Anti-Mullerian Hormone as a Marker of Oocyte Quantity, Developmental Potential, and Fetal Sex by Anja Stojsin Carter A Thesis Presented to The University of Guelph In partial fulfillment of requirements for the degree of Doctor of Philosophy in Biomedical Sciences Guelph, Ontario, Canada © Anja Stojsin Carter, December 2016 ABSTRACT ANTI-MULLERIAN HORMONE AS A MARKER OF OOCYTE QUANTITY, DEVELOPMENTAL POTENTIAL AND FETAL SEX Anja Stojsin Carter Advisor: University of Guelph, 2016 Professor W. A. King Delayed childbearing age, increased environmental pollutants accompanying modern life style, and an increase in inbreeding due to high demand for milk and meat yield have resulted in the decline in human and bovine reproduction respectively. Anti-Mullerian hormone (AMH) is expressed starting from 7-8 weeks of gestation in males and 20 weeks of gestation in females, in up to 100x higher concentration in males. AMH has been described as a non-invasive and accurate marker of ovarian quantitative fertility parameters in both humans and cattle. However, it is still unknown how AMH levels correlate to different fertility parameters in different cattle breeds; can AMH levels in the mother be used as a fetal sex marker during pregnancy in cows? In humans, can AMH collected in the single follicular fluid (mono FF), as well as granulosa cell (GC) AMH receptor 2 (AMHR2) be used as an embryo developmental potential marker? Here we present evidence of the higher levels of systemic and follicular AMH in Zebu compared to European cattle, corresponding to higher levels of AFC and oocytes. Cows pregnant with a male fetus, as compared to a female fetus, were observed to have a significantly different change in plasma AMH between day 35 and 135 during gestation. The placenta and cotyledon were found to express AMHR2 between day 38 and 80, however not significantly different between pregnancies with an opposite sex fetus. In the human study, systemic and average mono FF AMH, and average mono GC AMHR2 expression were significantly negatively correlated with the patient’s blastocyst rate. When patients were divided into normal and high groups based on the their systemic AMH levels, the following was observed: normal systemic AMH level patients had a lower average FF AMH level and a higher blastocyst rate compared to the higher systemic AMH level patients. Also, in normal systemic AMH patients, blastocysts corresponded to follicles with a lower level of AMH, while in high systemic AMH patients blastocysts corresponded to follicles with a higher level of AMH. iv DECLARATION OF WORK PERFORMED I declare that, with the exception of the items indicated below, all the work reported in the body of this thesis was performed by me. Cattle ovaries and reproductive tracts were collected by Pradeep Blaraju, Steven Huang, Heather Smale, Stephan Botha, and Brooklin Rushton during the course of these studies. Media used in Canada for oocyte in vitro maturation, in vitro fertilization, and in vitro culture were prepared by Liz St. John. Real time qPCR experiments in Chapter I were performed by Kiana Mahboubi. Cell culture and DNA extraction were performed by Dr. Olutobi Oluwole. Antral follicle count, ovary diameter measurements, serum collections, slaughterhouse ovary collection, bovine anti-Mullerian hormone ELISA measurements, and fetal serum collection in Brazil were performed with the help of Dr. Moyses Miranda, Dr. Otavio Ohashi, Dr. Nathalia Nogueira, Dr. Tiago de Bem, Rodrigo de Morais, Cinthia Lopes, Mayra Costa, Alessandra Ximenes, Tobias Sovernigo, and Marcus Filho. ONE Fertility patient consents were obtained by Melody Rasmussen and Maja Smrzlic. Human anti-Mullerian hormone ELISA measurements were performed by Alexandra Marinic-Cowden. Human follicular fluid collections were performed by Michael Neal. Human in vitro fertilization, culture, embryo assessment and transfer were performed by Katie Willoughby, Lisa Deys, and Lindsay Moffatt. Daniel Gillis performed most of the statistical analysis. v ACKNOWLEDGEMENTS First and foremost I would like to thank my supervisor Dr. Allan King. Allan provided me with the opportunity to work in his lab twice, doing a research project during the last year of my undergraduate degree, and then for my PhD, for which I was able to do a project highly relevant to my interests and goals. I was also given the opportunity to collaborate with a human fertility clinic and stay in close contact with the field in which my findings could have an impact. I also have to thank Allan for the opportunity to go on a research exchange to Brazil, to work with his collaborators in Belem, Para. Last but not least, Allan has been incredibly understanding of my personal life; I got married and had a baby after the second year of my degree. Allan organized the baby shower and allowed me to pursue motherhood in parallel with work on my thesis. The people Allan gathered around himself are