2 Specific aims of this thesis

272 Pages·2017·2.55 MB·English

by  Alison Coates

Optimizing IVF outcomes in the genomics era

Optimizing IVF outcomes in
the genomics era

A thesis submitted to the University of Kent for the degree of
DOCTOR OF PHILOSOPHY
in the Faculty of Science
2017
Alison Coates
School of Biosciences
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Optimizing IVF outcomes in the genomics era

Declaration

No part of this thesis has been submitted in support of an application for any degree or
qualification of the University of Kent or any other University or Institute of learning.

Alison Coates

15th June 2017
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Optimizing IVF outcomes in the genomics era

Acknowledgements

First, I would like to thank Professor Darren Griffin. Without his encouragement, I would have
never started this whole PhD process never mind finished it. Although we are 7,000 miles apart,
he has always been available remotely to bounce ideas off, edit papers, skype at the end of his
busy day and to keep me going when I had moments wondering if I would ever finish my
dissertation. An email from Darren always made my day, put me back on track and kept me
moving forwards. As I work full time, directing a busy IVF lab and have three children, a husband,
4 chickens and a cat, time to work on my thesis was often only found during weekends and
evenings and the goal of a final thesis seemed very distant at times.

I am grateful to the whole team at Oregon Reproductive medicine, Portland, Oregon, USA, for
supporting my work. Their thirst for innovation and always wanting to be better has enabled me
to conduct valuable clinical research thus evolving our field.

I would also like to thank: Gary Harton for introducing me to the idea of doing a PhD at my great
age, Allen Kung and Tyl Taylor who have been my stateside support group through the whole 5
year process, I could not have done this without you two! Emily Mounts for her ability to bridge
the gap between the patient and the complex nature of IVF in the era of genomics and her tireless
attention to detail. Rebecca Matthews, my embryology colleague and friend for almost 20 years,
for her contribution to the final flow and change of course that made the whole story of my thesis
come together. My excellent indispensable team of embryologists for creating an endless supply
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Optimizing IVF outcomes in the genomics era

of clinical data to work with and who are the most dedicated, compassionate and delightful group
of people to work with that one could ever ask for.

Finally, thank you to my husband Frank, for the last 30 years, for always believing in me and
encouraging me to fulfill my potential, and to my wonderful children, Elliot, Isadora and Ted, for
being great human beings.
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Optimizing IVF outcomes in the genomics era

This thesis encapsulates individual pieces of published or submitted manuscripts as follows:

Morphological and Kinetic Embryological Criteria and Association with Aneuploidy Rates: How
Might they be Used to Choose the Best IVF Embryo for Transfer? Coates A, Coate B, Holmes L,
Griffin DK. Human Genet Embryol 2015, 5:3 (Coates A, 2015)

Development and application of a novel strategy to explore blastocoel fluid and spent culture
media as a source of embryonic DNA. Babariya, D, Manoharan, A Welch, C, Spaeth, K, Munne, S,
Coates, A, Wells, D. abstract submitted to ESHRE 2017

The use of suboptimal sperm increases the risk of aneuploidy of the sex chromosomes in
preimplantation blastocyst embryos. Alison Coates, John S. Hesla, Amanda Hurliman, Breanne
Coate, Elizabeth Holmes, Rebecca Matthews, Emily L. Mounts, Kara J. Turner, Alan R. Thornhill,
Darren K. Griffin, Fertility and Sterility® Vol. 104, No. 4, October 2015 (Coates et al., 2015)

Differences in pregnancy outcomes in donor egg frozen embryo transfer (FET) cycles following
preimplantation genetic screening (PGS): a single center retrospective study. Alison Coates,
Brandon J. Bankowski, Allen Kung, Darren K. Griffin, Santiago Munne. Journal of assisted
reproduction and genetics, November 2016 (Coates et al., 2016a)
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Elective frozen-thawed vs fresh transfer of euploid embryos identified by PGD using next
generation sequencing; A randomized controlled trial. A.Coates, A. Kung, J.S. Hesla,
B.J.Bankowski, E.A.Barbieri1, E. Mounts, B. Ata, J.Cohen, S. Munné, Fertility and Sterility, Vol. 107,
No. 3, 723-730 March 2017 (Coates et al., 2017)

Mosaicism rates in embryos resulting in live birth or miscarriage
Authors: Tormasi, S, Capaldi, R. Gouw, F. Welch, C. Munne, S. Coates, A.
Poster Abstract PGDIS 2016 (Tormasi, 2016)

Planning for the future: how many oocytes do patients need to harvest to achieve their fertility
goals? Alison Coates Emily Mounts, Brandon Bankowski, Santiago Munne Abstract poster ASRM
annual meeting 2016 (Coates et al., 2016b)

Other publications generated during the timeframe of production of this thesis are listed below.
They have not been included as part of the general thesis but have been referenced if
appropriate. My contribution to each body of work is noted here.

