the pain of the process is their attempt to predict which decision they will best be able to live with. Healthcare professionals caring for the parents are most often extremely wary of appearing directive, which can mean parents feel very alone, struggling with one of the most harrowing decisions they are ever likely to face. Other familiar phrases that pepper media articles about prenatal testing are ‘designer babies’ and parents’ ‘pursuit of perfection’. Certain commentators accuse parents of terminating pregnancies for ‘minor’ or rectifiable conditions14 or warn that wider use of PGD will lead to parents seeking ‘the perfect baby’.15 All such sensationalist coverage is offensive to the parents who take their responsibilities very seriously and want nothing more than to deliver a healthy baby.

Private services As outlined previously, we at ARC can confidently predict that most parents will welcome the introduction of early non-invasive prenatal genetic diagnosis. Indeed, many are impatient for its arrival in practice and we must be careful not to raise unrealistic expectations as to how soon it might be widely available within the NHS. This impatience will lead some parents to the internet to access private provision from other parts of the world. This will be impossible to prohibit and it will therefore be important to provide accessible information to help parents to assess the quality of such services and to consider the possible consequences of this route.

Conclusion From ARC’s perspective, advances in NIPD and other forms of genetic testing that can offer a safe way to obtain diagnostic information will be of significant benefit to

204  |  JANE FISHER

parents and families. For some parents who carry genetic conditions, the availability of such tests may enable them to start or complete their families, which they may consider to be an untenable prospect without such provision. We would want to see new tests implemented into practice in a considered and entirely equitable way, with the excitement surrounding such new developments not blinding us to their consequences. High-quality pre-test information and, perhaps even more importantly, the opportunity to discuss testing with well-informed healthcare professionals are both crucial. ARC would also strongly advocate an emphasis on ensuring that individualised care and support pathways are firmly in place for the parents for whom test results bring difficult news about their pregnancy.

References 1.

2. 3. 4. 5. 6. 7.

8. 9. 10. 11. 12. 13. 14. 15.

Support after Termination for Abnormality (SATFA). SATFA News May 1991. [The woman quoted had a termination after the diagnosis of a chromosomal abnormality. SATFA was ARC’s original name. A newsletter now called ARC News is sent out to ARC members three times a year.] Statham H. Prenatal diagnosis of fetal abnormality: the decision to terminate the pregnancy and the psychological consequences. Fetal Matern Med Rev 2002;13:213–47. Healthtalkonline [www.healthtalkonline.org/Pregnancy_children/ Ending_a_pregnancy_for_fetal_abnormality/Topic/2001]. Association for Clinical Cytogenetics. UK National Audit Data 2006/7 [www.cytogenetics.org.uk]. Wolfson Institute of Preventive Medicine. The National Down Syndrome Cytogenetic Register Annual Report 2006. [www.wolfson.qmul.ac.uk/ndscr/reports/NDSCRreport06.pdf]. Marteau TM, Dormandy E. Facilitating informed choice in prenatal testing: how well are we doing? Am J Med Genet 2001;106:185–90. van den Heuvel A, Chitty L, Dormandy E, Newson A, Deans Z, Attwood S, et al. Will the introduction of non-invasive prenatal diagnostic testing erode informed choices? An experimental study of health care professionals. Patient Educ Couns 2009 [Epub ahead of print]. Human Fertilisation and Embryology Authority. Latest UK IVF Figures – 2006 [www.hfea.gov. uk/1269.html#1276]. Walsh F. IVF pregnancy from screened egg. BBC News website 26 January 2009 [news.bbc. co.uk/1/hi/health/7852292.stm]. Anderson RA, Pickering S. The current status of preimplantation genetic screening: British Fertility Society Policy and Practice Guidelines. Hum Fertil (Camb) 2008;11:71–5. Human Fertilisation and Embryology Authority. Pre-implantation genetic diagnosis (PGD) [www. hfea.gov.uk/69.html]. Lawson D. Shame on the doctors prejudiced against Down Syndrome. The Independent 25 November 2008. Salkeld L. Doctors told us to abort our disabled baby – but our son is proof that we were right to say no. Daily Mail 28 December 2007. Templeton SK. Babies aborted for minor disabilities. The Sunday Times 21 October 2007. Outcry as clinic offers ‘designer baby’ embryo screening for 200 diseases. London Evening Standard 13 November 2006.

16 Chapter 16

Informed consent: what should we be doing? Jenny Hewison and Louise Bryant

Introduction: consent, choices and decisions The concept of informed consent is central to the delivery of ethical healthcare services. Informed consent starts from the assumption that, following the provision of appropriate information, and based on his or her expertise, a clinician recommends a course of action to which the individual is asked to consent. Informing patients about the potential risks and consequences of a medical procedure is now considered essential to good practice and psychological preparation for such procedures has been shown to be linked to improved patient outcomes.1 Within reproductive genetic services, however, the term informed choice has come to be preferred over informed consent. Choosing to have prenatal screening, for example for Down syndrome, is not directly equivalent to consenting to a surgical procedure or even screening for conditions such as breast cancer, although many of the issues are related. In contrast to informed consent, an informed choice starts from the assumption that, once appropriate information has been provided, the course of action should be chosen by the patient rather than the clinician. In most cases, the only therapeutic intervention on offer following a diagnosis of abnormality via prenatal testing is termination of pregnancy. For this reason, most clinicians accept that prenatal testing choices should reflect the values of the individual woman and her partner. Testing policy and related guidelines reflect this viewpoint and require that information about the testing process should support individual choice by being balanced and nondirective.2,3 The emphasis on informed choice rather than consent reflects, among other things, the desire to distance prenatal testing programmes from unwanted eugenic associations.4 While informed choice and informed decision are often used interchangeably, a choice tends to refer to the outcome whereas a decision reflects the process of choosing between alternatives.5 As we shall discuss later, this is an important distinction when considering how to evaluate prenatal screening programmes. For these reasons, the term ‘informed decision’ is preferred. There are a number of definitions of informed decision making but one by Briss et al.6 works well within the prenatal screening context: An informed decision occurs when an individual understands the nature of the disease or condition being addressed; understands the clinical service © Jenny Hewison and Louise Bryant. Volume compilation © RCOG

206  |  JENNY HEWISON AND LOUISE BRYANT and its likely consequences, including risks, limitations, benefits, alternatives, and uncertainties; has considered his or her preferences as appropriate; has participated in decision making at a personally desirable level; and makes a decision consistent with his or her preferences and values.

This chapter will consider how healthcare services can support informed decision making within prenatal screening programmes. It then will then go on to discuss potential approaches to evaluating screening programmes that use ‘informed choice’ rather than test uptake as the outcome measure and discuss how these approaches cannot yet be considered satisfactory. Alternative ways that prenatal screening programmes could be evaluated will be suggested.

Practicalities: facilitating informed decisions To facilitate informed decision making in practice, both the constituent parts of this somewhat well-worn phrase need to be addressed. Information In the prenatal testing context, it is known that women and their partners welcome up-to-date, timely, accurate information delivered in a format that is understandable. For a screening decision to be informed, the following are considered essential areas of knowledge:7–9 n the purpose of the test n what the testing procedure involves n any risks associated with the test n the implications of the possible test results n the main features of the target condition(s). Therefore, as a minimum, information on these topics should be made available to pregnant women. In addition, a recent collaboration of members of the public, people with personal experience of genetic conditions, clinicians and information providers has suggested extensions to this list:10 n more information about the condition being tested for, including an explanation of the background and effects of the condition and any treatment and management options n an explanation of risk in simple terminology n the accuracy of test results n what happens after the test and potential follow-up tests n who will have access to test results, especially in relation to genetic information n an indication that decision making can be shared with clinicians or family. Information about the tested-for condition has often had a lower priority than information about the testing process and any such information provided has tended to have a medical or clinical focus. However, parents want to know what parenting a child with the testedfor conditions may be like and the need for balanced information on potential quality of life for a child with the tested-for condition has been found to be important.11 In reality, resource constraints make it unrealistic for healthcare professionals to provide in person all the information that a woman and her partner may need and

INFORMED CONSENT: WHAT SHOULD WE BE DOING?  |  207

those delivering prenatal screening may also not be best placed to provide information about the conditions being tested for. Language barriers may also reduce the ability of healthcare professionals to convey adequate information to support an informed decision. The UK National Screening Committee (NSC) prenatal screening leaflet for Down syndrome (now subsumed into the booklet Screening Tests for You and Your Baby) has been translated into 17 languages and is available through the NSC’s Screening Portal website.12 Healthtalkonline,13 the website of UK-based charity DIPEx, is supported by the NSC and provides information on prenatal screening, including carrier screening for sickle cell disease and thalassaemia. The modules contain interviews with women and couples who have undergone prenatal screening, diagnostic testing and termination. Website users can watch, listen to or read these interviews, some of which are in other languages (French, Mirpuri, Portuguese, Sylheti, Urdu). The AnSWeR website14 (Antenatal Screening Web Resource) was funded by the Wellcome Trust and provides information about the lives of people with cystic fibrosis, Down syndrome, neural tube defects, Klinefelter syndrome and Turner syndrome. It contains interviews with affected individuals and family members. Website users can listen to or read these interviews. Decision making There may be an assumption that if good-quality pre-test information is provided, an informed decision has been facilitated. While it is true that the provision of goodquality information is essential to support informed decisions, on its own this is insufficient.15 To meet the definition given earlier, the decision maker needs to be sufficiently engaged with the process so that their values and preferences can inform their screening choice. The evidence suggests that simple information giving does not guarantee this kind of engagement.16,17 In the situation where people are being asked to choose between different options using information containing unfamiliar terminology and complex ideas such as risk, the likelihood increases that their decisions will be influenced by the way the options themselves are presented rather than be the outcome of a deliberative decision process.18,19 For example, seemingly neutral interventions such as offering screening within an opt-out rather than an opt-in model or offering the test ‘today’ rather than at a future appointment are likely to increase screening uptake.20,21 This is because making difficult decisions is effortful and can produce emotional conflict. People thus prefer to make decisions using cognitive short cuts or heuristics: making one option ‘easier’ than another may override value-led decision making. Research into the psychology of decision making provides evidence that even presenting decisions as ‘choices’ can enhance the perceived value of whatever is on offer because choice is inherently attractive to humans.22 Therefore, it can be concluded that the way in which the offer of screening is presented, as well as the information people are given, can have a significant impact on the outcome in a way that may not always relate to a person’s underlying values about parenting a child with a disability, for example. Three simple strategies can be employed in any situation where the patient is involved in making decisions about their health care, including decisions about prenatal screening, to reduce these framing or biasing effects and to increase the likelihood that a more ‘thoughtful’ and value-consistent decision is made:22 n Change the language with which screening decisions are presented. Healthcare professionals should use language that emphasises decisions and potential consequences rather than choices and options. Instead of ‘offering’

