Molecular Cytogenetic Investigations into Infertility, Recurrent In Vitro Fertilisation failure and Multiple Miscarriage. by Calum Sinclair A thesis submitted for the degree of Doctor of Philosophy in the University of London November 1997 The Galton Laboratory Human Genetics Group Department of Biology University College London ProQuest Number: U643079 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest. ProQuest U643079 Published by ProQuest LLC(2015). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code. Microform Edition © ProQuest LLC. ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 Abstract This thesis is the cytogenetic study of three patient groups with different reproductive problems. The first patient group consisted of couples suffering from three or more miscarriages, who had previously been diagnosed as cytogenetically normal by a diagnostic laboratory. The group was analysed in greater depth, alongside fertile controls, for the presence of a low level mosaic 45,X cell line. Forty metaphase cells were analysed by G banding, and 450 interphase cells were analysed by FISH using a biotinylated chromosome X specific centromeric probe. The patient group consisted of thirteen couples and seven females, and the control group of fifteen couples. G-band analysis revealed more cells with sex chromosome aneuploidy from the females in the patient group than the females in the control group (weakly significant p<0.1). FISH analysis revealed a much stronger correlation between the patient group and XO mosaicism (p<0.05). When patients and controls were directly compared in a paired manner, 6 patients were identified as being XO mosaics. If this selection was graphically based (figure 5) the number of mosaic patients increased to nine. The percentages of XO cells from the patient group did not correlate with age, whereas there was an age dependant correlation in the control group. The second study was on patients who have had three or more failed I VF. attempts Cultured peripheral lymphocytes were analysed from the patient group (twenty two couples and twenty one females) and the control group (ten couples and five females). There were no constitutional abnormalities found, however three patients were found to be mosaic for three different abnormal cell lines (45,X, 47,XX+11 and 47,XX+ 18). Forty six preimplantation embryos (4 to 8 cell stage) obtained from the patient group were analysed, by FISH, using DNA probes for chromosomes 1, X and Y. 62.5% of these embryos were abnormal, with no embryos with constitutional abnormalities observed. The third study consisted of three couples carrying different balanced translocations. Probes were sought and evaluated for the possibility of preimplantation diagnosis using FISH. Suitable probes were found for two of the couples. Analysis of spare preimplantation embryos from one of the couples using the appropriate probes revealed all embryos to be chaotic. Acknowledgements I would like to thank my supervisors Drs Joy Delhanty and Jenny Farrington for their invaluable help both in the lab and in the preparation of this thesis. This work has been funded by the Birth Defects Foundation. I would also like to express my gratitude to Clare Conn, for her preimplantation diagnosis work, and all others who helped from lab 208. Many thanks are owed to the UCLH cytogenetic laboratory. I would like to thank both the patients and staff of the Hammersmith I VF clinic for the donation of embryo and blood samples used in this work. Particular thanks to Debbie Taylor for her help gathering histories and Drs Lass and Skull for organising the IVF samples. There are numerous statistics in this thesis and these would not have been possible without the kind help and infinite wisdom of Dr Santibanez-Koreb and Prof. Joliffe. I would also like to thank my family for their understanding in the past year and in particular, Kirsty for her help, support, understanding and patience. TABLE OF CONTENTS ABSTRACT 2 ACKNOWLEDGEMENTS 4 TABLE OF CONTENTS 5 LIST OF FIGURES 8 LIST OF TABLES 9 LIST OF ABBREVIATIONS 11 INTRODUCTION 1.1. REPRODUCTIVE FAILURE 14 1.1.1. The cytogenetics of reproductive success in normal couples 14 1.1.2. Infertility- incidence and causes 15 1.1.3. In Vitro Fertilisation (IVF) 16 1.1.4 Spontaneous Abortion- incidence and causes 17 1.1.5. Multiple miscarriage- incidence and causes 18 1.2. CYTOGENETICS OF REPRODUCTIVE FAILURE. 