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Minimally Invasive Prenatal Diagnosis of Inherited Disorders Employing Trophoblastic Cells Shed Into the Endocervical Canal

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Minimally Invasive Prenatal Diagnosis of Inherited Disorders Employing Trophoblastic Cells Shed Into the Endocervical Canal Minimally invasive prenatal diagnosis of inherited disorders employing trophoblastic cells shed into the endocervical canal. By Jon Kelvin Sherlock A thesis suhmittedfor the degree of Doctor of Philosophy at the University of London October 1998 The Galton Laboratory Departments of Biology and Obstetrics & Gynaecology University College London ProQuest Number: U644346 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 U644346 Published by ProQuest LLC(2016). 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 To Mum, Dad and Bob Acknowledgements Where to begin? If I were to mention by name all those who had helped, inspired and encouraged me during my scientific pursuits my acknowledgements would be longer than the thesis itself. Several people however stand out in my mind as having been essential to the completion of my doctorate. Firstly to my parents and brother; Lynne, Mike and Bob Sherlock. Without the love and encouragement of these, the most important people in my life, I doubt I’d even bother getting out of bed in the morning. From being motivated to learn from an early age, to my parents financial support allowing me to attend University, every step of my path to this achievement has been made possible by them. I really can’t thank you all enough. My very first thoughts of a career in science I owe to Professor Joy Delhanty and this is something for which I will be eternally grateful. As an undergraduate, my third year project, supervised by Prof. Delhanty, gave me the first taste of scientific research and was the basis of my subsequent placement as a Research Assistant at Guys Hospital. It is quite fitting that, by chance, I should return to her laboratory and embark upon this thesis at her suggestion and under her supervision. From the point I left University up to the present day my scientific mentor has been Professor Matteo Adinolfi who has taken me under his wing for the last 5 years, pushing me when required, chastising when necessary, but always encouraging. His love of science which is so evident, coupled with his diverse interests in so many other walks of life have been an inspiration to me. I could honestly not have wished for a fairer, more motivating boss than Prof., who has left me an infmitesimally better scientist than when he found me. In addition, without his guidance I would no doubt still be suffering from the delusion that Brighton and Hove Albion were a credible football team. I would also like to extend my thanks to Professor Charles Rodeck, who has not only looked after me financially with the continued procurement of grants, but has also been a great help with my research. For one so busy and important, I still wonder at how he can maintain such an approachable and warm disposition. This, together with his fantastically broad knowledge of science and medicine have made Prof. Rodeck a pleasure to work for. No matter how good the quality of one’s supervisors I do not believe that anyone could happily complete a research project without the support and friendship of other people in the laboratory. My sanity would long have departed if it were not for such comrades. I would particularly like to thank Dr. Dagan Wells who has been far more than just a friend and councillor. I consider myself privileged to have met, worked alongside, (and finally published with!) such a superb scientist and person. Cheers fella. The work in this thesis could not have been completed were it not for my numerous collaborators over the years. Starting with Angela and Barbara at Guys Hospital, then Boris and Ashutosh, and finally Molly and Vincenzo. It has been a real pleasure to work so closely with you all. I would like to also mention all my other colleges who have put up with all my moods (usually on a Monday morning after the latest BHA trouncing), helped me with problems and given me their friendship for which I am truly grateful; Dr. Joyce Harper, a friend and confident who had been so instrumental in my development as a scientist, Antonis, for his endless patience bearing the brunt of my taunts, Molly for her unfailing happiness and kindness, Vincenzo for his ideas and hard work and all others in our multicultural lab who have contributed to