Group Culture of Preimplantation Embryos Creates a Microenvironment That Determines Development, Viability, and Response to External Stressors

Home , Preimplantation factor

Group culture of preimplantation embryos creates a microenvironment that determines development, viability, and response to external stressors

Rebecca Lauren Kelley

ORCID 0000-0001-6868-0207

Submitted in total fulfilment of the requirements of the degree of Doctor of Philosophy

December 2018

School of BioSciences Faculty of Science

University of Melbourne Abstract Over 400,000 babies worldwide are born following in vitro fertilisation (IVF) treatment every year and millions of babies have been born from IVF over the last 40 years (Adamson et al. 2018,

Niederberger et al. 2018). Despite this, approximately half of couples undergoing IVF treatment do not achieve a successful pregnancy, even after multiple treatment cycles (Chambers et al.

2017, Fitzgerald et al. 2017). The period of in vitro culture is a crucial stage in determining not only the success of IVF treatment but also the long-term health of these babies (Feuer and

Rinaudo 2016).

Due to new embryo selection and tracking technologies, clinical embryologists increasingly culture embryos individually rather than in groups. However, individual culture can cause stress to the embryo (Reed 2012)because embryos are deprived of paracrine signals, which promote the development of embryos cultured in groups (Wydooghe et al. 2017). The identities and actions of most of these embryo-secreted molecules remain poorly understood.

Using a mouse model, this study compares the development of embryos cultured individually or in groups, and how other culture conditions influence the development of individually cultured embryos. Firstly, it was found that any period of individual culture was detrimental to development rates, cell numbers and percentage of inner cell mass (ICM), and the precompaction stage was no more sensitive to this stress than the post-compaction embryo.

Reducing the volume of culture media was beneficial to individually cultured embryos, but only under 5% oxygen, not atmospheric oxygen, indicating that there is an interaction between optimal embryo density and oxygen concentration. Reducing the media volume further by culturing embryos in microwells increased the % ICM and influenced the speed of cleavage and hatching. Addition of embryo-conditioned media to individually cultured embryos also increased hatching rate and cell numbers.

The interaction between individual culture and atmospheric oxygen was investigated in more detail. Compared to group culture under 5% oxygen, either individual culture (at 5% oxygen) or atmospheric oxygen (group culture) caused slower cleavages, lower development rates, and fewer cells, plus changes to glucose and amino acid metabolism. Embryos cultured individually under 20% oxygen were affected further, so that combining the two detrimental conditions resulted in earlier and longer delays to development, fewer and smaller blastocysts, and more exaggerated changes to metabolism compared to those cultured under 5% oxygen in groups.

There was very little effect of individual culture under 5% oxygen on fetal and placental development compared to group cultured embryos. However, individual culture under 20% oxygen resulted in smaller and skinnier fetuses, and lighter placentas compared to embryos cultured in groups. This may be because the further the culture conditions are from optimal, the more resources an embryo uses to adapt and survive, or there is an accumulation of trauma, and the manifestations of stress become more exaggerated.

Embryos in culture secrete high levels of IL-6, and supplementing culture media with IL-6 increased hatching rate and cell numbers, showing IL-6 to be an embryotrophic cytokine.

However, it is just one of hundreds of embryo-secreted molecules that may be involved in paracrine signalling during group culture.

This study demonstrates that individual culture is detrimental to mouse embryos, especially in combination with atmospheric oxygen. This raises concerns regarding the increasing prevalence of single embryo culture in human IVF. Furthermore, these data demonstrate the cumulative nature of stress during embryo culture and highlight the importance of optimising each element of the culture system.

Declaration

This is to certify that:

1. This thesis comprises only my original work towards the Doctor of Philosophy except where indicated in the preface.

2. Due acknowledgement has been made in the text to all other material used.

3. The thesis is fewer than 100,000 words in length, exclusive of tables, maps, bibliographies and appendices.

Rebecca Kelley

iii

Preface Rebecca Kelley (RK) was the primary author of all manuscripts within this thesis. These manuscripts were written with the assistance and editorial changes by supervisors David

Gardner (DG) and Alexandra Harvey. The ideas concerning the design of the experimental aspects of this thesis were conceived and developed by RK and DG. RK carried out the experimental work and collected and analysed the data used within this thesis. Analysis of amino acids in culture media in Chapter 5 was performed by Metabolomics Australia at the University of Melbourne. Histology in Chapter 5 was performed by Biomedical Sciences Histology Facility at the University of Melbourne.