208  |  JENNY HEWISON AND LOUISE BRYANT

the choice of having screening, be explicit that there is a decision to be made between not having screening and having screening. n Slow down the decision-making process. Heuristics are more likely to come into play in situations where the time for making a decision is constrained, or there appears to be pressure to make a quick decision.23 This means slowing down the decision-making process wherever appropriate. One way of doing this would be to state, where gestation permits, that the screening choice does not have to be made immediately. Ensuring that women are not only given the NHS screening information booklet but are shown the appropriate pages is important so that they can take time to read and discuss the options with their partner and family at home before reaching a decision. n Make explicit the need for women to ‘think actively’. Many women are unfamiliar with having to take responsibility for healthcare decisions, particularly in the antenatal setting.24 It is useful, therefore, to reinforce written information with verbal confirmation that there is a decision to be made with potential consequences, and to raise the idea that different consequences have different values for different people. One way to do this is to suggest that women write down the pros and cons of each option to help them see which option is the best decision for them and their family. Making decisions in this way is effortful, can be emotionally uncomfortable and may not always lead to greater satisfaction with the encounter, particularly if the woman has a preference for ‘physician-led’ decisions. However, if the goal of screening policy is to increase informed choice, rather than ease of choice, this is a trade-off that may have to be accepted. In other areas of health care, use of strategies such as these has been shown to lead to more informed decisions because they promote attention to relevant information, help reduce decisional conflict and increase value-consistent decisions.25

Evaluating the performance of prenatal screening and testing programmes The emphasis on increasing informed decisions rather than promoting test uptake brings an additional challenge to the evaluation of fetal anomaly screening programmes. Outcome measurement is a cornerstone of evidence-based practice because of its role in establishing the effectiveness of different kinds of care. In addition to providing the best evidence of an intervention’s effectiveness, outcome measures have the distinct advantage that they allow a focus on the service user rather than the service provider. All other things being equal, therefore, it would be desirable for the various strands of NSC activity to be measured in the same way; that is, tables or graphs showing the proportion of eligible individuals having a screening test in any one year, and how that proportion changes over time. An evaluation of a screening programme aimed at reducing deaths from breast cancer, for example, would in the long term need to examine outcome data to see whether a fall in mortality had occurred. In the short term, however, an acceptable proxy outcome may be figures demonstrating an increasing proportion of eligible women attending for mammograms. Since it can be assumed that avoiding death from cancer is universally desirable, it has become acceptable shorthand to assume that having a cancer screening test is the ‘right’ behaviour, provided of course that test performance has been formally examined and

INFORMED CONSENT: WHAT SHOULD WE BE DOING?  |  209

judged to be of an adequate standard. However, as we have already identified, it is axiomatic in prenatal testing that there is no such thing as the ‘right’ behaviour. How then is the ‘right’ outcome for a particular woman to be determined in relation to prenatal screening? Can a measure be found that acts as a proxy outcome for effectiveness purposes? In a response to this, Marteau and colleagues26 developed the Multi-dimensional Measure of Informed Choice (MMIC), its aim being to allow informed choice to be treated as an alternative to test uptake in terms of a patientrelated outcome. The MMIC is based on the premise is that it is possible to work out what an individual woman ‘should’ do, using a psychological theory called the Theory of Planned Behaviour.27 This theory, which is widely used in health psychology, aims to understand, predict and explain behaviour, particularly in terms of the relationship between a person’s attitudes towards engaging in a behaviour (for example, having a screening test) and actually engaging in that behaviour. According to the MMIC, a person’s test choice can be considered informed (a) if their test-related knowledge is good (dimension 1), they hold a favourable attitude towards undergoing the test (dimension 2) and then undergo that test (dimension 3); or (b) if their knowledge is good, they hold an unfavourable attitude towards undergoing the test and then do not undergo that test. Any other combination, that is, low knowledge and/or an inconsistent attitude–behaviour relationship, would constitute an uninformed choice. This three-dimensional definition of informed choice is usually represented diagrammatically as a 2 × 2 × 2 cube.26 In research studies using the MMIC, knowledge and attitudes have been captured using a self-completion questionnaire given to women following consultation with their healthcare provider; test behaviour is then captured via the patient notes. The developers of the measure have suggested that, by using the measure, screening services can be ‘classified in terms of the proportions of populations making informed choices’, in other words, informed choice can be used as an outcome instead of test uptake.28 However, while this is an important first step in the move away from the ‘high uptake equals success’ approach, the MMIC and similar measures developed within the same theoretical framework are limited in some important ways that are sufficient to make them inappropriate for measuring the outcomes of fetal anomaly screening programmes. Knowledge In the latest version of the MMIC, ten knowledge items are measured.29 Of these ten, five are concerned with understanding the screening risk result and two with assessing knowledge of Down syndrome (association with learning disability and lower life expectancy). The other three items assess whether the woman knows the conditions the test screens for, whether she knows the ‘possible risks’ associated with an invasive test and whether that woman would be offered a termination of pregnancy following a positive diagnosis. A measure based on the MMIC and developed by van den Berg and colleagues5,30 for research in the Netherlands uses a similar knowledge measure but has fewer risk-related items and does not make any reference to the potential outcome of the offer of termination. There are a number of potential difficulties with these knowledge measures as they stand. Altering the content of the items has significant implications for assessing whether or not a woman is considered informed or not. For example, numerical risk is known to be widely misunderstood, so scores will be higher if the measure includes fewer risk items – as in the van den Berg measure. If the scale contains a higher proportion of risk items – as in the MMIC – numbers of informed women

210  |  JENNY HEWISON AND LOUISE BRYANT

will decrease. Although various instruments measuring test-related knowledge exist within the literature, there is no agreement about the knowledge items that are necessary and sufficient to support informed choice.31 For example, women who are intending to decline a test may pay less attention to, or retain less information about, the meaning of different risk cut-off figures (which are not salient for them), making them more likely to appear in the ‘less informed’ group. In fact, women who actively decline tests have been found to be more likely to have thought about the decisionrelevant issues than many women who accept an offer of testing.5 There are also problems with the way in which that level of knowledge is operationalised in this type of measure. Knowledge scores are dichotomised, enabling women to be classified as ‘informed’ and ‘not informed’. Different studies have used the sample median, the scale midpoint of five, or a scale midpoint figure corrected for guessing, but in each of these cases it is possible to be classified as informed while having major gaps in understanding. Although a median split identifies those who have an above-average level of knowledge, this is not equivalent to being informed if the average is low. Using a midpoint score of five, or even a guess-corrected score of seven, still allows for the possibility that women considered to be well informed may not understand some key information that most would consider essential in any meaningful definition of informed choice. It is argued, therefore, that while current measures of knowledge in relation to the measurement of informed choice may be of value in a research setting, they cannot yet be considered satisfactory in relation to assessing the outcome of screening programmes. Knowledge measures could be improved by further research to establish how best to capture the knowledge most important for informed test decisions, and by addressing the issues with measurement. Values and attitudes Central to the notion of informed choice, and to the definition on which the MMIC is based, are personal values. Personal values are psychological constructs that are fundamental to the belief systems of an individual. A person’s underlying values will influence their specific attitudes – their evaluations of a particular social object or act. For example, values about the relative worth of people with disabilities will be reflected in attitudes, say towards the rights to employment, housing and education of those people. Prenatal testing can give potential parents information about the health status of their baby – information that can, if they wish, be used to justify a termination of a pregnancy under current UK law (with the exception of Northern Ireland), in some cases up until full term. The values that underlie attitudes towards termination of pregnancy for fetal abnormality, or indeed continuation of such a pregnancy, can be subtle, differentiated and complex, and they are very difficult to measure. Because of this complexity, the developers of the MMIC have chosen to measure attitudes towards a specific action – that of undergoing a screening test, for example for Down syndrome. This attitude was selected, in line with the theoretical model on which it is based, on the assumption that, as attitudes reflect values, measurement of this specific attitude ‘will encompass one or more salient values’, for example a person’s perceived ability to parent a disabled child.26 The version of the measure used by van den Berg et al.30 is less specific and measures attitudes towards ‘testing for congenital defects during my pregnancy’. Depending on the version of the measure, around six items are used to measure attitude towards undergoing the screening test. The items ask women to use a rating scale to indicate whether ‘having the screening

