APPROACHES TO IMPROVING EMBRYO IMPLANTATION by JUSTIN JAMIE CHU A thesis submitted to the University of Birmingham for the degree of DOCTOR OF PHILOSOPHY School of Clinical and Experimental Medicine, College of Medical and Dental Science, University of Birmingham, May 2016 University of Birmingham Research Archive e-theses repository This unpublished thesis/dissertation is copyright of the author and/or third parties. The intellectual property rights of the author or third parties in respect of this work are as defined by The Copyright Designs and Patents Act 1988 or as modified by any successor legislation. Any use made of information contained in this thesis/dissertation must be in accordance with that legislation and must be properly acknowledged. Further distribution or reproduction in any format is prohibited without the permission of the copyright holder. Abstract Embryo implantation represents a complex process vital in ensuring the normal development of pregnancy. Whether embryo implantation is the goal of natural conception or assisted reproductive treatment, the environment within the uterine cavity must be optimised in order to increase the chance of pregnancy. This thesis uses a mixture of research methods to investigate potential approaches to improving embryo implantation. Below are the key findings from this thesis: 1. The vitamin D status in women undergoing assisted reproductive treatment is important. An interventional trial would prove or disprove the merits of vitamin D deficiency treatment in these women. 2. There is not enough evidence to suggest a clear association between vitamin D and recurrent miscarriage, however there is a strong argument for biological plausibility. 3. The use of endometrial fluid collected at the time of embryo transfer in women undergoing assisted reproductive treatments for metabolomics analysis is possible. 4. Women with hydrosalpinx associated tubal infertility should be offered salpingostomy as a treatment option as the natural conception rates are similar to that achieved in in vitro fertilisation treatment. i Dedication I would like to dedicate this thesis to Anneke, Lily and Rolo – my wife, daughter and labradoodle who give me the greatest joy and happiness. I also dedicate this thesis to my parents, Paul and Sylvia Denham, who have supported me throughout my career and have been there for me in good times and in bad. ii Acknowledgments I wish to issue great thanks to Professor Arri Coomarasamy, who has been my supervisor, mentor and friend throughout my journey so far in medical research. I thank Ioannis Gallos for guiding me through my statistical analyses and enhancing my knowledge of research methodology. I also wish to thank Jackson Kirkman-Brown for his supervision and mentorship in the endometrial fluid metabolomics chapters included in this thesis. Thank you to Warwick Dunn and his metabolomics team at the University of Birmingham for the assistance and guidance with the endometrial fluid metabolomics work. I would like to acknowledge the kind team at the Birmingham Women’s Fertility Centre for allowing me to recruit patients and collect samples there. Thank you to Bee Tan, Abey Eapen and Hoda Harb who acted as second reviewers for the systematic reviews. Thank you to Rima Smith and Hoda Harb for helping with recruitment of patients into my cohort studies. Lastly, I would like to thank all of the patients that consented to participation in my studies. iii Abbreviations 1,25[OH]2D 1,25-dihydroxyvitamin D / Calcitriol 25[OH]D 25-hydroxyvitamin D / Calcidiol ART Artificial Reproductive Treatment BMI Body Mass Index CCG Clinical Commissioning Group ChRS Centre of Human Reproductive Sciences CI Confidence Interval CSF2 Colony Stimulating Factor-2 DIMS Direct Infusion Mass Spectrometry FET Frozen Embryo Transfer HFEA Human Fertility and Embryology Authority HOX10A Homeobox-leucine zipper protein 10 A ICSI Intra-cytoplasmic Sperm Injection IF-ϒ Interferon gamma IL Interleukin IQR Interquartile Range IVF In Vitro Fertilisation LC-MS/MS Tandem Liquid Chromatography Mass Spectrometry LIF Leukaemia Inhibiting Factor MeSH Medical Subject Heading NHS National Health Service NICE National Institute for Health and Care Excellence NRES National Research Ethics Service PCOS Polycystic Ovarian Syndrome PID Pelvic Inflammatory Disease SD Standard Deviation TNF-α Tumour Necrosis Factor-alpha TGF-β Transforming Growth Factor Beta iv UK United Kingdom VDBP Vitamin D Binding Protein v Contents APPROACHES TO IMPROVING EMBRYO IMPLANTATION ........................................................... i Abstract ....................................................................................................................................... i Dedication ................................................................................................................................... ii Acknowledgments ..................................................................................................................... iii Abbreviations ............................................................................................................................. iv List of Tables ............................................................................................................................ xiv List of Figures ............................................................................................................................ xv CHAPTER 1: THESIS INTRODUCTION .......................................................................................... 1 Embryo implantation .............................................................................................................. 2 Entry of the morula into the endometrial cavity ............................................................... 2 Blastocyst adherence and penetration .............................................................................. 3 The importance of the endometrium in embryo implantation ............................................. 5 Infertility as a disease ............................................................................................................. 7 Causes of infertility ................................................................................................................. 7 Treatment of infertility ........................................................................................................... 8 Fertility treatment funding ................................................................................................... 10 IVF and embryo implantation ............................................................................................... 10 Vitamin D and IVF treatment ................................................................................................ 12 Vitamin D and recurrent miscarriage ................................................................................... 13 vi The use of endometrial fluid in predicting IVF treatment outcome .................................... 13 Salpingostomy in the treatment of hydrosalpinx ................................................................. 15 Conclusions drawn ................................................................................................................ 16 CHAPTER 2: VITAMIN D AND ASSISTED REPRODUCTIVE TREATMENTS – SYSTEMATIC REVIEW AND META-ANALYSIS................................................................................................................ 17 Introduction .......................................................................................................................... 18 Methods................................................................................................................................ 21 Literature Search .............................................................................................................. 21 Study Selection ................................................................................................................. 22 Validity Assessment .......................................................................................................... 23 Statistical Analysis ............................................................................................................ 23 Results................................................................................................................................... 24 Study characteristics ......................................................................................................... 26 Clinical pregnancy ............................................................................................................. 35 Clinical pregnancy rates according to source of oocyte used .......................................... 37 Clinical pregnancy rates according to biofluid used to assess vitamin D status .............. 40 Live birth ........................................................................................................................... 40 Implantation ..................................................................................................................... 41 Miscarriage ....................................................................................................................... 44 Discussion ............................................................................................................................. 46 vii Summary ..............................................................................................................................