Chapters 3, 4 and 6 have been published and are presented in this thesis unchanged from their published form. Chapter 5 has been submitted for publication to Reproductive BioMedicine

Online in December 2018 and is under review.

Publications produced as part of this thesis Kelley RL, Gardner DK Combined effects of individual culture and atmospheric oxygen on preimplantation mouse embryos in vitro. Reprod Biomed Online 2016 33(5):537-549

Kelley RL, Gardner DK In vitro culture of individual preimplantation embryos in the mouse: the role of embryo density, microwells, oxygen, timing and conditioned media. Reprod Biomed

Online 2017 34(5):441-454

Kelley RL, Gardner DK Addition of interleukin-6 to mouse embryo culture increases blastocyst cell number and influences the inner cell mass to trophectoderm ratio. Clin Exp Reprod Med

2017 44(3):119-125

Additional publications resulting from work during candidature Gardner DK, Kelley RL Impact of the IVF laboratory environment on human preimplantation embryo phenotype. J Dev Orig Health Dis 2017 8(4):418-435

Pankhurst, M. W., Kelley, R. L., Sanders, R. L., Woodcock, S. R., Oorschot, D. E., Batchelor, N. J.

Anti-Mullerian hormone overexpression restricts preantral ovarian follicle survival. J Endocrinol

2018 237(2):153-163

Conference abstracts arising from this thesis Kelley RL and Gardner DK. Consequences of culturing preimplantation embryos individually. The

Annual Scientific Conference of the Society for Reproductive Biology, Adelaide, 2015.

Kelley RL and Gardner DK. In vitro culture of mouse preimplantation embryos individually delays cleavage rate and increases sensitivity to stress. Australian Society for Medical Research (Vic)

Scientific Meeting, Melbourne, 2015

Kelley RL and Gardner DK. A combination of individual culture and atmospheric oxygen in mouse embryo culture is more detrimental to preimplantation and postimplantation development than either treatment alone. Alpha Scientists in Reproductive Medicine Biennial Conference,

Copenhagen, 2016.

Kelley RL and Gardner DK. Individual culture and 20% oxygen are detrimental to preimplantation mouse embryo development, but the combination of the two is more damaging than either treatment alone. Scientists in Reproductive Technology Annual Meeting, Adelaide, 2016.

Kelley RL Culture conditions and stress. Fertility Society of Australia Annual Conference,

Melbourne, 2018 (invited speaker)

Awards, grants and scholarships 2014 Melbourne Research Scholarship and Top-up Scholarship

2015 Jasper Loftus Hills Award

2016 Society for Reproductive Biology Early Career Researcher collaborative travel award

2016 Norma Hilda Schuster (nee Swift) Scholarship

2016 Best Free Communication – Alpha Scientists in Reproductive Medicine 11th Biennial Conference, Copenhagen

2016 F.H. Drummond Travel Award

2016 University of Melbourne Science Abroad Travelling Scholarship

Acknowledgements Firstly, thank you to my supervisors David and Alex. Thank you, David, for convincing me to start on this challenging journey and for being unwaveringly supportive of me the whole way. Your guidance and enthusiasm were essential and much appreciated, and I am grateful for the all opportunities you have provided and the doors you have opened. Thank you, Alex, for your advice and friendship along the way, and especially for helping me improve my written work.

Thank you also to the other members of our lab for your camaraderie and teamwork, especially

Mai Truong, without whom the lab would probably fall apart. Thank you also to Lisa Lee and

Petra Wale for your advice and friendship, which makes the journey much easier.

I am grateful for the financial support from the University of Melbourne and the School of

BioSciences which allowed me to undertake this PhD. The grants I received from Jasper Loftus

Hills Award and the Norma Hilda Schuster (nee Swift) Scholarship helped to fund my research, and the Drummond travel scholarship and Science Abroad Travelling Scholarship allowed me to present my work at an international conference. Receiving the SRB ECR collaborative travel award also made possible a productive trans-Tasman collaboration.

Thank you so much to my parents, who have supported me not only through the PhD but in every endeavour I’ve undertaken so far. I am incredibly lucky to know that I can always rely on you when I need it most. In particular, I couldn’t have finished this thesis without your grandparental skills, thank you so much for looking after me and my babies so I could place my focus elsewhere for a while.