INFORMED CONSENT: WHAT SHOULD WE BE DOING?  |  211

test’ will be ‘a bad thing’ or ‘not a bad thing’, ‘beneficial’ or ‘not beneficial’, for example. The median score is used by the MMIC developers as the cut-off between a favourable attitude and an unfavourable attitude, although van den Berg et al.5,30 split attitudes into three groups (favourable, neutral and unfavourable). However, a screening test is a means not an end. Different testing technologies (for example, serum screening and ultrasound) can produce the same risk information, while the same technology can produce information about different conditions (for example, risk figures for Down syndrome and neural tube defect). People may hold different attitudes towards undergoing different testing procedures despite the same risk information they provide, for example ultrasound scanning is viewed very favourably by most pregnant women and their families who often look forward to the opportunity of ‘seeing their baby’.32 Of course, one test may not be enough: another procedure may need to be offered, and, even then, definitive information may not be obtained. Attitudes towards undergoing screening do not necessarily reflect attitudes towards undergoing invasive procedures or termination of pregnancy, the latter being most closely linked with the underlying values and attitudes associated with parenting a disabled child.33 The question must therefore be asked: does an attitude towards undergoing a specific test – or to undergoing tests more generally – act as an adequate proxy for underlying values for parenting a child with Down syndrome? For these measures to work as planned, the answer must be a convincing ‘yes’. In reality, the picture is more complicated. First, it is of course necessary that a woman consents to a procedure (for example, a scan or a blood test). Second, it is accepted that choosing to have a screening test does not imply that a woman will choose to have other tests that may subsequently be offered to her: each test is recognised as a separate choice. The important question is whether or not a woman offered a test is aware of and understands the potential consequences – harms as well as benefits – of both of the options available to her, that is, not undergoing a test as well as undergoing a test, and understands the relevance of the information to herself and her personal values. Because screening tests pose no risk to the pregnancy, it could perhaps be argued that the decision to have one is not really a decision at all. Potential subsequent decisions can then be rationalised as ‘we’ll cross that bridge when we come to it’. If the screening procedure involves an ultrasound scan then it becomes even less likely that attitudes towards undergoing a particular procedure can ever be treated as a proxy for underlying values about reproductive choice and the purposes of prenatal testing. It is therefore argued that the very specific measurement of attitudes towards undergoing a particular type of screening cannot be said to be a satisfactory measure of the decision maker’s values. Behaviour and the predictive power of attitudes The third dimension within the MMIC and related measures is behavioural implementation: did the woman have the offered screening test or not? Information from the three dimensions, all in binary form, is combined to produce a new binary outcome classification: the choice was informed, or it was uninformed. As described earlier, there are two different ways in which women can be classified as having made an uninformed choice. In the first of these, the underlying argument is that people whose knowledge scores fall below a certain level should always be classified as having made an uninformed choice, whatever their values and whatever the choice. There are no problems in principle with this approach, although it does depend on the quality of the method used to assess whether knowledge is adequate.

212  |  JENNY HEWISON AND LOUISE BRYANT

A woman whose knowledge is judged adequate can also be classified as having made an uninformed choice if her behaviour seems to be discrepant with her attitudes. The behaviour is uptake of the screening test on offer and it is measured unambiguously from clinic records. It follows, therefore, that the robustness of the discrepancy/no discrepancy conclusion depends on the quality and adequacy of the attitude measure as a predictor of who ‘should’ and ‘should not’ have the screening test. In effect, the attitude measure is used to ‘diagnose’ those who should and should not have Down syndrome screening, so its performance in that role needs to be assessed according to the usual methods and criteria. No test is ever 100% accurate, so test developers are required to calculate indicators such as the false positive and false negative rates and demonstrate their relationship to different scale cut-off points so that the best cut-off point can be chosen. Receiver operating characteristic (ROC) curves are frequently drawn up to illustrate and compare test performance. ROC curves can be constructed from clinical prediction rules or their psychological equivalents and provide a full picture of test accuracy and the possible sensitivity/specificity trade-offs that underlie it. The NSC requires that a test reaches certain standards of accuracy, with explicit levels of sensitivity and specificity, before it can be recommended for use. Within the current UK Down syndrome screening programme, screening methods are intended to achieve a detection rate greater than 75% with a relatively low level of false positives. Measures such as the MMIC use attitude questions and apply them, together with a cut-off point chosen by the authors, to classify women into two groups: those who ‘should’ and ‘should not’ have such a test. Actual uptake is then compared with the results of this classification rule. In another setting, such discrepancies would be characterised as false positives and false negatives; in the MMIC context, however, failures of prediction are all described as uninformed choices. This logic is not stated explicitly but it is clear: given adequate knowledge, if a woman’s choice is not explicable using the attitude assessment component, then that choice will be classified as uninformed. A corollary of the above is that improvements in attitude measurement or selection of settings that result in better prediction of behaviour will apparently lead to improved levels of informed choice, because the numbers of false positives and false negatives will have been reduced. Most psychological models such as those underlying the MMIC that use attitudes to predict behaviour have relatively modest predictive power and that power tends to be variable across samples and settings.34 Work continues on the development of the models but one of the better ways to improve predictive power seems to be to ask more-specific questions, for example how well can attitudes towards having a particular test, measured today, predict uptake of that same test, today? While having theoretical appeal in terms of behaviour prediction, these developments can leave values about parenting a child with a disability behind. Reliance on the individual patient’s attitude–behaviour relationship to determine informed choice (once knowledge has been established) can mean that important differences in service delivery can also be obscured. A number of studies have identified that offering a test at a routine visit is likely to increase uptake compared with sites where a separate appointment is required.21,35 However, other factors may also affect uptake across services. For example, in some sites, very active, testoriented staff may stress the benefits of testing to women or at least make sure the test opportunity is clearly signposted. In other sites, a more passive or ‘aloof ’ stance may be the cultural norm. These different service cultures may result in women holding different attitudes towards undergoing prenatal testing. There is very little research in

INFORMED CONSENT: WHAT SHOULD WE BE DOING?  |  213

this area, however, as most has focused on the individual attitudes of health providers. In either of these scenarios, as long as uptake (high or low) is ‘explicable’ in terms of attitude–behaviour consistency, levels of informed choice can be apparently high and important contributions to between-site differences will remain unscrutinised. Including a measure of decision making As discussed previously, some authors have drawn attention to the distinction between a choice and a decision, defining a decision as a process of choosing between two or more options and a choice as the outcome of a decision.5 In this scenario, measuring only the choice (the outcome) does not take account of whether or not people use their knowledge to think about the alternatives before making their final choice.36 Van den berg and colleagues5 have suggested that attitude–behaviour consistency may be achieved because some women believe testing is a ‘self-evident’ behaviour and not one that requires a decision; in this case, the outcome cannot be said to be an informed decision. As a result, they have added a measure of deliberation to their existing measures of knowledge and attitudes. The measure of deliberation assesses how well women have visualised and thought through the potential consequences of undergoing and not undergoing screening. In the one published study using this measure, substantially fewer women were classified as having made an informed decision than would have been classified as having made an ‘informed choice’ using measures of knowledge and attitude–behaviour consistency alone. While the addition of a measure of the deliberation process is an interesting development, it requires further work to test its reliability and validity in this setting. In addition, because van den Berg and colleagues’ measure incorporates the problematic elements discussed previously, it cannot be considered appropriate as a measure of the effectiveness of a screening programme.

Evaluating screening programmes: what else can we do? It is suggested that informed choice, as operationalised within existing measures, is not an adequate outcome measure for prenatal testing screening programmes, either at individual or clinic level. The ‘right’ choice for a particular woman and her family will be determined by a complex mixture of individual, social and serviceprovision factors. Future research should, therefore, increase understanding of how these interact. Theoretically derived instruments will continue to have a place in the research context but it must always be borne in mind that increasing the ability to predict what people will do is not at all the same as increasing understanding of what they should do. For the foreseeable future, programmes offering tests for which there can be no single right course of action should not be evaluated in terms of uptake or other proxy outcome indicator but rather in terms of the quality of their processes. Crucially, measures of service effectiveness or quality should be unrelated to the choices made because people who accept and decline tests should both have received the same high-quality support in making their choices. We should therefore be measuring quality of care at the site level and quality of decision making at the individual level. There are three main areas that can be targeted to achieve this: n The staff level. Providing good-quality written information, whether through a leaflet or the internet, is only one of the elements necessary to facilitate an informed decision. We have seen that the healthcare professionals with whom women interact have a crucial role to play, as the way in which the offer of

214  |  JENNY HEWISON AND LOUISE BRYANT

screening is framed can bias the decisions women make. The simple strategies outlined above can help in this respect. However, while healthcare professionals must guard against influencing individual choice, it is acknowledged that many women would prefer a more shared supportive approach to decision making.37 There is growing evidence on shared decision making within healthcare settings, mostly from the USA and Canada, showing that decision support can be given in a way that also facilitates value-consistent individual choice. Staff education and training programmes, together with quality monitoring in relation to decision support protocols, are already operating in some areas of healthcare.38 n The service level. There are many settings and circumstances outside of the healthcare context in which there can be no pre-specified ‘right answer’ and no single best outcome of the service being provided. Arbitration services and custody hearings are two obvious examples. The quality of these kinds of service can only be measured in process terms, for example have the staff been properly trained? Are they adequately supervised? Are all clients given the time and opportunity to express their views? Are agreed procedures followed? Are there systems for ensuring comparability between different service providers? It is suggested that the concept of providing services fairly to people with different needs and points of view also applies to prenatal screening and testing programmes. The objective is ‘fairness’ to all and it is important to the reputation of the service that it is both known, and shown, to be fair. It is suggested that this concept could also be profitably investigated with respect to prenatal screening and testing. n The individual level. There are now a number of well-validated measures of decision making, for example the Decisional Conflict Scale, that may be suitable for evaluating quality of decision making in prenatal screening programmes at the individual level.39 These, together with measures of satisfaction with the fairness of the process, could be administered to a sample of women and used, in conjunction with the other approaches outlined, to assess whether a prenatal screening service is achieving high-quality decision support and fairness of process.

References 1. 2. 3. 4. 5. 6. 7. 8.