Validation of next-generation sequencing for preimplantation genetic screening of embryos.
Kung, A. Munne, S. Bankowski, B. Coates, A. Wells, D. Reprod Biomed Online. 2015 Dec;
31(6):760-9 (Kung et al., 2015)
Personal contribution to the manuscript: I biopsied all embryos for use in the validation study.
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Optimizing IVF outcomes in the genomics era

No diagnosis after day 3 biopsy: indicative of embryo prognosis or biopsy error

O-147 ASRM annual meeting (Yeboah et al.)

Personal contribution to the manuscript: I advised the author and edited the text.

High Variability in chromosome abnormality rates in embryos from young infertile women. S. S.
Sawarkar, J. Zhang, D. L. Hill, J. S. Hesla, A. Coates, L. Ribustello, S. Ghadir, S. Munne.P-102
Tuesday, October 18, 2016 (Sawarkar et al.)
Personal contribution to the manuscript: Chromosome data from blastocysts biopsied in my
own laboratory was used as part of a multicenter retrospective study by the reference genetics
laboratory (Reprogenetics) to illustrate variability of aneuploidy rates within populations of
young infertile women

The four published manuscripts where I was first author have been inserted as chapters mainly
as they appeared in print. The methods sections for stimulation, culture and DNA testing
protocols for each paper have been consolidated into one general methods section at the end of
the specific aims section and referenced in each chapter to prevent repetition. Parts of this thesis
consist of unpublished work using data generated from IVF cycles in my own laboratory.
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Table of contents page
Declaration 2
Acknowledgements 3
Incorporation of published work 5
List of abbreviations 13
Thesis Abstract 14
List of Tables 17
List of Figures 18
1.0 General Introduction 19
1.1 Infertility 19
1.1.1 Definition and incidence 19
1.1.2 Etiology and treatments of infertility 19
1.1.3 In-vitro Fertilization (IVF) 22
1.1.4 Evolution of IVF 23
1.2 Factors contributing to the success or failure of a cycle of IVF 27
1.2.1 Factors affecting uterine receptivity 27
1.2.1.1 Preparation of the uterus for conception in a natural cycle 27
1.2.1.2 Preparation of the uterus for transfer in a stimulated IVF cycle 28
1.2.1.3 Preparation of the uterus for a frozen embryo transfer 29
1.2.1.4 Uterine receptivity 30
1.2.2 Factors affecting embryo development in-vitro 31
1.2.2.1 Culture media 31
1.2.2.2 Temperature 33
1.2.2.3 pH 34
1.2.2.4 Oxygen tension 35
1.3 Determining Embryo viability 36
1.3.1. Embryo Morphology 37
1.3.2 Pronuclear position and Nucleoli patterns 40
1.3.3 Morphokinetics 43
1.3.4 Combining morphokinetics and spent media analysis 44
1.3.5 Analyzing spent culture media 45
1.3.5.1 Metabolomics 45
1.3.5.2 Proteomics 46
1.3.6 Cumulus gene expression 47
1.3.7 MicroRNA (miRNA) in follicular fluid 48
1.3.8 Mitochondrial DNA quantification 49
1.3.9 Summary of factors contributing to the success or failure of an IVF cycle 52
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1.4 Aneuploidy 53
1.4.1 Mechanisms of chromosome mal-segregation 53
1.4.1.1 Non-disjunction 54
1.4.1.2 Anaphase lag 55
1.4.1.3 Precocious separation of sister chromatids 56
1.4.2 Oocyte aneuploidy 57
1.4.2.1 Advanced maternal age and oocyte aneuploidy 57
1.4.2.2 Environmental effects on oocyte aneuploidy 58
1.4.3 Sperm aneuploidy 59
1.4.3.1 Advanced paternal age and its’ effect on sperm aneuploidy 60
