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Proquest Dissertations GENETIC ANALYSIS OF EMBRYONIC AND FETAL TISSUES by Pornpimol Ruangvutilert A thesis submitted for the degree of Doctor of Philosophy in the University of London July 2000 Department of Obstetrics and Gynaecology University College London ProQuest Number: U642216 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 U642216 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 Pornpimol Ruangvutilert Abstract ABSTRACT In this thesis, genetic analysis has been performed on embryonic and fetal material. Firstly, it was used to demonstrate the presence of fetal cells in the cervical mucus of pregnant women in the first trimester. Several studies have shown that fetal cells are present in the cervical canal but some reports have not identified these cells. In the present study, fetal genetic material was demonstrated in the cervical mucus from 66 of 193 (34%) women who underwent termination of pregnancy and from 7 in 37 (19%) on-going pregnancies. The polymerase chain reaction (PCR) was more efficient in detecting fetal cells than fluorescent in situ hybridisation (FISH). Secondly, FISH was used to determine the level of the trisomie cell line in different tissues, including placenta, from first and second trimester trisomie fetuses obtained from termination of pregnancy. Several studies have suggested that there are correlations between the degree of mosaicism in a chromosomally abnormal fetus, the severity of the disease and the chance of survival to term. The level of abnormal cells obtained from this study appeared not to be correlated with the clinical manifestations and the survival potential. As a side-line of this work, the efficiency of FISH on metaphase and interphase nuclei from skin fibroblast cultures from a trisomie and a triploid fetuses was determined. Finally, FISH was used to determine the mosaicism in human embryos on day 5 post-insemination, both arrested and blastocyst stage embryos. Mosaicism was found in 80% of the arrested embryos and 90% of the blastocysts. This may have an implication for preimplantation genetic diagnosis using blastocyst biopsy. In conclusion, FISH and PCR were used to study fetal genetic material in cervical mucus; FISH was used to study percentages of abnormal cell lines in trisomie fetal tissues and blastomeres from day-5 embryos. Pornpimol Ruangvutilert Acknowledgements ACKNOWLEDGEMENTS I would like to express my deep gratitude to my supervisors, Professor Charles H Rodeck and Dr Joyce C Harper, for their supervision, patience, help, guidance and support throughout my period as a PhD student. I extend my thanks to Professor Matteo Adinolfi and Professor Joy DA Delhanty for their additional help and advice. Also, I would like to acknowledge Dr Ray Noble, the Departmental Postgraduate Advisor (now also the Sub-Dean of Faculty of Clinical Sciences) for his support. I have been funded for the study by the Royal Thai Government and Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand. I am very grateful to Dr Jon K Sherlock, Dr Dagan Wells, Vincenzo Cirigliano, Clare M Conn, Agnes Greer, and Dr Margaret Fox for their patience and help in introducing me to the laboratory techniques and in answering my questions. Also, I owe a great deal to Dr Wirawit PiyamongkoTs wonderful computer skill. I would like to thank Dr Eric Jauniaux, Mr RHT Ward, Dr Robert Ogle, Dr Donald Peebles, all the staff of the operating theatre, Elizabeth Garrett Anderson Hospital and the staff of the Fetal Medicine Unit, University College Hospital for the cooperation in transcervical sample collection. I would like to thank Dr Gudrun E Moore, Gillian Bell and Katerina Chatzimeletiou, Queen Charlotte’s and Chelsea Hospital, Imperial College School of Medicine, London, for the samples and the initiation of fetal tissue study. I thank Mr Paul Serhal, Sally Marchant, Bhavna Raikundalia, Alpesh Doshi, Paula Almeida and all the staff of the Assisted Conception Unit, University College Hospital for the help in embryo samples. Also, I am grateful to Dr James Stanger, Lingard Fertility Centre, New South Wales, Australia for providing frozen-thawed embryos for FISH work up. My deep appreciation goes to Dr Natalie Greenwold, Dr Bianca Masturzo, Dr Karim Kalache and Dr Phakphum Phophong for helping me retrieve some patients’ clinical details from the database at the Antenatal Clinic, Fetal Medicine Unit and Assisted Conception Unit. Pornpimol Ruangvutilert Acknowledgements I acknowledge Dr Doreen Perera, Dr Susanne Cupisti, Dr