Johnston M, Vogele C. Benefits of psychological preparation for surgery: a meta-analysis. Ann Behav Med 1993;15:245–56. NHS Antenatal and Newborn Screening Programmes. Down’s Syndrome Screening Programme Objectives. London: NHS; 2006. National Institute for Health and Clinical Excellence. Antenatal Care: Routine Care for the Healthy Pregnant Woman. NICE clinical guideline 62. London: NICE; 2008. Human Genetics Commission. Making Babies: Reproductive Decisions and Genetic Technologies. London: Human Genetics Commission; 2006. van den Berg M, Timmermans DRM, ten Kate LP, van Vugt JMG, van der Wal LKG. Informed decision making in the context or prenatal screening. Patient Educ Couns 2006;63:110–17. Briss P, Rimer B, Reilley B, Coates RC, Lee NC, Mullen P, et al. Promoting informed decisions about cancer screening in communities and healthcare systems. Am J Prev Med 2004;26:67–80. Reid M. Consumer-oriented studies in relation to prenatal screening tests. Eur J Obstet Gynecol Reprod Biol 1988;28 Suppl:79–92. Royal College of Physicians. Prenatal Diagnosis and Genetic Screening: Community and Service Implications. London: Royal College of Physicians; 1989.

INFORMED CONSENT: WHAT SHOULD WE BE DOING?  |  215 9. 10. 11.

12. 13. 14. 15.

16. 17. 18. 19. 20. 21. 22. 23.

24. 25.

26. 27. 28.

29.

30. 31. 32. 33. 34.

Royal College of Obstetricians and Gynaecologists. Termination of Pregnancy for Fetal Abnormality in England, Wales and Scotland. London: RCOG Press; 1996. Shepperd S, Farndon P, Grainge V, Oliver S, Parker M, Perera R, et al. DISCERN-Genetics: quality criteria for information on genetic testing. Eur J Hum Genet 2006;14:1179–88. Ahmed S, Atkin K, Hewison J, Green JM. The influence of faith and religion and the role of religious and community leaders in prenatal decisions for sickle cell disorders and thalassaemia major. Prenat Diagn 2006;26:801–9. UK National Screening Committee. UK Screening Portal [www.screening.nhs.uk/]. DIPEx. Healthtalkonline [www.healthtalkonline.org/]. Antenatal Screening Web Resource. AnSWeR website [www.antenataltesting.info/]. Bryant LD, Ahmed S, Hewison J. Conveying information about screening. In: Rodeck C, Whittle M, editors. Fetal Medicine: Basic Science and Clinical Practice. 2nd ed. Edinburgh: Churchill Livingstone; 2008. Seror V, Ville Y. Prenatal screening for Down syndrome: women’s involvement in decisionmaking and their attitudes to screening. Prenat Diagn 2009;29:120–8. Stapleton H, Kirkham M, Curtis P, Thomas G. Framing information in antenatal care. Br J Midwifery 2002;10:197–201. Tversky A, Kahneman D. The framing of decisions and the psychology of choice. Science 1981;211:453–58. Jones SK, Frisch D, Yurak TJ, Kim E. Choices and opportunities: another effect of framing on decisions. J Behav Decis Making 1998;11:211–26. Johnson EJ, Goldstein D. Do defaults save lives? Science 2003;302:1339. Bekker H, Modell M, Denniss G, Silver A, Mathew C, Bobrow M, et al. Uptake of cystic fibrosis testing in primary care: supply push or demand pull? BMJ 1993;306:1584–6. Bryant LD, Bown N, Bekker HL, House A. The lure of ‘patient choice’. Br J Gen Pract 2007;57:822–6. Maule AJ, Edland AC. The effects of time pressure on judgement and decision making. In: Ranyard R, Crozier WR, Svenson O, editors. Decision Making: Cognitive Models and Explanation. London: Routledge; 1997. p. 189–204. Ahmed S, Green J, Hewison J. Antenatal thalassaemia carrier testing: women’s perceptions of “information” and “consent”. J Med Screen 2005;12:69–77. Bekker HL. Using decision making theory to inform clinical practice. In: Elwyn G, Edwards A, editors. Shared Decision Making – Achieving Evidence-based Patient Choice. London: Open University Press; 2009. Marteau TM, Dormandy E, Michie S. A measure of informed choice. Health Expect 2001;4:99–108. Ajzen I. The theory of planned behavior. Organ Behav Hum Decis Process 1991;50:179–211. Marteau TM. Informed choice: a construct in search of a name. In: Edwards A, Elwyn G, editors. Shared Decision-Making in Health Care: Achieving Evidence-based Patient Choice. 2nd ed. Oxford: Oxford University Press; 2009. p. 87–94. Dormandy E, Michie S, Hooper R, Marteau TM. Informed choice in antenatal Down syndrome screening: A cluster-randomised trial of combined versus separate visit testing. Patient Educ Couns 2006;62:56–64. Van den Berg M, Timmermans DR, ten Kate LP, van Vugt JMG, van der Wal G. Are pregnant women making informed choices about prenatal screening? Genet Med 2005;7:332–8. Green JM, Hewison J, Bekker HL, Bryant LD, Cuckle HS. Psychosocial aspects of genetic screening of pregnant women and newborns: a systematic review. Health Technol Assess 2004;8:iii,ix–x,1–109. Garcia J, Bricker L, Henderson J, Martin MA, Mugford M, Nielson J, et al. Women’s views of pregnancy ultrasound: a systematic review. Birth 2002;29:225–50. Bryant LD, Green JM, Hewison J. The role of attitudes towards the targets of behaviour in predicting and informing prenatal testing choices Psychol Health 2009 (in press). Cooke R, French DP. How well do the theory of reasoned action and theory of planned behaviour predict intentions and attendance at screening programmes? A meta-analysis. Psychol Health 2008;23:745–65.

216  |  JENNY HEWISON AND LOUISE BRYANT 35.

36.

37. 38.

39.

Dormandy E, Hooper R, Michie S, Marteau TM. Informed choice to undergo prenatal screening: a comparison of two hospitals conducting testing either as part of a routine visit or requiring a separate visit. J Med Screen 2002;9:109–14. Bekker HL. Genetic screening: facilitating informed choices. In: Cooper DN, Thomas N, editors. Encyclopaedia of the Human Genome. New York: Nature Publishing Group – Macmillan Publishers Ltd; 2003. p. 926–30. Garcia E, Timmermans DRM, van Leeuwen E. Rethinking autonomy in the context of prenatal screening. Prenat Diagn 2008;28:115–20. Stacey D, Pomey MP, O’Connor AM, Graham ID. Adoption and sustainability of decision support for patients facing health decisions: an implementation case study in nursing. Implement Sci 2006;1:17. O’Connor AM. Validation of a decisional conflict scale. Med Decis Making 1995;15:25–30.

17 Chapter 17

Consensus views arising from the 57th Study Group: Reproductive Genetics

Clinical practice 1. Genetic testing should be delivered within a framework of health care and appropriate pre- and post-test counselling should be available. 2. Laboratories should make available to service users evidence-based information on the analytical and clinical validity and utility of tests offered. 3. There should be increased capacity for improved high-throughput diagnostics for karyotyping, single-gene disorders and complex disorders (multigenic/copy number variants) with the ability to transition research genetic tests into the clinical arena as rapidly as possible when deemed appropriate. 4. Complex disorders of sex development should be managed by a multidisciplinary team with experience of relevant issues throughout life (fetal/newborn/ childhood/transition/adulthood). 5. Carefully coordinated care pathways need to be in place when diagnoses are made. 6. It should be acknowledged that prenatal gene transfer may be the only therapeutic approach for treatment of certain severe life-threatening diseases. 7. Fetal karyotyping should remain the ‘gold standard’ test following invasive prenatal diagnosis until appropriately tested and evaluated higher-resolution whole-genome analytical methods can be introduced.

Health policy 9. The National Institute for Health Research’s remit should be extended to genetic test evaluation studies and funding for translation of tests into NHS service (for example, test development and validation studies) should be more readily available through this route. 10. Tests for low-penetrance biomarkers should be subjected to a similar, though not necessarily identical, process of scrutiny to those rare high-penetrance genetic conditions currently dealt with by the UK Genetic Testing Network.

218  |  CONSENSUS VIEWS

11. Assuming the successful introduction of non-invasive prenatal diagnosis for the detection of Down syndrome and other common aneuploidies, future fetal karyotyping will focus on cases with ultrasound-detected structural abnormalities, in which case: n The use of prenatal array comparative genomic hybridisation (aCGH) should be focused in rapid-turnaround and high-throughput laboratories with the proven infrastructure to cope with the high levels of fast parental follow-up studies that will be required. n Cytogenetic laboratories need to change training infrastructure now to reflect the significant bioinformatic and molecular components involved with aCGH and other whole-genome analytical methodologies (for example, shotgun sequencing). n aCGH should replace fetal kayotyping as soon as the data proving its effectiveness are available. 12. Programmes offering tests for which there can be no recommended ‘right’ course of action should be measured not by test uptake or other outcome indicator but by the quality of their processes. 13. There is a need for a comprehensive programme of education for professionals and the public with regard to novel genetic tests and technologies. 14. Access to and the optimum service configuration of genomic technologies in consolidated specialised laboratory services should be specifically addressed by the new national clinical director for pathology services. 15. Fetal D testing of alloimmunised women should be extended to the whole of the UK and not just be for those with high levels of anti-D. 16. Fetal D testing for all D-negative pregnant women should be introduced as soon as the best time has been identified. 17. The public, healthcare professionals and policy makers need to be kept informed of the progress and limitations of fetal D testing, both to avoid raising unreasonable expectations and to enable preparation for expeditious implementation into NHS service when it is safe and appropriate to do so. 18. Couples should be seen by a genetics expert before PGD. 19. Guidelines need to be developed to define the criteria for referral for screening for Y chromosome microdeletions in male infertility. Saviour siblings 20. Preimplantation genetic diagnosis (PGD) for saviour siblings should be permitted or denied based on the permissibility or impermissibility of live-organ donation by children. 21. Prospective PGD and selection may be warranted in some cases. 22. PGD centres should closely integrate the services of assisted reproductive technology (ART) and genetics, and be accredited by appropriate organisations. 23. Paediatric follow-up should be a mandatory part of a PGD service. 24. PGD and preimplantation genetic screening (PGS) should be recognised as separate clinical services and PGS should only be offered as part of a randomised controlled trial.