I am also extremely grateful to my parents-in-law Barbara and Lindsay, who are so generous with their time and have helped us so much over the last few years. I didn’t know you when I started this PhD, but I am very fortunate to be a member of your family now.

vii

Finally, thank you to my wonderful husband Tim. We met right at the start of this PhD and we’ve had an amazing ride since, squeezing in a wedding, two babies, and lots of other adventures. I’m so excited to start the next part of our lives together. And to Clara and Eli, who did not make the

PhD easier, but make life infinitely better.

viii

Table of contents Abstract ...... i Declaration ...... iii Preface ...... iv Publications produced as part of this thesis ...... iv Additional publications resulting from work during candidature ...... v Conference abstracts arising from this thesis ...... v Awards, grants and scholarships ...... vi Acknowledgements ...... vii Table of contents ...... ix Lists of tables and figures ...... xi Figures ...... xi Tables ...... xiii List of Abbreviations ...... xv Chapter 1: General Introduction ...... 1 Infertility and assisted reproduction ...... 1 The mammalian preimplantation embryo ...... 2 The embryo’s environment in vivo...... 5 Embryo culture in vitro ...... 8 Preimplantation environment influences postnatal development...... 21 Multiple stresses ...... 23 Summary ...... 24 Chapter 2: Materials and methods ...... 26 Chemicals and reagents ...... 27 Animals ...... 27 Quality control and quality assurance in the embryology laboratory ...... 27 Generation and collection of embryos ...... 28 Embryo culture ...... 29 Assessment of preimplantation embryo development ...... 36 Assessment of peri-implantation and postimplantation development ...... 41 Analysis of embryo-conditioned media ...... 49 Statistical analysis ...... 51 Chapter 3: In vitro culture of individual mouse preimplantation embryos: the role of embryo density, microwells, oxygen, timing and conditioned media...... 54 Introduction……………………………………………………………………………………………………………………….54

Materials and methods……………………………………………………………………………………………………...56

Results………………………………………………………………………………………………………………………………..58

Discussion…………………………………………………………………………………………………………………………..60

References………………………………………………………………………………………………………………………….64

Supplementary data ...... 69 Chapter 4: Combined effects of individual culture and atmospheric oxygen on preimplantation mouse embryos in vitro ...... 73 Introduction……………………………………………………………………………………………………………………….74

Materials and methods……………………………………………………………………………………………………...75

Results………………………………………………………………………………………………………………………………..76

Discussion…………………………………………………………………………………………………………………………..79

References………………………………………………………………………………………………………………………….81

Supplementary Data ...... 87 Chapter 5: Individual culture and atmospheric oxygen during culture affect mouse preimplantation embryo metabolism and postimplantation development ...... 88 Abstract ...... 89 Introduction ...... 90 Materials & Methods ...... 93 Results ...... 99 Discussion ...... 113 Supplementary data ...... 124 Chapter 6: Addition of interleukin-6 to mouse embryo culture increases blastocyst cell number and influences the inner cell mass to trophectoderm ratio ...... 125 Introduction……………………………………………………………………………………………………………………..126

Materials and methods…………………………………………………………………………………………………….127

Results………………………………………………………………………………………………………………………………128

Discussion…………………………………………………………………………………………………………………………129

References………………………………………………………………………………………………………………………..130

Chapter 7: General discussion ...... 133 Conclusions ...... 144 Appendices ...... 146 Appendix A ...... 147 References ...... 150

Lists of tables and figures Figures Chapter 2

Figure 1. Diagram of microwells 34

Figure 2. Developmental stages of the preimplantation mouse embryo 37

Figure 3. Interior of MCOK-5M[RC] time-lapse incubator. 39

Figure 4. A blastocyst after nuclear staining with bisBenzamide 40

Figure 5. Blastocyst stained using differential staining technique 41

Figure 6. Blastocyst outgrowths 42

Figure 7. Day 15 placenta stained with Masson trichrome 49

Chapter 3

Figure 1. Microwells 56

Figure 2. Effect of single culture during precompaction or post-compaction 59

stages

Figure 3. Effect of embryo density and oxygen on single embryo development 60

Figure 4. Effect of culture in microwell dishes on morphokinetics, relative to 61

single culture in a 2 µl drop

Figure 5. Effect of culture in microwell dishes on cell numbers 62

Figure 6. Effect of embryo-conditioned media on individual embryos 63

Supplementary Figure 1. Correlation between cell number of group cultured 69

embryos used to make conditioned media, and cell number of single embryos

cultured in embryo-conditioned media

Chapter 4

Figure 1. Development of embryos in 5% or 20% oxygen, in groups of 10 or 76

individually

Figure 2. Cell numbers on day 4 and 5 of culture in 5% or 20% oxygen, in groups 77 of 10 or individually