the enjoyable atmosphere over the years. I’d like also to thank Prof. Yvonne Edwards for her printer, Mark Griffin who somehow managed to plod through all this bumf to proof read it for me, and Shelley for her love and support. Lastly, I’d like mention my late Nan, who I know would have been extremely proud, and also my later Granddad ‘Fishes’, a man fascinated by science, who had the forethought to instigate the family tradition of Kelvin as a middle-name. It’s just a shame I didn’t get into low temperature physics. Abstract Fetal (trophoblastic) cellular elements have been detected in transcervical cell (TCC) samples collected from the uterine cavity and cervical canal of pregnant women between 6 and 15 weeks of fetal gestation. These cells, present in a background of maternal material, have been identified utilising various molecular techniques. Chromosome Y specific DNA sequences have been detected using both fluorescent in situ hybridisation (FISH) and polymerase chain reaction (PGR) assays. Fetal specific short tandem repeat (STR) allele sizes have been identified in TCC samples indicating the presence of fetal DNA. Trophoblast cells have been identified in TCC samples using monoclonal antibodies specific for trophoblast antigens. Using these methods various TCC sampling procedures have been compared for their efficiency of fetal cell retrieval. The safety of TCC sampling has also been assessed by the collection of samples from ongoing pregnancies prior to CVS, and comparison with a control group. The fetal Rh(d) type has been successfully diagnosed from TCC samples collected from Rh(d) negative mothers. Fetal genetic errors have been identified including trisomy 18, trisomy 21, XYY and triploidy. In attempts to identify recessive fetal disorders and X linked diseases, the isolation of trophoblastic elements has been attempted. Numerous methods have been employed to this end including micromanipulation, trophoblast specific mRNA detection, and magnetic activated cell sorting using trophoblast specific antibodies. Multiplex quantitative fluorescent PCR techniques have been developed to test these isolated cell clumps and individual cells for various inherited disorders. These include the detection of sex chromosome complement, chromosome aneuploidies, the delta-F 508 three base pair deletion causing cystic fibrosis, the single base change causing sickle cell anaemia, and a single base change and two deletions causing beta-thalassaemia. To test separated trophoblast cells, and with a view to potential preimplantation diagnosis, large numbers of isolated single cells were tested with these methods and the results assessed. Table of Contents Title 1 Dedication 2 Acknowledgements 3 Abstract 4 Table of Contents 5 List of Tables 16 List of Figures 17 Abbreviations 19 Chapter 1 INTRODUCTION. 21 Part 1. Prenatal Diagnosis 21 1.1. Invasive Prenatal Diagnosis Methods 22 1.1.1 Amniocentesis 22 1.1.1.1 Background 22 1.1.1.2 Risks & Drawbacks 25 1.1.1.2.a. Procedure related pregnancy loss 25 1.1.1.2.b. Additional Fetal risks 25 1.1.1.2 c. Time of the procedure 26 1.1.1.3. Early amniocentesis 26 1.1.1.4. Filter amniocentesis 29 1.1.2. Chorionic villus sampling (CVS) 30 1.1.2.1. Background 30 1.1.2.2. Risks 32 1.1.2.2.a. Procedure Related Abortions 32 1.1.2.2.b. Bleeding 32 1.1.2.2.C. Infection 33 1.1.2.2.d. Rupture of membranes 33 1.1.2.2.e. Rhesus Sensitisation 33 1.1.2.2.f. Placental Mosaicism 33 1.1.2.2.g. Maternal contamination 34 1.1.2.2.h. Fetal Abnormalities 35 1.1.2.3. Transverse versus Transabdominal CVS. 36 1.1.3. Fetal chord blood sampling 36 1.1.4. Fetal biopsy 37 1.1.5. Coelocentesis 37 1.2. Existing Non-invasive Prenatal Diagnostic methods 39 1.2.1. Ultrasound scanning 39 1.2.2. Biochemical screening 40 Part 2. Development of the Placenta 43 1.3. Implantation. 43 1.4. The uterus 46 1.5. The Placenta 46 1.5.1. Chorionic villi 47 1.5.2. Cell columns and cell islands 48 1.5.3. Intervillous space 52 1.5.4. Uteroplacental circulation 52 1.5.5. Chorionic plate 53 1.5.6. Trophoblasts at the decidual basalis 54 1.5.6.1. Syncytial knots 54 1.5.6.2. Syncytial sprouts 54 1.5.7. Trophoblasts at the decidual capsularis 55 Part 3. Fetal Cells in the Maternal Peripheral Blood Circulation 57 1.6. Studies on whole blood. 59 1.7. Trophoblasts at the site of the decidua basalis 62 1.8. Fetal leukocytes in the maternal peripheral blood circulation 69 1.8.1. Lymphocytes 69 1.8.1.1. Cultures 69 1.8.12. Quinacrine mustard staining 69 1.81.3. Paternal HLA selection 70 1.8.2.
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