CONSENSUS VIEWS  |  219

25. Future developments should continue to involve medical professionals, user groups, regulatory bodies and ethicists. 26. Fetal medicine units need to have a genetics team that is available to discuss atrisk pregnancies on a daily basis. 27. Teaching programmes should endeavour to include mutidisciplinary aspects whereby joint teaching between genetic and fetal medicine specialties occurs. Fetal gene and stem cell therapy 28. Policy development is needed in relation to procedures that do not fall neatly into either the research or ‘normal clinical practice’ categories. 29. There is a need to consider consent policy in relation to termination of pregnancy if an attempt at therapy fails. 30. There is a need to consider consent policy for highly uncertain new therapies.

Research Breakthrough technologies Embryonic stem cell-derived germ cells 31. Focused basic research is needed to understand the normal genetic and epigenetic events that occur during primordial germ cell specification from embryonic stem (ES) cells and in the differentiation of primordial germ cells. Fetal stem cell and gene therapy 32. Improvements in vector design and safety will be needed before safe targeted delivery to the fetus can be achieved; studies into long-term safety in large animal models (non-human primates) should be supported 33. There is a need for research into the acceptability of prenatal gene therapy. 34. Fetal stem cell therapy requires considerable preclinical development and research before clinical introduction, although rescue treatment may be justified in continuing pregnancies with osteogenesis imperfecta on a case-by-case basis subject to ethical, safety and oversight restrictions. Epigenetic regulation 35. Further research is needed into understanding changes in methylation and in histone modifications in the genome. Development of existing technology 36. Existing technology should be developed to: n promote the development of bioinformatics resources for data analysis n promote the development of biobanks into reproductive disease to facilitate appropriate research.

220  |  CONSENSUS VIEWS

Implementation of technologies 37. The effectiveness of aCGH should be formally evaluated in the UK with a large prospective trial. 38. Full health technology and economic assessments should be carried out on the emerging new genetic and genomic technologies, including the potential benefits and savings in other areas of healthcare spending. 39. Research into the development and implementation of technologies associated with the analysis of free fetal nucleic acids for diagnostic purposes is recommended. 40. Research is needed into the long-term outcomes of novel therapeutic interventions.

Other recommendations 41. It would be helpful and appropriate if the impossibility of objectively defining ‘serious’, at least in relation to disabilities, illnesses or conditions of ‘midspectrum’ severity, were recognised and reflected upon in the prenatal diagnosis (PND) and PGD contexts. This is complicated by uncertainties in relation to prognosis. 42. It would be helpful and appropriate if possible parental and family interests were recognised and reflected upon as being of particular relevance in the interpretation of ‘serious’ in the criteria for PND and PGD in the many cases where a condition would nevertheless mean that a future child will have a life he or she thinks worth living. 43. Measures of service quality should be unrelated to decisions made because people who accept and decline tests should both have received the same highquality support in making their choices. 44. There is a need to be more explicit about the challenges for parents and professionals in dealing with uncertain outcomes. .

i Index

Abortion Act 1967 183, 186, 187–90 ACCE framework 193, 194–7 achondrogenesis 136, 137 achondroplasia PGD selection for 52 prenatal diagnosis 132, 135, 137, 164 adeno-associated virus (AAV) vectors 105, 106 immune responses 102, 113 prenatal gene therapy 108, 109 safety 114 specific-organ targeting 109 adeno-retro hybrid vectors 106 adenovirus vectors 106 immune response 113 prenatal gene therapy 105, 108–9 safety 114 Advisory Committee on Genetic Testing (AGCT) 184 African populations, Rh D polymorphism 174, 175 AIRE mutations 3 alkaline phosphatase 137 allele drop-out (ADO) 43 Alzheimer’s disease, PGD 51–2 ambiguous genitalia 15, 26, 27–8 antenatal detection 140–1 early postnatal management 29–30 American Association for Reproductive Medicine 50 amniocentesis 159 decision making by parents 199–200 fetal karyotyping 147 fetal Rh D typing 173–4 rapid aneuploidy testing 148–9 amniotic fluid, mesenchymal stem cells 86–7 androgen insensitivity syndrome 3, 6, 27 antenatal detection 140 complete (CAIS) 6, 15, 21, 27 partial (PAIS) 6, 27 androgenotes 71, 72 androgen receptors 17 gene (AR) mutations 3, 6, 25

androgens disorders of synthesis and action 24–5, 27 excess states 25, 28 aneuploidies see chromosomal anomalies; Down syndrome; trisomies Angelman syndrome 77–8 animal models of human disease fetal stem cell therapy 89–91, 92–3 prenatal gene therapy 107–9 anovulation 2 AnSWeR (Antenatal Screening Web Resource) 207 Antenatal Results and Choices (ARC) 199–200, 202, 203–4 antenatal screening 131–2 Down syndrome 149–50, 201 evaluating programme outcomes 208–13, 220 informed consent, choices and decisions 205–14 ultrasound scanning 131–43, 202, 211 anti-D immunoglobulin prophylaxis 173, 178–9 antimüllerian hormone (AMH) 17, 20 impaired secretion 26 ‘anti-testis’ genes 17 Antley–Bixler syndrome 27 Apert syndrome 165 aromatase insufficiency 25, 28 array comparative genomic hybridisation (aCGH) 150–7, 218, 220 role in prenatal diagnosis 152–7 technique 150–2 ART see assisted reproductive technologies arthrogryposis 138–9 ARX/X-linked lissencephaly 23, 24 assisted reproductive technologies (ART) childhood growth and development 76 genomic imprinting and 73–6 genomic imprinting syndromes and 77–8 see also in vitro fertilisation atelosteogenesis type II 136

222  |  INDEX ATRX deletion 24 attitudes measuring 210–11 predicting behaviour 211–13 auditing, genetic test effectiveness 196 AURKC gene mutations 6 autonomy, reproductive, right to 61–2, 63–5 AZF microdeletions 5–6 bacterial artificial chromosomes (BACs) 150, 151 Bardet–Biedel syndrome 143 Batten disease 111 Beckwith–Wiedemann syndrome 77–8, 134–5 behaviour, predictive power of attitudes 211–13 bilirubin uridine diphosphate (UDP) glucuronosyltransferase 108, 113 biobanks 219 bioinformatics 219 Blackfan–Diamond anaemia 59 blastomere biopsy 38–9 blepharophimosis-ptosis-epicanthus inversus syndrome (BPES) 3 blood groups, non-invasive fetal typing 173–80 BMP15 gene mutations 3 bone marrow adult stem cells 83–4 fetal stem cells 84–6 bone marrow transplantation (BMT) 84, 87, 91, 92 bowel obstruction, antenatal diagnosis 141–2 bradycardias, fetal 139–40 brain malformations 133–4, 135 branchio-oto-renal syndrome 132, 143 BRCA1/BRCA2 mutations, PGD 51–2 British Fertility Society (BFS) 50, 202 CAG triplet repeats, male infertility 6 campomelic dysplasia 23, 24, 26, 140 carbohydrate-deficient glycoprotein syndrome 140 cardiac abnormalities, fetal 132, 135, 137–8 cardiac dysrhythmias fetal hydrops 139–40 inherited 196 care pathways 201, 202, 217 cat eye syndrome 138 CBX2 gene 24 CCR5 gene 177, 178 CDY gene 5 cell-free fetal DNA (cffDNA) 159, 173, 198 diagnosis of aneuploidy 163–6

diagnosis of single-gene disorders 163, 164–5 fetal genotyping for other blood groups 179–80 fetal rhesus D genotyping 174, 175–9, 180 fetal sex determination 160–3 limitations 168 see also non-invasive prenatal diagnosis cell-free fetal mRNA 166, 167 cffDNA see cell-free fetal DNA CFTR gene mutations 6 prenatal transfer 105, 114 CGH see comparative genomic hybridisation CHARGE syndrome 140–1, 142 children live organ donation by 66–7, 68 proxy consent 127–8 saviour see saviour siblings choice informed see informed choice uninformed 209, 211–12 cholesterol side-chain cleavage deficiency (CYP11A1) 18, 24, 27 chorioamnionitis 114 chorionic villus sampling (CVS) 159, 168 decision making by parents 199–200 fetal karyotyping 147 fetal mesenchymal stem cells 86 fetal Rh D typing 173–4 rapid aneuploidy testing methods 148–9 chromosomal anomalies infertility 4–5, 8 PGD 44, 47, 48–9 prenatal diagnosis 147–57 array CGH and 152–4, 155, 156 current service delivery models 149–50 karyotyping reporting times 147, 148 new technologies 150–2 non-invasive 154–7, 163–7 potential service reconfiguration 154–7 rapid aneuploidy testing 147–9 ultrasound scan 138 see also fetal karyotyping recurrent miscarriage 4 chromosome rearrangements, sex selection 47–50 cleft lip and palate 138, 187 clinical geneticists PGD services 36