Figure 3. Time of cleavage events relative to group culture at 5% oxygen 79

Chapter 5

Figure 1. Developmental stage of embryos in metabolism assays 100

Figure 2. Glucose consumption per embryo on Days 3, 4 and 5 of culture 102

Figure 3. Overall amino acid utilization on day 5 104

Figure 4. Amino acid consumption/production 107

Figure 5. Fetal and placental development 109

Chapter 6

Figure 1. Supplementation of individual mouse embryo culture with 127 recombinant mouse IL-6

Figure 2. Supplementation of individual mouse embryo cultures with 128 recombinant human IL-6

Chapter 7

Figure 1. The effects of individual culture and 20% oxygen on mouse blastocysts 140

xii

Tables Chapter 1

Table 1. Group culture vs individual culture of human embryos. 13

Chapter 2

Table 1. Composition of embryo culture media 31

Table 2. The morphological scale used to assess fetal development 46

Table 3. Sakura Tissue-Tek VIP6 Processing Schedule (short cycle) 47

Table 4. Masson Trichrome stain protocol 48

Chapter 3

Table 1. Effect of culture in microwell dishes on morphokinetics 61

Table 2. Effect of culture in microwell dishes according to cleavage times normalised 62

to the two-cell cleavage time

Supplementary Table 1. The effect of precompaction or post-compaction single 70

embryo culture

Supplementary Table 2. The effect of embryo density and oxygen on singly cultured 71

embryo development

Supplementary Table 3. Development of single embryos in embryo-conditioned 72

media

Chapter 4

Table 1. Time of cleavage events of embryos cultured in 5% or 20% oxygen, in groups 78

of 10 or individually post-hCG

Table 2. Time of cleavage events of embryos cultured in 5% or 20% oxygen, in groups 78

or individually post-t2

Supplementary Table 1. Culture of embryos in atmospheric or reduced oxygen, in 87

groups of 10 or individually.

Chapter 5

xiii

Table 1: Metabolism 104

Table 2. Postimplantation development after culture in 5% oxygen 110

Table 3. Postimplantation development after culture in 20% oxygen 112

Supplementary table 1. Developmental stage of embryos in metabolism assays 124

xiv

List of Abbreviations Acetyl-CoA - Acetyl coenzyme A

ApoA1 - Apolipoprotein A1

ART – Assisted reproductive technology

ATP – Adenosine triphosphate

BSA – Bovine serum albumin cc2 - The duration of the second cell cycle of the embryo, i.e. the interval between t2 and t3 cc3 - The duration of the third cell cycle of the embryo, i.e. the interval between t4 and t5

CRH - Corticotrophin-releasing hormone

DNP - 2,4-Dinitrophenol

DOHaD - Developmental Origins of Health and Disease

EBSS - Earle’s balanced salt solution

EGA - Embryonic genome activation

EGF - Epidermal growth factor

ELISA - Enzyme-linked immunosorbent assay

FCS – Fetal calf serum

GM-CSF - Granulocyte-macrophage colony-stimulating factor

H2O2 - Hydrogen peroxide

HAT - Histone acetyltransferase

HB-EGF - Heparin-binding EGF-like growth factor hCG – Human chorionic gonadotrophin

HIF – Hypoxia-inducible factor

HSA – Human serum albumin

ICM – Inner cell mass

ICSI - Intracytoplasmic sperm injection

IFN - Interferon

IGF - insulin-like growth factor

IL - Interleukin

ISP1 - Implantation serine protease 1

IU – International units

IVF – In vitro fertilisation

KSOM - potassium simplex optimized medium

LIF - Leukaemia inhibitory factor

MAPK - Mitogen-activated protein kinase miRNA – microRNA

MMP - Matrix metalloproteinase proteinase

MOPS - 3-(N-Morpholino) propanesulfonic acid mtDNA - Mitochondrial DNA mTOR - Mechanistic target of rapamycin