Richa Sudd, Dr Puteri Baharuddin, Dr Rosemary Ekong, Dr Radma Mahmood, Angela Apessos, Patrick AbouSleiman, Antonis loulianos, Chun Kai Chen, Barbara Smith, Susana Tamaz and Mara Simoupoulou for their friendship and help. With regard to statistical aspect, I am in debt to Ms Mai Stafford, Department of Epidemiology and Public Health, UCL Medical School for her valuable advice. I also thank Dr Yongyut and Dr Vorapan Sirivatanauksorn for additional help in my writing up. I would also like to thank Mrs Irene Hopton-Scott and her team in the Departmental Office for the administration and financial matters. In particular, I wish to express my deep gratitude to all the patients for their cooperation and participation in the study. Pornpimol Ruangvutilert Dedication To my late parents, my dearest brother and sister, and all the patients Pornpimol Ruangvutilert Table of contents TABLE OF CONTENTS ABSTRACT 2 ACKNOWLEDGEMENTS 3 DEDICATION 5 TABLE OF CONTENTS 6 LIST OF TABLES 13 LIST OF FIGURES 16 ABBREVIATIONS 18 CHAPTER 1 INTRODUCTION 1.1 CHROMOSOME ABNORMALITIES 23 1.1.1 NUMERICAL ABERRATIONS 23 1.1.1.1 Polyploidy 23 1.1.1.2 Aneuploidy 24 1.1.2 STRUCTURAL ABERRATIONS 30 1.1.3 OTHER ABERRATIONS 33 1.2 PRENATAL SCREENING 36 1.2.1 BIOCHEMICAL TESTS 36 1.2.1.1 Screening for neural tube defects 36 1.2.1.2 Screening for chromosome abnormalities 37 1.2.2 ULTRASONOGRAPHY 40 1.2.2.1 Ultrasound markers for aneuploidies in the second trimester 40 1.2.2.2 Ultrasound markers for aneuploidies in the first trimester 42 1.3 EMBRYOLOGY RELATED TO PRENATAL AND PREIMPLANTATION GENETIC DIAGNOSIS (PGD) 44 1.3.1 PREIMPLANTATION EMBRYONIC DEVELOPMENT 44 1.3.2 THE AMNIOTIC AND COELOMIC (CHORIONIC) CAVITIES 46 1.3.3 THE CHORIONIC VILLI 53 1.4 PRENATAL DIAGNOSIS 57 1.4.1 AMNIOCENTESIS 57 1.4.2 CHORIONIC VILLUS SAMPLING (CVS) 60 1.4.2.1 Confined placental mosaicism 62 1.4.3 FETAL BLOOD SAMPLING (FBS) 64 Pornpimol Ruangvutilert Table of contents 1.5 LABORATORY TECHNIQUES 67 1.5.1 KARYOTYPING 67 1.5.2 FLUORESCENT /A S’/YU HYBRIDISATION (FISH) 68 1.5.2.1 Probe labelling (nick translation) 69 1.5.2.2 Fluorescent in situ hybridisation (FISH) 69 1.5.3 POLYMERASE CHAIN REACTION (PCR) 73 1.6 NON-INVASIVE PRENATAL DIAGNOSIS 77 1.6.1 FETAL CELLS IN MATERNAL BLOOD CIRCULATION 77 1.6.2 FETAL CELLS IN TRANSCERVICAL CELL (TCC) SAMPLES 82 1.7 PREIMPLANTATION GENETIC DIAGNOSIS (PGD) 87 1.7.1 IN VITRO FERTILISATION (IVF) 87 1.7.2 OBTAINING CELL(S) FOR ANALYSIS 88 1.7.2.1 Cleavage stage biopsy 88 1.7.2.2 Polar body biopsy 88 1.7.2.3 Blastocyst biopsy 89 1.7.3 GENETIC ANALYSIS IN PGD 90 1.8 AIMS OF STUDIES IN THE THESIS 91 1.8.1 PRESENCE OF FETAL CELLS IN TCC SAMPLES 91 1.8.2 FISH ANALYSIS ON TRISOMIC FETAL TISSUES 91 1.8.3 FISH ANALYSIS ON DAY-5 EMBRYOS 91 Pornpimol Ruangvutilert Table of contents CHAPTER 2 MATERIALS AND METHODS 2.1 MATERIALS 94 2.2 POLYMERASE CHAIN REACTION (PCR) 99 2.2.1 DNA EXTRACTION 99 2.2.1.1 DNA extraction from maternal blood 99 2.2.1.2 DNA extraction from the placenta (method 1) 100 2.2.1.3 DNA extraction from the placenta (method 2) 101 2.2.1.4 DNA extraction from TCC samples 101 2.2.2 PCR PROCEDURE 101 2.2.2.1 PCR for the amelogenin region 104 2.2.2.2 PCR for short tandem repeats (STRs) 105 2.2.3 PCR ANALYSIS 105 2.2.3.1 Gel electrophoresis 105 2.2.3.2 A utomated laser DNA analyser (ABI Prism ™310) 106 2.3 FLUORESCENT IN SITU HYBRIDISATION (FISH) 107 2.3.1 SAMPLE AND CONTROL LYMPHOCYTE PREPARATION 107 2.3.1.1 Placental tissue and TCC preparation 107 2.3.1.2 Fetal tissue preparation 108 2.3.1.3 Day-5 embryo spreading 109 2.3.1.4 Control lymphocyte culture 112 2.3.1.5 Slide preparation 113 2.3.2 NON-COMMERCIAL PROBES 114 2.3.2.1 DNA preparation and purification 114 2.3.2.2 Probe labelling (Nick translation) 116 2.3.3 COMMERCIAL PROBES 118 2.3.4 FISH PROCEDURE (Non-commercial probes) 121 2.3.5 FISH PROCEDURE (Commercial probes) 122 Pornpimol Ruangvutilert Table of contents CHAPTER 3 FETAL CELLS IN TCC SAMPLES 3.1 INTRODUCTION 125 3.2 MATERIALS AND METHODS 129 3.2.1 PATIENTS AND SAMPLE COLLECTION 129 3.2.1.1 Collection by the Pipelle catheter 129 3.2.1.2 Collection by lavage and syringe aspiration 131 3.2.1.3 Collection from on-going pregnancies 131 3.2.2 FISH AND PCR 131 3.2.2.1 FISH 131 3.2.2.2 PCR for the amelogenin region 132 3.2.2.3 PCRfor the STRs 132 3.3 RESULTS 136 3.3.1 RESULTS FROM TERMINATION OF PREGNANCY (TOP) CASES 136 3.3.1.1 Cases with FISH results 13 7 3.3.1.2 Cases with PCR for the amelogenin alone 140 3.3.1.3 Patterns o f fetal DNA evaluation using STR markers
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