INDEX  |  223 role in fetal medicine unit 131–43, 218 clinical practice, consensus views 217 clitoromegaly 26 collagenopathies, type II 137 common complex disorders, genetic testing 197 comparative genomic hybridisation (CGH) 202 array (aCGH), prenatal diagnosis 150–7, 218, 220 PGD 35, 37, 47 complete androgen insensitivity syndrome (CAIS) 6, 15, 21, 27 confined placental mosaicism (CPM) 168 conflict theory, fetal growth 76 congenital abnormalities see fetal abnormalities congenital adrenal hyperplasia (CAH) 23, 27, 28 cffDNA for fetal sex determination 160, 162–3 genetic test evaluation 194 lipoid (StAR deficiency) 24, 27, 140 non-invasive prenatal diagnosis 165 prenatal diagnosis and treatment 28–9, 140 steroidogenesis defects causing 18 see also 21-hydroxylase deficiency congenital bilateral absence of vas deferens (CBAVD) 6, 36 consensus views 217–20 consent (informed) 205–14 fetal/early childhood stem cell and gene therapies 127–8 live organ donation by children 66–7 non-invasive prenatal diagnosis 201–2 contractures, fetal 138–9 copy number changes 150, 152–3 copy number variants (CNVs) 150, 151, 153 cord blood haematopoietic stem cells 84 mesenchymal stem cells 85, 86, 87 Cornelia de Lange syndrome 141, 142 cost-effectiveness fetal Rh D testing 178–9 new genetic tests 195 stem cell and gene therapies 128 Costello syndrome 133 costs, array CGH 153 counselling genetic testing 194 invasive prenatal diagnosis 199–200 non-invasive prenatal diagnosis 168, 201–2

Crigler–Najjar type 1 syndrome 108, 113 Crouzon syndrome 165 CYP11A1 deficiency 18, 24, 27 CYP17A1 see 17α-hydroxylase cystic fibrosis antenatal ultrasound detection 141 genetic testing 196 information provision 207 non-invasive prenatal diagnosis 163, 165 preimplantation genetic diagnosis 36, 38 prenatal gene therapy 104 severity of illness 185 cytogenetic laboratories 147, 155–7, 218 Database of Genomic Variants 150, 153 DAX1 gene 17, 24 DAZ gene 5–6 deafness, PGD selection for 52 Decisional Conflict Scale 214 decision making facilitation in practice 207–8 invasive prenatal diagnostic tests 199–200 measuring 213 quality of 213–14 shared 214 termination of pregnancy, legal aspects 187–90 see also informed decisions Denys–Drash syndrome 24, 26 desert hedgehog (DHH) gene defects 24 designer babies 203 development ART children 76 PGD/PGS children 41 developmental delay, prenatal diagnosis 150, 153, 155 dexamethasone, prenatal treatment 28–9, 162 diaphragmatic hernia, congenital 104, 105, 133 diastrophic dysplasia 136 differentially methylated regions (DMRs) 72–3, 74 DiGeorge/velocardiofacial 22q11.2 microdeletion 132, 138, 153 dihydrotestosterone (DHT) 17, 18 DIPEx 207 disability/impairment ground for termination of pregnancy 187–90 impact on parents 186 PGD for selection for 52

224  |  INDEX disorders of sex development (DSD) 15–16, 20–31 46,XX 21, 22, 25, 27–8 46,XY 21, 22, 24–5, 26–7 classification and diagnosis 21–3 consensus views 217 early postnatal management 29 fertility management 30–1 long-term outcome 30 molecular diagnosis and highthroughput analysis 30 multidisciplinary team (MDT) approach 15, 29–30 nomenclature 21 ovotesticular 26, 27–8 prenatal diagnosis 28–9 sex chromosome 21, 22, 23–6 testicular 27–8 DMRT1 deletion 24 DNA cell-free fetal see cell-free fetal DNA vectors 106, 107 DNA methylation controlling genomic imprinting 72–3 differential, fetal/maternal genes 160–1, 166–7, 177 effects of ART 74–6 male and female germlines 73, 74 male infertility and 7–8 Down syndrome (trisomy 21) antenatal screening 149–50, 201 decision making by parents 199–200 evaluating screening programmes 209, 210–11, 212 information provision 207 invasive prenatal diagnosis 147–50 non-invasive prenatal diagnosis 154–7, 163–7, 168, 201 severity of illness 185 termination of pregnancy 189 Duchenne muscular dystrophy 89, 104 ductus venosus, absent 140 duodenal atresia 141 echocardiography, fetal 137–8 ectrodactyly 141, 142 education non-invasive prenatal diagnosis 168, 218 see also counselling Ehlers–Danlos syndrome type IV 132 EIF2B gene mutations 3 Ellis–van Creveld syndrome 135–6 embryonic stem (ES) cells 83, 84 differentiation into germ cells 219 ethical issues 124–5

embryos biopsy 38–9, 50 genomic imprinting of cultured 74–5 number transferred 40 transfer 39 endocrine disruptors 7 endometriosis 1, 2 epidermolysis bullosa 104 epigenetics 71–9, 219 ART and 73–9 male infertility 7–8 see also DNA methylation; genomic imprinting equine infectious anaemia virus (EIAV) vector 107, 112–13 ESR1 gene 8 estrogens 20 ethical aspects eugenics and designer babies 203 genetic test evaluation 195 legal criteria for termination of pregnancy and PGD 183–92 non-invasive prenatal diagnosis 168, 201–2 prenatal gene therapy 114–15 saviour siblings 59–68 stem cell therapy and gene therapy 123–9 ethnic variations, causes of female infertility 2, 3 eugenics 203, 205 European Society of Human Reproduction and Embryology (ESHRE) 36, 40–1, 43, 47, 50 evil acts, benefiting from 124–5 factor VII deficiency 103, 104, 111 factor IX gene transfer 102, 107–8, 112 Fanconi anaemia 84, 141 female infertility 1–4 femur fibula ulna (FFU) syndrome 136 fertility treatment, disorders of sex development 30–1 fetal abnormalities 11–13 week ultrasound scan 133–5 18–20 week ultrasound scan 135–43 after PGD/PGS 41 invasive prenatal diagnosis 150, 153 termination of pregnancy for 183–90, 191–2, 200–1 see also prenatal diagnosis fetal autologous stem cell gene therapy 102 fetal cells, maternal circulation 159 fetal dysmorphology 131–43, 150 fetal growth restriction, prenatal gene therapy 103–5, 111–12

INDEX  |  225 fetal karyotyping 147–57 comparison with array CGH 153, 156 consensus views 217, 218 current service delivery models 149–50 future role 154–7 potential new technologies replacing 150–3, 154 rapid aneuploidy tests 147–9 fetal medicine units, role of clinical geneticists 131–43, 218–19 fetal somatic gene therapy 102 see also prenatal gene therapy fetal stem cells ethical issues 124–5 sources 84–7, 88 fetal stem cell therapy 83–94, 102 consensus views 218–19 consent issues 127–8 rescue therapy 93–4 routes to clinical translation 92–4 sources of stem cells 83–7, 88 see also in utero transplantation fetus interests, termination in 190 moral status 189–90 fluorescence in situ hybridisation (FISH) PGD 37, 47, 48–9, 50 prenatal diagnosis 147–9, 157 foamy virus vectors 107 follicle-stimulating hormone (FSH) 20 follicle-stimulating hormone (FSH) receptor mutations 3 FOXL2 gene mutations 3 fractures, intrauterine 136–7 fragile X mental retardation (FMR) gene 3, 194 fragile X syndrome 168, 194, 196 Frasier syndrome 24, 26 Free DNA Fetal Kit RhD® 175 GALT gene mutations 3 gastrointestinal disorders, antenatal diagnosis 141–2 gender assignment 26 gene dossiers 193, 194–7 gene therapy adult and neonatal 102 consent for fetuses and children 127–8 cost-effectiveness and resource allocation 128 ethical aspects 123–9 prenatal 101–17 reproductive effects 126 research–treatment overlap 126–7 stem cell-based 83

treatment–enhancement overlap 127 genetic association studies, infertility 2–3, 6–7, 8 Genetic Commissioning Advisory Group (GenCAG) 193, 196 genetic test evaluation 193–8, 217 ACCE framework 193, 194–7 changes/new systems required 197–8 common complex disorders 197 international frameworks 194 UK approach 193 genetic tests analytical validity 194 auditing effectiveness 196 clinical utility 195 clinical validity 195 consensus views 217, 220 counselling before and after 194 definition 194 ethical, legal and social issues 195 gatekeeping 196 information for commissioning 196 service provision 193, 217–19 genomic imprinting 71–9 effects of ART 73–6 growth and 76–8 mechanism 72–3, 74 placenta 76–7 postnatal, significance 78 syndromes 77–8 germ cells 18–19 embryonic stem cell-derived 219 see also primordial germ cells germline changes after prenatal gene therapy 111 ethical issues 126 Glover, Jonathan 184, 185 glucocorticoid resistance 25 α-glucosidase (GAA) 109 glycogen storage disease type II 109 gonadal dysgenesis 24, 26 complete 26 mixed 22, 24, 26 partial 26 gonads, development 17, 19–20 growth ART children 76 genomic imprinting and 76–8 PGD/PGS children 41 gynogenotes 71, 72 H19 gene 75–6, 77 haematopoietic stem cells (HSC) in utero transplantation (IUT) 88–9, 91 sources 84