20 1.2.1. Cytogenetic T echniques. 20 1.2.1.1. Culture and metaphase spreads 20 1.2.1.2. Chromosome B anding 21 1.2.1.3. Molecular Cytogenetics 24 1.2.1.3.1. History 25 1.2.1.3.2 Fluorescent detection system 25 1.2.1.3.3. DNA Probes and their applications. 26 1.2.1.3.4. Applications of FISH technology 27 1.2.1.3.4.1 Interphase FISH. 27 1.2.1.3.4.2 Metaphase FISH 28 1.2.1.3.4.3 Mapping and Fibre FISH 29 1.2.2. Cytogenetic abnormalities 30 1.2.2.1. Aneuploidy 30 1.2.2.1.1. Turner syndrome (45,X) 31 1.2.2.1.1.2 History 31 1.2.2.1.3. Mechanism of loss 32 1.2.2.1.4. Prenatal survival 32 1.2.2.1.5. Reproductive Physiology of Turner Syndrome. 34 1.2.2.1.6. The role of Turner Syndrome in Reproductive Failure 36 1.2.2.2. Mosaicism 38 1.2.2.2.1. Phenotypic effect and uniparental disomy 38 1.2.2.2.2 Low level mosaicism/ single aneuploid cells 41 1.2.3. Stmctural Abnormalities 43 1.2.3.1. Deletions 43 1.2.3.2. Insertions and duplications 43 1.2.3.3. Inversions 44 1.2.3.4. Translocations 44 1.2.3.5. Incidence 45 1.2.3.6. Mitosis and Meiosis 46 1.2.3.6.1 Mitosis 46 1.2.3.6.2. Meiosis 47 1.2.3.7. Effect on Reproduction 49 1.2.3.8. Pachytene Segregation Patterns 50 1.2.3.9. The role of structural rearrangements in reproductive failure 53 1.2.4. Cytogenetic studies in reproductive failure. 55 1.2.4.1. Infertility 55 1.2.4.2. In Vitro Fertilisation 56 1.2.4.3. Fetal loss 57 1.3. PREIMPLANTATION GENETIC DIAGNOSIS (PGD) 62 1.3.1. Approaches 62 1.3.2. Chromosomal Mosaicism in Embryos 63 1.4. AIMS 66 1.4.1. Multiple Miscarriage study 66 1.4.2. Recurrent IVF failure study 66 1.4.3. Preimplantation Diagnosis for carriers of chromosome 66 translocations MATERIALS AND METHODS 2.1 SAMPLE PREPARATION 68 2.1.1 Lymphocyte preparation 68 2.1.1.1. Culture 68 2.1.1.2. Harvest 68 2.1.1.3. Slide preparation 69 2.1.2 Embryo preparation 69 2.1.2.1. Oocyte collection and embryo culture 69 2.1.2.2 Embryo spreading and slide preparation 69 2.2 CHROMOSOME VISUALISATION 70 2.2.1 Giemsa banding 70 2.2.2. DNA probes for Fluorescent in situ hybridisation 71 2.2.2.1 Probe construction and extraction 71 2.2.2.2. E.coli transformation 71 2.2.2.3. Cosmid and Plasmid maxipreps 72 2.2.2.4. Yac minipreps 72 2.2.2.5. Measuring DNA Concentration 73 2.2.2.6. ALU Polymerase Chain Reaction (PCR) 74 2.2.2.1. Indirect labelling of probes 74 2.2.2.8. Direct labelling 74 2.2.2.9. Probe precipitation and in situ suppression 75 2.2.3. Fluorescent in situ hybridisation 76 2.2.3.1. Pre-hybridisation slide treatment 76 2.2.3.2. Dénaturation 76 2.2.3.3. Hybridisation 77 2.2.3.4. Post Hybridisation washes 77 2.3 STUDY PROTOCOL 79 2.3.1. Multiple miscarriage study 79 2.3.1.a Multiple miscarriage Study Controls 79 2.3.2. Recurrent IVF failure study 79 2.3.2.a I.V.F. Study Controls 80 RESULTS 3.0.1 STUDY NOMENCLATURE 82 3.1 MULTIPLE MISCARRIAGE STUDY 83 3.1.1. Description of results 83 3.1.2. G-band results 89 3.1.3. FISH results. 94 3.1.4. Summary of results 100 3.2. RECURRENT IVF FAILURE STUDY 101 3.2.1 Number of samples successfully tested 101 3.2.2. G-band results 101 3.2.3. Embryo Results 112 3.2.4. Summary of embryo results 126 3.3.1 PREIMPLANTATION GENETIC DIAGNOSIS STUDY 127 3.3.2. Preimplantation Diagnosis Case 1 (PD 1 127 3.3.3. Preimplantation Diagnosis Case 2 (PD 2) 132 3.3.4. Preimplantation Diagnosis Case 3 (PD 3) 141 3.3.5. Summary of Results 144 DISCUSSION 4.1 MULTIPLE MISCARRIAGE (MM) STUDY 146 4.1.1. G-band analysis 146 4.1.2. FISH analysis 148 4.1.3. Age related aneuploidy 149 4.2 RECURRENT IVF FAILURE STUDY 153 4.2.1. G-band analysis 153 4.2.2. Mosaicism in peripheral lymphocyte cultures 153 4.2.3. Analysis of preimplantation embryos 154 4.2.4. Cell division cycle (cdc) checkpoints 157 4.2.5. Embryos with XX and XY nuclei 159 4.3. PREIMPLANTATION GENETIC DIAGNOSIS 162 CONCLUSIONS 5.1 Multiple Miscarriage Patients 165 5.2.