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Identifying Biomarkers for Non-Invasive Diagnosis of Endometriosis

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Identifying Biomarkers for Non-Invasive Diagnosis of Endometriosis Identifying biomarkers for non-invasive diagnosis of endometriosis Stella Irungu Department of Women’s Cancer Institute for Women’s Health University College London PhD in Biomarker Discovery and Proteomics This thesis is submitted in partial fulfilment of the requirements for the degree of Doctor of Philosophy from University College London I, Stella Irungu confirm that the work presented in this thesis is my own. Where information has been derived from other sources, I confirm that this has been indicated. 2 Abstract Endometriosis is a gynaecological disorder occurring when endometrial cells are shed through the fallopian tubes and implant on surfaces in the abdomen and pelvis. There they form lesions that respond to hormones of the cycle and stimulate inflammation. Women with endometriosis experience painful debilitating periods, pain on intercourse and defecation, and may have difficulties conceiving. It is a common disorder, affecting 5-10% of women of reproductive age. Diagnosis of endometriosis is difficult and is often delayed by 5-11 years. Symptoms do not correlate with disease severity and imaging techniques are only sensitive for diagnosing ovarian endometriomas. Definitive diagnosis is surgical, requiring laparoscopy under general anaesthetic, exposing patients to potentially serious complications. With these facts in mind, the aim of this project was to identify biomarkers for the non-invasive diagnosis of endometriosis. This was achieved by defining the protein expression profiles of tissue samples collected from women diagnosed with endometriosis and from control patients who underwent surgery for investigation of chronic pelvic pain or who underwent prophylactic surgery because of familial cancer history. Discovery work involved the use of complementary, quantitative proteomic profiling by 2D difference gel electrophoresis and multiplex mass tagging linked to liquid chromatography-based separation and tandem mass spectrometry. Selected candidate biomarkers (LUM, CPM, TNC, TPM2 and PAEP) were verified using ELISA in serum samples collected from the same women. Biomarkers reported in the literature were also tested. Diagnostic performance of each marker was established. The best single marker in discriminating endometriosis and controls was CA125 (AUC=0.724, P=0.002). Multi-marker models were also constructed and the best model in discriminating between endometriosis and healthy controls by cross-validation was CA125, ICAM (AUC=0.744). CA125, ICAM, FST model (AUC=0.75) gave the performance in discriminating between endometriosis and both controls by cross-validation. 3 Acknowledgements Firstly, I would like to express my sincere gratitude to my supervisor Dr John Timms for his continuous patience, guidance and support throughout my PhD research and writing of this thesis. I would also like to thank my secondary supervisor Mr Ertan Saridogan and Dr Dimitri Mavrelos for sharing their knowledge and expertise in endometriosis research. I am immensely grateful to Dr Jenny Worthington for her valuable practical assistance in mass spectrometry and for her patience and encouragement for the most part of my research. To all the staff of the UCLH Reproductive Medicine Unit, thank you for the assistance during patient recruitment and sample collection. To my colleagues at the proteomics lab (Joy, Harry, Oleg, Richard and Tendayi), thank you for your friendship and support. I would also like to express my special gratitude to Prof Sam Kariuki (KEMRI-CMR) who encouraged me to pursue a PhD. My sincere gratitude also goes to the Commonwealth Scholarship Commission, whose funding enabled me to undertake my research. Last but not least, I would like to thank my parents Mwangi Irungu and Jane Irungu for their unconditional support and for being a constant source of encouragement and inspiration throughout my many years of studying. To my brother Dr Eric Irungu, thank you for the unwavering encouragement and support especially the late nights spent on skype editing parts of this thesis. To my sister Nana Irungu, thank you for being a constant source of love and laughter. Finally, I would like to express my sincere gratitude to my friends Zilper, Christine, Maria, Rehema, Caroline, Cynthia, Justin, Job and Brian for the emotional support as well as the fun and happy memories. A special thank you goes to Lizah, Debbie and Anne. Words alone are not enough to express how grateful I am for the kindness and support you have shown me. 4 TABLE OF CONTENTS Abstract .................................................................................................................................. 3 Acknowledgements .............................................................................................................. 4 List of tables ......................................................................................................................... 12 List of figures ....................................................................................................................... 14 Abbreviations ...................................................................................................................... 16 CHAPTER ONE: INTRODUCTION ................................................................................ 18 1.1 Endometriosis- A history of the disease ....................................................................... 18 1.1.1 Vincent Knapp .......................................................................................................... 18 1.1.2 Carl von Rokitansky ................................................................................................. 19 1.1.3 Thomas Cullen .......................................................................................................... 20 1.1.3 John Sampson ........................................................................................................... 20 1.2 Disease characterisation ................................................................................................ 21 1.2.1 Peritoneal endometriosis........................................................................................... 21 1.2.2 Ovarian endometriosis .............................................................................................. 23 1.2.3 Deep infiltrating endometriosis (DIE) ...................................................................... 24 1.3 Pathogenesis ................................................................................................................... 25 1.3.1 Transplantation theory .............................................................................................. 25 1.3.2 Retrograde menstruation........................................................................................... 25 1.3.3 Lymphatic/haematogenous spread (Halban’s theory). ............................................. 28 1.3.4 Iatrogenic transplantation ......................................................................................... 29 1.3.5 In situ development theory ....................................................................................... 29 1.3.6 Coelomic Metaplasia Theory.................................................................................... 30 5 1.3.7 Immune abnormalities .............................................................................................. 31 1.4 Epidemiology .................................................................................................................. 33 1.5 Risk factors..................................................................................................................... 34 1.5.1 Genetic risk ............................................................................................................... 34 1.5.2 Demographic risk factors.......................................................................................... 35 1.5.3 Menstrual factors ...................................................................................................... 36 1.5.4 Oral contraceptive use .............................................................................................. 36 1.5.5 Family History .......................................................................................................... 37 1.5.6 Environmental factors............................................................................................... 37 1.5.7 Diet and other lifestyle factors .................................................................................. 37 1.6 Diagnosis ......................................................................................................................... 38 1.6.1 Surgical diagnosis ..................................................................................................... 38 1.6.2 Laparoscopy ............................................................................................................. 38 1.6.3 Imaging techniques ................................................................................................... 40 1.7 Treatment of endometriosis .......................................................................................... 41 1.7.1 Medical therapies for endometriosis ......................................................................... 41 1.7.1.1 Danazol .................................................................................................................. 42 1.7.1.2 Non-steroidal anti-inflammatory drugs ................................................................
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