1.4.3.2 Sperm quality and sperm aneuploidy 61
1.4.3.3 Environmental effects on sperm aneuploidy 62
1.4.4 Embryonic (post-zygotic) aneuploidy 63
1.4.4.1 Advanced maternal age and its effect on embryonic aneuploidy 63
1.4.4.2 Environmental effects on embryonic aneuploidy 64
1.5 How do we detect aneuploidy in pre-implantation embryos? 66
1.5.1 Non-invasive PGS using spent culture media 66
1.5.2 Blastocoel fluid biopsy 68
1.5.3 Embryo Biopsy 71
1.5.3.1 polar body biopsy 71
1.5.3.2 blastomere biopsy 72
1.5.3.3 trophectoderm biopsy 75
1.6 The evolution of Pre-Implantation Genetic Screening (PGS) 78
1.6.1 specific single gene mutation detection 79
1.6.2 aneuploidy detection 80
1.6.2.1 Fluorescence In-situ Hybridization (FISH) 80
1.6.2.2 quantitative Polymerase Chain Reaction (qPCR) 81
1.6.2.3 Whole Genome Amplification (WGA) 82
1.6.2.4 Comparative Genomic Hybridization (CGH) 83
1.6.2.5 Array Comparative Genomic Hybridization (aCGH) 83
1.6.2.6 Single Nucleotide Polymorphism (SNP) array 85
1.6.2.7 Next Generation Sequencing 85
1.6.2.8 Summary of aneuploidy detection technologies 86
1.7 Is aneuploidy always unequivocal? 87
1.7.1 What is a mosaic embryo? 87
1.7.2 Detecting mosaicism in blastocysts 88
1.7.3 Rate of mosaicism in blastocysts and in pregnancy 90
1.7.4 Where do the cells of the trophectoderm originate from? 91
1.7.4.1 Risk of discordant cell lines in the ICM versus trophectoderm 93
1.7.5 Does the ratio of euploid to aneuploid cells matter to the
“severity” of the mosaic diagnosis? 93
1.7.6 Should patients transfer mosaic embryos and if so does the
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affected chromosome matter? 94
1.7.7 Reanalyzing DNA samples using NGS from transferred embryos
diagnosed as euploid with aCGH 98
1.8 Supplementing PGS with non-invasive testing 100
1.8.1 Combining Morphokinetics and PGS 100
1.8.2 Combining static morphology and PGS 101
1.9 Limitations of the embryo biopsy and testing process: 102
1.9.1 Risks of misdiagnosis 102
1.9.2 Risks of using Whole genome amplification for PGS 104
1.9.2 Risks of biopsy to the embryo 104
1.10 Referral categories for PGS 106
1.10.1 Higher risk patients 106
1.10.1.1 Advanced maternal age 106
1.10.1.2 Previous history of aneuploid conception 108
1.10.1.3 Failed IVF cycles 108
1.10.1.4 Recurrent pregnancy loss 108
1.10.2 Lower risk patients 109
1.10.2.1 Young maternal age 109
1.10.2.2 PGS before cryopreservation 110
1.11 Perspectives on the potential to further improve IVF 110
2.0 Specific aims of this thesis 111
2.1 General Methods - common to all publications 113
2.1.1 Timeline of data collection 113
2.1.2 changes to protocols during data collection timeline 113
2.1.3 Stimulation and embryo culture protocols 115
2.1.4 Embryo grading and morphological analysis 116
2.1.5 Embryo biopsy 117
2.1.6 reducing contamination of samples 117
2.1.7 DNA analysis 118

2.2 Specific aim 1: To determine if we can accurately predict aneuploidy without
biopsying embryos by the following methods: 120
2.2.1 Method 1a: To establish if static morphology correlates with ploidy status 120
2.2.1.1 My Personal contribution to the work 120
2.2.1.2 Chapter summary 120
2.2.1.3 Introduction 122
2.2.1.4 Materials and methods 126
2.2.1.5 Results 126
2.2.1.6 Discussion 133
2.2.2 Method 1b: To determine if we can use cell free DNA in spent media
to quantify ploidy status 139
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