226  |  INDEX haemoglobinopathies antenatal screening 201 fetal stem cell therapy 91 non-invasive prenatal diagnosis 164, 165 haemolytic disease of fetus and newborn (HDFN) 173, 179 haemophilia postnatal gene therapy 102, 105 prenatal gene therapy 103, 107–8, 111 harm principle 62, 63–4, 65 Hashmi saviour sibling case 59 healthcare professionals attitudes to screening tests 212–13 measuring quality of care 213–14 presentation of screening decisions 207–8 health policy, consensus views 217–19 health risk, ground for termination of pregnancy 188, 190 Healthtalkonline 207 herpes simplex virus vectors 106 HLA-matched siblings see saviour siblings holoprosencephaly 133–4 Holt–Oram syndrome 141 HPA-1a platelet antigen, fetal genotyping 180 HRAS mutations 133 human chorionic gonadotrophin (hCG) 17, 18 Human Fertilisation and Embryology (HFE) Act 1990 42, 183 Human Fertilisation and Embryology (HFE) Act 2008 47, 52, 183, 184, 191 Human Fertilisation and Embryology Authority (HFEA) 42, 59, 184, 190–1 Human Genetics Commission (HGC) 184, 189, 190, 191 Huntington disease non-invasive prenatal diagnosis 163, 165 PGD 51 Hurler syndrome 91 hydrocephalus 135 hydrops, fetal 139–40 11β-hydroxylase deficiency 18, 28 17α-hydroxylase (CYP17A1) deficiency 18, 24, 27 21-hydroxylase deficiency 15, 18, 25, 28 prenatal diagnosis 140 17-hydroxyprogesterone (17-OHP) 28 3β-hydroxysteroid dehydrogenase 2 (3β-HSD 2) (HSD3B2) deficiency 18, 24, 28 17β-hydroxysteroid dehydrogenase 3 (17β-HSD 3) deficiency 18, 25, 27 hypophosphatasia 137 hypospadias 23, 26

hypothalamic–pituitary–gonadal axis 17, 20 hypoxic ischaemic encephalopathy 104 Igf2 gene 75–6, 77 IGF2R/Igf2r gene 75, 76, 77 immune responses, vectors and transgenes 102, 108, 113 immune tolerance fetal stem cell therapy 85–6, 88 gene therapy 102–3 impairment see disability/impairment imprint control regions (ICRs) 73, 75–6 imprinting, genomic see genomic imprinting inducible pluripotent stem cells (iPS) 83, 84 infertility disorders of sex development 15 genetic aetiology 1–8 information, provision 206–7 informed choice definition 209 measurement 209–13 vs informed decisions 205, 213 informed consent see consent informed decisions definition 205–6 facilitation in practice 205–8 measuring outcomes 208–13 vs informed choice 205, 213 see also decision making inhibin A/B 20 insertional mutagenesis, integrating gene vectors 112–13 Ins gene 76 Institut de Biotechnologies Jacques Boy 175 insulin-like 3 (INSL 3) 20 interests child’s 184–5 parent’s 185–6 International Blood Group Reference Laboratory (IBGRL) 176–7 International Society of Blood Transfusion (ISBT) 180 International Standard Cytogenomic Array (ISCA) Consortium 151–2 intersex disorders see disorders of sex development intracytoplasmic sperm injection (ICSI) 38, 73–4, 78 in utero transplantation (IUT) of stem cells 87–94, 102 clinical development 91–2 preclinical development 88–91 rationale 88

INDEX  |  227 routes to clinical translation 92–4 see also fetal stem cell therapy; mesenchymal stem cells in vitro fertilisation (IVF) 38, 73 childhood growth and development 76 genomic imprinting syndromes and 78 PGD 203 PGS 50, 202 saviour siblings see saviour siblings in vitro maturation of oocytes 74 Jepson v. the Chief Constable of West Mercia Police Constabulary 186–8, 189 Jeune’s asphyxiating dystrophy 136 joint dislocations, fetal 139 karyotype/phenotype discordance 29 karyotyping disorders of sex development 21 fetal see fetal karyotyping Kell (K) antigen, fetal typing 179–80 Kennedy disease 6 kidney donation, live, by children 67 Klinefelter syndrome (XXY) 4, 22, 23, 207 fertility treatment 30–1 Kniest dysplasia 137 knowledge, measurement 209–10 language barriers 207 large offspring syndrome (LOS) 74, 75 Larsen syndrome 139 Leber congenital amaurosis (LCA) 108, 165 lentivirus vectors 106, 107, 108 reversion to wild type 113–14 silencing 112 Lesch–Nyhan syndrome 184 Leydig cells 17, 20 life, worth/not worth living 183–6 lifestyle genetics 194 limb abnormalities, isolated 141, 142 lissencephaly, X-linked 23, 24 live organ donation by children 66–7, 68 saviour siblings 65–6 lobster claw hand 141, 142 locked nucleic acids (LNAs) 179 long QT syndrome 140 luteinising hormone (LH) 17, 18, 20 receptor mutations 3 resistance 24 lysosomal storage disorders fetal stem cell therapy 91 prenatal gene therapy 104, 108–9 male infertility 4–8, 218

Marfan syndrome 197 MASPIN gene 160–1 MassARRAY® system 175 mass spectrometry 166, 167, 175, 180 Mayer–Rokitansky–Küster–Hauser (MRKH) syndrome 1, 3 Meckel–Gruber syndrome 143 membranes, stem cells 86 menopause genetic influences 3 premature see primary ovarian insufficiency mental health, ground for termination of pregnancy 188, 190 mesenchymal stem cells (MSCs) 84–7, 88 adipose tissue (AT-MSCs) 85, 86 adult 84, 85, 88 amniotic fluid (AF-MSCs) 86–7 barriers to engraftment 92–3 cord blood 85, 86, 87 fetal (fMSCs) 84–7, 88 in utero transplantation 88, 89–92 production of clinical grade 93 Wharton’s jelly (WJ-MSCs) 87 see also fetal stem cell therapy; in utero transplantation (IUT) of stem cells MEST/Mest gene 75–6, 77 microarrays, PGD 35, 47, 52 microtubule-associated protein (MAPT) 155 Mill, John Stuart 62, 63–4 miscarriage recurrent 4 risk, and decision making 199–200 mixed gonadal dysgenesis 22, 24, 26 monosomy X see Turner syndrome mucopolysaccharidosis (MPS) type VII 108–9 Müllerian inhibiting substance (MIS) see antimüllerian hormone Multi-dimensional Measure of Informed Choice (MMIC) 209–13 multiple pregnancy non-invasive prenatal diagnosis 163, 168 PGD cycles 40 murine leukaemia virus (MLV) vectors 107, 112 muscular dystrophy 104 congenital 139 fetal stem cell therapy 89 myasthenia gravis 139 myeloablation, prior to stem cell therapy 92 myotonic dystrophy PGD 37–8 prenatal diagnosis 139, 165 myotubular myopathy 139

228  |  INDEX nail patella syndrome 139 nano-carriers 107, 117 National Institute for Health and Clinical Excellence (NICE) 178 National Institute for Health Research 196, 217 National Screening Committee (NSC), UK 147, 148–9 evaluation of screening programmes 208, 212 information provision 207 neural tube defects 207, 211 neuronal ceroid lipofuscinosis 111 next-generation technologies, evaluation 197, 198 Niemann–Pick disease 91 NOBOX mutations 3 non-invasive prenatal diagnosis (NIPD) 159–69, 197–8 aneuploidies 154–7, 163–7 consensus views 218, 220 ethics, education and counselling 168, 201–2 fetal blood group status 173–80 fetal sex determination 160–3, 168 limitations 168 parent perspective 200–1, 203–4 private services 203 single-gene disorders 163, 164–5 see also cell-free fetal DNA Noonan syndrome 133, 140 NR5A1 gene defects 24, 26 nuchal translucency (NT), increased, with normal karyotype 133, 136 nutrigenomics 194 oesophageal atresia 142 off-label treatment 127 oligohydramnios 139 oligonucleotides, array CGH 151–2 omphalocele 134–5 O’Neill, Onora 64 oocytes 19 superovulated, imprinting defects 75–6 in vitro maturation 74 oogonia 19 organ donation, live see live organ donation Organisation for Economic Co-operation and Development (OECD) 194 organ transplant recipients, fetal rhesus D typing 177 ornithine transcarbamylase deficiency 114 osteogenesis imperfecta (OI) antenatal diagnosis 136–7 fetal stem cell therapy 90–2, 93–4

postnatal stem cell therapy 91 osteopetrosis, fetal stem cell therapy 89 outcome measurement 208–9 ovarian stimulation 38 ovaries development 17, 19 development defects 25, 27–8 germ cell fate 19 primary insufficiency see primary ovarian insufficiency ovotesticular disorder of sex development (DSD) 26, 27–8 ovulatory infertility 1–2 P450 oxidoreductase (POR) deficiency 18, 25, 27, 28 P450 side-chain cleavage (P450scc) (CYP11A1) 18, 24, 27 palmarplantar hyperkeratosis 25 parents consideration of views on PGD 190–1 deciding on termination of pregnancy 187–90 freedom to determine child’s life 66 impact of child’s disability on 186 informed consent, choices and decisions 205–14 interests, termination of pregnancy and PGD 185–6, 191–2 perspective on prenatal diagnosis 199–204 reasons for having children 60, 61–5 partial androgen insensitivity syndrome (PAIS) 6, 27 paternity testing, prenatal 168 Peg3 gene 76 Pennings, G 60, 65–6, 68 PGD see preimplantation genetic diagnosis PGS see preimplantation genetic screening PHLDA2 gene 76, 77 phthalates 7 PLAC4 mRNA 166, 167 placenta confined mosaicism (CPM) 168 genomic imprinting and 76–7 stem cells from 84, 86 pleural effusions, fetal 140 pluripotent stem cells, inducible (iPS) 83, 84 POF1, 2 and 3 loci 3 polar body biopsy 38, 50 polycystic kidney disease adult 194, 195 prenatal diagnosis 143 polycystic ovary syndrome (PCOS) 2–3 polyhydramnios 139

