Application of Minimally-Invasive Uterine Fluid Aspiration to Identify Candidate Biomarkers of Endometrial Receptivity through a Transcriptomic Approach by Crystal Chan A thesis submitted in conformity with the requirements for the degree of Master of Science Institute of Medical Science University of Toronto © Copyright by Crystal Chan 2012 Application of Minimally-Invasive Uterine Fluid Aspiration to Identify Candidate Biomarkers of Endometrial Receptivity through a Transcriptomic Approach Crystal Chan Master of Science Institute of Medical Science University of Toronto 2012 Abstract The endometrium is receptive to the embryo during a restricted window in the mid-secretory phase. My objectives were to develop a minimally-invasive endometrial sampling method for gene expression profiling, and to identify genes differentially expressed in the receptive phase. Twenty-three normo-ovulatory women underwent uterine fluid aspiration during the pre- receptive (LH+2) and receptive (LH+7) phase of the same natural cycle. RNA was extracted, reverse transcribed, amplified and hybridized to whole-genome microarrays. Unsupervised hierarchical clustering revealed self-segregation of pre-receptive and receptive samples. Importantly, profiling by uterine fluid aspiration was representative of biopsy. An unpaired t-test with a false discovery rate of 0.05 and a Δ threshold of 4-fold identified 245 unique transcripts as differentially expressed in the receptive phase. NanoString analysis validated 96% of these genes. This approach will now allow us to correlate expression of these candidate biomarkers to implantation outcomes, towards the development of clinical assays predictive for endometrial receptivity. ii Acknowledgments I would first and foremost like to thank Dr. Ted Brown and Dr. Ellen Greenblatt, supervisors extraordinaire, who gave me the resources, inspiration, and unconditional support to transform this research from a pipe dream to reality. Early on in my residency training, I did not envision taking two years off my clinical training to pursue a Master’s degree. I also could not foresee that I would discover such a passionate interest in Reproductive Endocrinology and Infertility. Dr. Greenblatt and Dr. Brown were instrumental in helping me to define my academic goals, explore my interests, and complete a successful research project. Their supervision was invaluable, and words cannot fully express my gratitude. My sincere appreciation goes out to my Program Advisory Committee members, Dr. Robert Casper and Dr. Stephen Lye, who were supportive yet challenging enough to help spur my research forward. Thank you for asking me tough questions and training me to think on my feet. I am also grateful to Dr. Lye for believing in my research and contributing to the final validation steps. I would also like to thank the “Genome Canada” crew – Carl Virtanen, Neil Winegarden, Natalie Stickle, Julie Tsao, Dr. Allan King, and Dr. Pavneesh Madan. The level of bioinformatic support from Carl was absolutely above and beyond, except for the time BBM was down. Carl made bioinformatics make sense, and though the learning curve was steep and slippery, I am extremely grateful for all his teaching. Neil provided much needed clarity and guidance on everything “high throughput”, “multiplexed”, “-omic”, and really anything that needed to be translated from Science Jargon to English. Our Genome Canada grant-writing sessions will always have a special place in my heart. iii I truly appreciate the time and effort that Dr. Terence Colgan contributed to this project, despite a busy clinical practice. His expertise in gynecologic pathology was integral to the success of this project. My thanks also go out to Dr. Robert Riddell for his expertise in gastrointestinal pathology. Thanks to these pathologists, I no longer fear the microscope. Special thanks go out to the heart and soul of the Rogers Lab and Brown Lab, i.e. Dr. Shawn Chua and Dr. Alexandra Kollara. They trained me for hours in the fundamentals of molecular biology, while expecting nothing in return. Their teaching and support were crucially important, and the skills I acquired from them helped me thrive in the lab. In fact, I believe I can now out- animate Dr. Chua on Microsoft Powerpoint. In the Department of Obstetrics and Gynaecology, there are many to thank. Thank you to the staff of the Mount Sinai Centre for Fertility and Reproductive Health, who made patient recruitment and sampling possible. I would like to thank Dr. Alan Bocking and Dr. Heather Shapiro, who whole-heartedly endorsed my decision to enroll in the Clinician Investigator Program and were supportive and understanding throughout. I would also like to thank all the gynecologic surgeons who welcomed me into their ORs on a weekly - biweekly basis and never stigmatized me for being “the one doing research”. The fact that I still feel confident about my surgical skills after two years is a testament to their teaching and mentorship. Penultimate thanks go out to my family. Thank you for watching me regress into a grad student, with grad student hours and habits, without too much criticism. Thank you for adopting Daisy, especially during the hard times of thesis-writing. Thank you for the prepared food, the shoulder to cry on, and for not saying “I told you you should have gone into Dentistry.” iv Finally, none of this would have been possible without the contributions of the patients enrolled in this study. It always amazes and inspires me to see patients contribute to science out of pure altruism. I hope that this research comes to fruition and ultimately benefits couples suffering from infertility. v Table of Contents Acknowledgments .......................................................................................................................... iii Table of Contents ........................................................................................................................... vi List of Tables ................................................................................................................................. ix List of Figures ................................................................................................................................. x List of Abbreviations ..................................................................................................................... xi Chapter 1 INTRODUCTION .......................................................................................................... 1 1.1 INFERTILITY .................................................................................................................... 1 1.1.1 Assisted reproductive technology ........................................................................... 2 1.1.2 Embryo transfer practices and multi-foetal pregnancy in ART .............................. 4 1.2 IMPLANTATION .............................................................................................................. 7 1.2.1 Apposition and adhesion ......................................................................................... 8 1.2.2 Invasion and placentation ..................................................................................... 10 1.3 ENDOMETRIAL RECEPTIVITY ................................................................................... 12 1.3.1 The window of implantation ................................................................................. 13 1.3.2 Current methods of assessing endometrial receptivity ......................................... 14 1.3.3 The role of hormones in endometrial receptivity .................................................. 18 1.3.4 Molecular factors implicated in endometrial receptivity ...................................... 20 1.3.4.1 Cytokines ................................................................................................ 20 1.3.4.2 Growth factors ........................................................................................ 23 1.3.4.3 Molecules involved in cell adhesion ...................................................... 25 1.3.4.4 Cell cycle regulators ............................................................................... 28 1.3.4.5 Glycodelin-A .......................................................................................... 29 vi 1.4 GENE EXPRESSION STUDIES OF ENDOMETRIAL RECEPTIVITY ...................... 31 1.4.1 Endometrial sampling techniques ......................................................................... 33 1.5 THESIS HYPOTHESIS AND RATIONALE .................................................................. 35 1.6 OBJECTIVES ................................................................................................................... 37 1.7 DESCRIPTION OF COLLABORATIONS AND ROLES .............................................. 37 Chapter 2 MATERIALS AND METHODS ................................................................................. 39 2.1 PATIENT SELECTION ................................................................................................... 39 2.2 TISSUE COLLECTION ................................................................................................... 40 2.3 RNA EXTRACTION ........................................................................................................ 43 2.4 RNA INTEGRITY TESTING .......................................................................................... 43 2.5 REVERSE TRANSCRIPTION, AMPLIFICATION OF