all brilliant and I had the honor of being part of his team. I would like to thank Dr. Pavneesh Madan for his support throughout my project and his valuable input on the project progression and experiment planning. Dr. Madan also helped me troubleshoot through my immunofluorescence and confocal microscopy work, and was encouraging throughout my manuscript writing. I would like to thank Dr. Neil MacLusky for guidance and support during my committee meetings and for his help with some of my statistics questions. Neil is a very sweet, approachable and genuine person, finding time to talk to students and allowing them to feel like one of his colleagues. I would like to thank Mike Neal, ONE Fertility Burlington scientific director, for giving me the opportunity to collaborate, for his enthusiasm, for help with writing the ethics protocols, vi abstracts and manuscript, and for enabling me to collect valuable sample material, attend conferences, and extend my research opportunities by including a human study. I would like to thank Dr. Julang Li, my external committee member, for kind words, support and guidance. I would like to give a special Thank You to Dr. Laura Favetta. Dr. Favetta was my advisor, my friend, my sister, my parent. I met Laura during my undergraduate degree project and she has provided me with infinite help since then. It was an honor knowing Dr. Favetta, with her devoted and passionate approach to students and teaching. Dr. Favetta’s knowledge on qPCR and endocrinology kept me going in the right direction! I would like to thank Dr. Daniel Gillis for his immense help and patience in performing majority of the statistical analysis in this thesis. I would like to thank Liz St. John for her help with ovary and placenta collections, support in any lab related issues, and who, with her kindness, never intimidated the students around her. I would like to thank Dr. Monica Antenos for sharing her knowledge, and providing incredible support in the lab, where she created a friendly, relaxed, but very stimulating atmosphere. I would like to thank Ed Reyes and Allison MacKay for their technical support and patience, dealing with ordering supplies and questions at, often, inconvenient times. I would like to thank Helen Coates for her patience and help with confocal microcopy. I would like to thank Dr. Tamas Ravay for his valuable input in qPCR and immunofluorescence and molecular troubleshooting. Dr. Ravay was also more than just a colleague; he was a friend and a support throughout this project. vii I would like to thank Kiana Mahboube, a Masters student, who worked on part of my project and helped incredibly with qPCR work, and who has also been a close friend and support. I would like to thank to Dr. Stalker, Kata Ozs, and Dr. Petrik for their help with troubleshooting experiments and cell material. I would also like to thank our collaborators and extended family in Brazil, Dr. Miranda, Dr. Ohashi, Dr. Costa, Dr. de Bem, and many others. Without your input this project would have been so much more boring. I loved my experience in Brazil, and thank you for providing me with so many opportunities to examine and learn so much about your beautiful cattle: Bos taurus indicus. I would also like to extend my gratitude to the team at ONE Fertility, Burlington. Thank you to the embryologists: Katie, Lindsay, and Lisa for their patience to have me in the lab during their busy schedules, to have patients answer my questions, and to trust me to help them with some of their media preparations and case setups. I also would like to thank the andrology team: Tiffany and Leanne for allowing me to use some of their space to prepare research samples. I would like to thank the staff: Melody, Maja and Alexandra for their help with patient consent forms and running the human ELISA. Last but not least, I would like to thank Dr. Karnis, Dr. Amin, Dr. Huges and Dr. Faghih for their trust and the opportunity to collaborate. This research would have not been possible without the financial support from the OVC Doctoral Scholarship, The Natural Sciences and Engineering Research Council of Canada (NSERC), Canada Research Chair (CRC), and North/South Animal Biology and Reproductive Biotechnology Consortium- Canada/Brazil Doctoral Student Research Exchange. viii I would now like to thank my friends and family all around the world. I would like to thank my friends in Canada: Carolyn, Sarah, Jacky, Tobi, Kayla, Allison, Carmon, Leslie, Stewart, Graham, Faz, Anh, Nayoung, Jyoti, Nina, Anuja, Moez, Ari and many others in the Biomedical Sciences Department, including staff Kim Best and Frances Graziotto, for their friendship and support. I would like to thank especially my friend Gilan Abdelaal, who was there for me through the good, bad and the ugly. I would like to thank my former high school teacher Dragan Gajic, for his love and passion for science that molded my undergrad and grad scientific path.