INDEX  |  229 polymerase chain reaction (PCR) 37 digital 163, 166 non-invasive prenatal diagnosis 160, 164, 165 quantitative fluorescence (QF-PCR) 148–9, 154, 155 real-time fluorescence (f-PCR) 43, 160 real-time quantitative (RQPCR), fetal D testing 175, 176 postnatal test (Pennings), saviour siblings 60, 65–6 Prader–Willi syndrome 78 pregnancy-associated plasma protein A (PAPP-A) 141 preimplantation genetic diagnosis (PGD) 35–52, 203 chromosome rearrangements 47, 48–9 clinical service 35–6 consensus views 218–20 disability selection 52 ethical aspects of legal criteria 183–6, 190–2 funding 42–3 future prospects 52 late-onset disorders 51 non fully penetrant disorders 51–2 paediatric outcomes 41 process 37–40 reasons for requesting 36–7 regulation 41–2 selection of tissue-matched siblings 50–1, 59 sex selection 47–50 single-gene disorders 43–7 success rates 40–1 suitable conditions 42, 44–5 Preimplantation Genetic Diagnosis International Society (PGDIS) 36, 42, 43 preimplantation genetic haplotyping (PGH) 43–7, 50 preimplantation genetic screening (PGS) 35, 50, 202–3 consensus views 218 distinction from PGD 47, 50 paediatric outcomes 41 prenatal diagnosis (PND) chromosomal anomalies see chromosomal anomalies, prenatal diagnosis consensus views 217, 220 disorders of sex development 28–9 ethical concerns 203 evaluating outcomes 208–13, 220 legal criteria for termination of pregnancy 183–90, 191–2

parent perspective 199–204 PGD as alternative to 37 structural fetal anomalies 131–43 prenatal gene therapy 101–17 advantages 102–3 candidate diseases 103–5, 111–12 consensus views 217, 218–19 consent issues 127–8 delivery techniques 110–11, 114 duration of expression 112–13 ethical concerns 114–15 fetal and maternal immune responses 113 future 116–17 human application 115 in practice 115, 116 preclinical studies 107–9 reversion to wild type vector 113–14 safety of fetus, mother and future progeny 114 targeting to correct organ 109–11 vectors 105–7, 116–17 prenatal stem cell therapy see fetal stem cell therapy prenatal treatment congenital adrenal hyperplasia (CAH) 28–9, 162 consent issues 127–8 primary ovarian insufficiency (POS) 1–2, 3, 194 primordial germ cells (PGCs) 17, 18–19 genomic imprints 73 private services, non-invasive prenatal diagnosis 203 propionic acidaemia 165 psychological harms, saviour children 63 PTPN11 mutations 133 puberty 20 quality assurance, non-invasive fetal blood group testing 180 quality of care, evaluating 213–14 quality of life, judging future child’s 184–6 radial ray defects 141 RASSF1A gene 160–1, 177 receiver operating characteristic (ROC) curves 212 5α-reductase type 2 17 deficiency 25, 27 regional genetics centres, multidisciplinary 193 relative mutation dosage (RMD), digital 163 renal agenesis 142–3 renal anomalies, antenatal diagnosis 142–3

230  |  INDEX research consensus views 219–20 overlap with treatment 126–7 resource allocation, stem cell and gene therapies 128 resource inhibiting (RI) and enhancing (RE) genes 78 retinal guanylate cyclase-1 gene 108 retinal pigment epithelium-specific 65 kD protein (RPE65) gene 108 retinitis pigmentosa 165 retinoblastoma 195 RET mutations 142–3 retrovirus vectors 106, 107, 112 rhabdomyomas, cardiac 138 RHCE gene 174, 175, 179 RHD gene 174–5 rhesus (Rh) C (RH2) antigen, fetal typing 179 rhesus (Rh) c (RH4) antigen, fetal typing 179 rhesus (Rh) D antigen alloimmunisation 173, 174 invasive fetal testing 173–4 molecular basis of polymorphism 174–5 non-invasive fetal testing 174, 175–9, 218 alloimmunised pregnant women 175–7 assessing need for antenatal anti-D prophylaxis 178–9 internal controls 177–8 quality assurance 180 rhesus (Rh) E (RH3) antigen, fetal typing 179 risk, evaluating knowledge 209–10 Royal College of Obstetricians and Gynaecologists (RCOG) 187–8, 189, 190 RSPO1WNT4 gene defects 17, 24, 28 saviour siblings (HLA-matched siblings) 59–68 consensus views 218 harms to 62, 63, 67–8 Pennings’ postnatal test 60, 65–6 reasons for having 60, 61–5 selection using PGD 50–1, 59 third-party intervention requirement 60–1 treatment of 60, 65–7 value to parents 62–3 Sendai virus 105 Sequenom 175 serious condition criterion 183–4, 192 consensus views 220 interests of child vs parent 184–6

PGD 183, 190–1 termination of pregnancy 183, 187–90 Sertoli cells 17, 20 serum autologous 93 fetal bovine/calf 74, 93 severe combined immunodeficiency (SCID) fetal stem cell therapy 91 postnatal gene therapy 84, 107, 112 prenatal gene therapy 103, 104 sex ‘brain’ 16 chromosomal 16 fetal, determination 160–3, 168, 194 gonadal and phenotypic 17 reversal 140–1 selection, PGD 47–50 sex chromosome disorders of sex development (DSD) 21, 22, 23–6 sex development, human 16–20 disorders see disorders of sex development early postnatal changes 20 germ cell fate and gonad position 18–20 sheep, prenatal gene therapy 103, 108, 109, 110–11 Sheldon, Sally 185, 187 short-rib polydactyly syndromes 133, 136 SIDDT syndrome 24 single-gene disorders genetic test evaluation 194–7 non-invasive prenatal diagnosis 163, 164–5 PGD 43–7 single-nucleotide polymorphisms (SNPs) 151–2 Down syndrome 166, 167 single-gene disorders 164, 165 skeletal dysplasias 132, 133, 135–7 Slc22 genes 77 sleeping beauty (SB) transposons 107 SMCY gene 5 Smith–Lemli–Opitz syndrome 24, 140 Snrpn gene 75–6 SOX9 gene 17 defects see campomelic dysplasia duplication 4, 25 SPATA16 gene mutations 6 special non-invasive advances in fetal and neonatal evaluation (SAFE) network 180 spina bifida 185 spinal and bulbar muscular atrophy (SBMA) 6 spinal muscular atrophy 104 spondyloepiphyseal dysplasia congenita 137

INDEX  |  231 SRY gene 4, 16, 17 defects 24 fetal sex determination 160 translocation 25, 28 staff see healthcare professionals StAR see steroidogenic acute regulatory protein stem cell-based gene therapy 83 stem cells 83–7 stem cell therapy benefiting from an evil act 124–5 consent for fetuses and children 127–8 cost-effectiveness and resource allocation 128 ethical aspects 123–9 osteogenesis imperfecta 91 prenatal see fetal stem cell therapy reproductive effects 126 research–treatment overlap 126–7 treatment–enhancement overlap 127 see also bone marrow transplantation steroidogenesis 18 steroidogenic acute regulatory protein (StAR) 18, 24, 27, 140 steroidogenic factor 1 deficiency 24, 26 superovulation, genomic imprinting defects 75–6 talipes 136, 137, 138–9 Tay–Sachs disease 184–5 teratogenicity, virus vectors 114 teratomas ovarian 71 stem cell-related 83, 84 termination of pregnancy after 24 weeks 189–90 ethical aspects of legal criteria 183–90, 191–2 measuring attitudes to 210 parent perspective 200–1 PGD as means of avoiding 36–7 testes descent 19–20 determination 16 development 17 germ cell fate 18–19 postnatal function 20 steroidogenesis 18 undescended 23 testicular disorders of sex development (DSD) 27–8 testicular dysgenesis 7, 26 testicular germ cell cancer 7 testosterone 17, 18, 20 Tet-dependent transgene expression 113

tetralogy of Fallot 132 thalassaemia non-invasive prenatal diagnosis 163, 164 prenatal therapy 91, 103, 104 saviour sibling 59 thanatophoric dwarfism 137 Theory of Planned Behaviour 209 thrombocytopenia, neonatal alloimmune 180 tissue-matched siblings see saviour siblings torsion dystonia 165 total anomalous pulmonary venous drainage 138 tracheo-oesophageal fistula 142 transgenes immune responses 108, 113 regulation of expression 113, 117 see also gene therapy translocations, chromosome infertility 5 PGD 47, 48–9 Treacher Collins syndrome 132 trinucleotide repeats, infertility 3, 6 trisomies invasive prenatal diagnosis 147–57 non-invasive prenatal diagnosis 163–7 trisomy 4p 138 trisomy 13 133, 138 trisomy 18 134, 141 trisomy 21 see Down syndrome trophectoderm biopsy 38 TSPY gene, DSY14 marker sequence 159, 160 tubal infertility 1 tuberous sclerosis 138 Turner syndrome (monosomy X) 2, 3, 22, 23 fertility treatment 30–1 information provision 207 ultrasound detection 140 22q11.2 microdeletion syndrome 132, 138, 153 UK Genetic Testing Network (UKGTN) 193, 194–7, 217 ultrasound antenatal screening 131–43, 202, 211 11–13 week scan 133–5 18–20 week scan 135–43 guided injection, fetal gene therapy 110–11, 114 umbilical cord blood see cord blood mesenchymal stem cells 87 uniparental disomy (UPD) 71–2 USP26 gene variants 6–7

232  |  INDEX uterine abnormalities 1, 3, 139 UTY gene 5 VACTERL association 141, 142 values, measuring personal 210 vascular endothelial growth factor (VEGF), prenatal gene therapy 103–5, 111–12 vas deferens, congenital bilateral absence (CBAVD) 6, 36 vectors, gene immune responses 102, 113 insertional mutagenesis risk 112–13 maternal and fetal safety 114 organ-specific targeting 109 prenatal gene therapy 105–7, 116–17 reversion to wild type 113–14 silencing 112 WAGR syndrome 24 Wharton’s jelly mesenchymal stem cells (WJ-MSCs) 87 Whitaker saviour sibling case 59, 61, 63 Wilkinson, Stephen 185, 187 Williams, Glanville 187 Wilms’ tumour 135 WNT4/RSPO1 gene defects 17, 24, 28 women deciding on termination of pregnancy 187–90 risk to, as ground for termination of pregnancy 188, 190 see also parents WT1 gene defects 24, 26 45,X/46,XY mosaicism 24, 26 xenotransplantation 126 X-linked disorders cffDNA for fetal sex determination 160, 161–3 PGD 43, 44, 47–50 46,XX/46,XY karyotype 26 46,XX disorders of sex development (DSD) 21, 22, 25, 27–8 XX males 4, 25 46,XY disorders of sex development (DSD) 21, 22, 24–5, 26–7 Y chromosome 16 haplogroups 5 microdeletions 5–6, 218