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Effects of Ovarian Stimulation on Oocyte Development and Embryo Quality

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Effects of Ovarian Stimulation on Oocyte Development and Embryo Quality EFFECTS OF OVARIAN STIMULATION ON OOCYTE DEVELOPMENT AND EMBRYO QUALITY By Kimberley Marie Swann BSc (Hons), MMedSci Thesis submitted to the University of Nottingham for the degree of Doctor of Philosophy May 2014 School of Clinical Sciences Queens Medical Centre Nottingham NG7 2UH DECLARATION I hereby declare that the work in this thesis has been devised, conducted and composed by myself and has not been submitted for any other degree, in Nottingham or elsewhere. The work presented herein is my own, where other sources have been used, the authors have been duly acknowledged. Kimberley Marie Swann Abstract ABSTRACT Ovarian stimulation plays a pivotal role in assisted reproductive therapies, to increase the number of embryos available for treatment; however, there is no clear consensus from meta-analyses in the literature which, if any, of the preparations in use are superior in terms of clinical outcomes. The aim of this thesis was to examine the effect of common human gonadotrophin preparations with different half lives and LH activity (hMG, rFSH and Pergoveris) on embryo quality and resulting offspring, compared to non- stimulated negative controls and positive PMSG treated controls, using the mouse model. The studies in this thesis indicated that an LH ceiling threshold is evident during folliculogenesis, where the use of long acting LH preparations resulted in higher numbers of fragmented oocytes, absent of cumulus cells (P<0.001), reduced expression of the pro and anti-angiogenic factors, MYHII and PEDF in cumulus cells (P<0.05), increased embryonic developmental arrest (P<0.001) and perturbed IGF2 (P<0.05) and VEGFA gene expression in resulting blastocysts (P<0.01), compared to negative controls. Use of preparations containing LH bioactivity resulted in offspring with altered total body weight trajectories and internal organ weight abnormalities (P<0.05), which were, in some instances, compounded by in vitro culture. In addition, we elucidated a relationship between FSH half life differences between urinary and recombinant preparations and embryo quality. The urinary human gonadotrophin preparation, hMG, could yield developmentally competent embryos at lower concentrations, than the recombinant Pergoveris treatment. In addition to FSH, these preparations contain LH and both low doses of preparations composed of short half life rFSH and rLH and high doses of preparations containing long acting LH bioactivity, resulted in the highest rates of developmental arrest. These groups were observed to have complete absence of H19 expression. The results of this thesis provide clear evidence that ovarian stimulation does negatively impact the embryo and subsequent offspring and provides support for an LH ceiling threshold, above which detrimental effects occur, both on in vitro embryo development and in vivo foetal development, which later effects postnatal growth. i Publications PUBLICATIONS The relationship between luteinising hormone in ovarian stimulation and subsequent embryo development and viability K. Swann , J. Meadows , K. Jayaprakasan , B.K. Campbell and W. Maalouf Accepted for Oral Presentation at The 8th Biennial Fertility Conference of the UK Fertility Societies, Liverpool, United Kingdom, January 2013 September 2013, Vol. 16, No. 3 , Pages 218-227 The effects of gonadotropin stimulation and 2-cell embryo biopsy in mice on embryo quality and blastocyst development P. Ghosh , K. Swann , B.K. Campbell and W. Maalouf Accepted for Oral Presentation at the 8th Biennial Fertility Conference of the UK Fertility Societies, Liverpool, United Kingdom, January 2013 September 2013, Vol. 16, No. 3 , Pages 218-227 The effect of different ovarian stimulation protocols on imprinted gene expression and live birth rates in mice K. Swann, B.K. Campbell, N. Raine-Fenning, K. Jayaprakasan and W. Maalouf Accepted for Oral Presentation at the 29th ESHRE annual meeting, London, United Kingdom, July 2013 Runner up for conference and at ESHRE 2013 where I was pre-selected for two awards: the Basic Science Award for Oral Presentation and for the Fertility Society of Australia Exchange Award. Hum. Reprod. (2013) 28 (suppl 1): i73-i75 ii Acknowledgements AKNOWLEDGEMENTS I would like to start by thanking my supervisors Professor Bruce Campbell, Dr Walid Maalouf and Dr Kannamannadiar Jayaprakasan. Thanks goes to Anne Skinner and Nicky Farrar for their help when I first started, by helping me find my feet in the lab and showing me how to handle the mice. Thanks goes to the BMSU animal unit, specifically to Sally-Anne Edwards, Mark Trussell, Anne-Marie Kelly and Neil Yates, who without which none of the mice work would have been possible. Thanks goes to everyone who touched my life during these past few years and made them that more bearable. Specifically for the friendships I developed with Miriam Baumgarten, Lukasz Polanski, Kirstyn Sewell, Yee Yin and Amarin Narkwichean, who were always there to offer a shoulder to lean on and an ear to listen. I would like to thank Lessly Sepulveda, who although came to the department later in my studies, was a good friend and companion and also helped with the printing and sorting of the initial draft of this thesis. I would like to thank Dr Peter Marsters, who helped immensely with the PCR in this project but also was a pillar of support and a kind ear during some difficult patches in my PhD. For this I will be forever thankful. As will I be to Dr Cecilia Sjoblom, whose initial laboratory and theoretical training in embryology put me in good steam for my PhD and future career. Her help and unwavering faith in me is truly appreciated and deeply touching and I can only dream of following in her footsteps someday. I would like to thank all my family, mum, nan and grandad for their support and encouragement not to give up. Last but not least I would like to thank the University of Nottingham and NURTURE fertility for providing the funding that gave me the opportunity to complete this project. iii Table of contents TABLE OF CONTENTS ABSTRACT..............................................................................i PUBLICATIONS........................................................................ii AKNOWLEDGEMENTS.............................................................iii TABLE OF CONTENTS...............................................................iv LIST OF FIGURES ..................................................................ix LIST OF TABLES....................................................................xix LIST OF ABBREVIATIONS...... .................................................xxi CHAPTER 1: LITERATURE REVIEW ...................................1 1.1 THE IMPORTANCE OF OVARIAN STIMULATION.....................1 1.2 OOGENESIS AND FOLLICULOGENESIS.... ...........................3 1.2.1 Primordial follicle formation...............................................3 1.2.2 Gonadotrophin independent follicles........ ..........................4 1.2.3 Gonadotrophin responsive follicles.. ..................................5 1.2.4 Gonadotrophin dependant follicles.. ..................................6 1.2.5 Mouse folliculogenesis.. .................................................7 1.2.6 Endocrinology of folliculogenesis.... ...................................9 1.2.6.1 Follicular Phase...................................................10 1.2.6.2 Ovulation...........................................................11 1.2.6.3 Luteal Phase......................................................12 1.2.7 Oogenesis and oocyte maturation....................................13 1.2.8 Factors involved in folliculogenesis and oogenesis..............14 1.2.9 Angiogenesis in folliculogenesis and oogenesis...................16 iv Table of contents 1.3 EMBRYO DEVELOPMENT..................................................17 1.4 EPIGENETICS IN GAMETOGENESIS AND EMBRYOGENESIS..19 1.4.1 The role of IGF2 and H19................................................23 1.5 IMPLANTATION..............................................................25 1.5.1 Implantation in human...................................................25 1.5.2 Mouse implantation........................................................27 1.5.3 The Renin-angiotensin system.........................................27 1.6 OVARIAN STIMULATION.................................................30 1.6.1 Clinical applications in ART..............................................30 1.7 GONADOTROPHINS........................................................32 1.7.1 Gonadotrophin structure.................................................32 1.7.2 Purified gonadotrophins ..................................................33 1.7.3 Recombinant gonadotrophins..........................................36 1.7.4 Efficacy of different gonadotrophins for COS......................37 1.7.5 Ovarian stimulation in mice.............................................40 1.8 CONSEQUENCES OF SUPEROVULATION............................41 1.8.1 Oocyte yield..................................................................41 1.8.2 Developmental manifestations.........................................43 1.8.2.1 Embryo development alterations...........................43 1.8.2.2 Postnatal development alterations.........................44 1.8.3 Genetic perturbations.....................................................46 1.8.3.1 ART and imprinted disorders.................................46 1.8.3.2 The association of methylation and COS.................46 1.8.4 Disruption of implantation...............................................50
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