NAD - Nicotinamide adenine dinucleotide

NADPH - Nicotinamide adenine dinucleotide phosphate

NEAAs – Non-essential amino acids

OXPHOS – oxidative phosphorylation

PAF - Platelet activating factor

PBS - Phosphate-buffered saline

PDGF - Platelet-derived growth factor

PFK – Phosphofructokinase

PGT – Preimplantation genetic testing

PI3K - Phosphoinositide-3-kinase

PIF - Preimplantation Factor

PMSG – Pregnant mare’s serum gonadotrophin

PVP - Polyvinylpyrrolidone

ROS – Reactive oxygen species

SAM - S-adenosyl methionine

SSR - Synthetic serum replacement

Syn - Syngamy s2 - The duration of the second synchrony, i.e. the interval between t3 and t4 s3 - The duration of the third synchrony, i.e. the interval between t5 and t8. t2, t3 etc - Cleavage time to 2 cells, 3 cells, etc

xvi

TCA - tricarboxylic acid

TE - Trophectoderm

TGF - Transforming growth factor

TNBS - 2,4,6-trinitrobenzenesulfonic acid

TNF - Tumour necrosis factor uPA - Urokinase-type plasminogen activator

xvii

Chapter 1: General Introduction

Chapter 1: Introduction

Infertility and assisted reproduction Approximately 12% of couples worldwide suffer from infertility or subfertility, many of whom seek treatment with assisted reproductive technologies (ART), resulting in more than 1.3 million cycles of in vitro fertilisation (IVF) worldwide per year (Mascarenhas et al. 2012, Dyer et al. 2016).

In Australia, nearly 4% of babies born in 2015 were conceived using ART, and the number of couples undergoing treatment continues to increase (Fitzgerald et al. 2017). However, approximately half of couples undergoing IVF do not achieve a successful pregnancy, even after multiple treatment cycles (Malizia et al. 2009, Chambers et al. 2017, Fitzgerald et al. 2017), resulting in psychological stress for patients (Lande et al. 2015, Haimovici et al. 2018). There are also concerning reports of both short- and long-term health consequences for IVF babies, including increased risk of pregnancy complications, vascular dysfunction, altered body fat composition, high blood pressure, high fasting glucose, and increased risk of cancer, although many of these remain controversial (Romundstad et al. 2006, Ceelen et al. 2007, Ceelen et al.

2008, Kallen et al. 2010, Scherrer et al. 2012, Qin et al. 2017). Infertility treatment involves many different procedures, and techniques vary between clinics, making it difficult to determine the reason why an embryo does not result in a pregnancy, or why there may be later health problems. The underlying subfertility and health of the parents, ovarian stimulation, and procedures such as intracytoplasmic sperm injection (ICSI), biopsy or freezing may all contribute to IVF pregnancy rates and the health of IVF children (Davies et al. 2012, Raatikainen et al. 2012,

Green et al. 2013, Seggers et al. 2014, Pontesilli et al. 2015, Litzky et al. 2017). Another contributor is the environment the preimplantation embryo is exposed to during in vitro culture, since this is crucial in determining not only the ability of the embryo to form a blastocyst, but also long-term health (Kwong et al. 2000, Fernandez-Gonzalez et al. 2004, Watkins et al. 2007,

Fernandez-Gonzalez et al. 2010, Scott et al. 2010). It is therefore imperative to optimise every element of the culture environment in order to achieve the best outcomes for IVF patients and their children.

Chapter 1: Introduction

The mammalian preimplantation embryo Fertilisation to blastocyst A fertilised oocyte undergoes dramatic changes over several days to become a blastocyst

(Piliszek and Madeja 2018, Wamaitha and Niakan 2018). An oocyte is a single large cell

(approximately 70 µm diameter in the mouse and 120 µm in humans (Griffin et al. 2006)) surrounded by a glycoprotein extracellular matrix called the zona pellucida. Fertilisation triggers the reactivation of meiosis, resulting in the extrusion of the second polar body and pronucleus formation (Cuthbertson and Cobbold 1985, Saunders et al. 2002, Wakai et al. 2011). The male and female pronuclear envelopes then break down and the pronuclei fuse, known as syngamy, then the mitotic spindle assembles and mitosis begins in the newly formed zygote.

The zygote undergoes a series of symmetrical cleavage divisions which increases the number of cells from one to eight. The cytoplasm is divided evenly between the cells, which become increasingly smaller and remain uniform (Aiken et al. 2004). During these cleavage stages, the precompaction embryo undergoes embryonic genome activation (EGA), which begins at the 1- to 2-cell stage in the mouse and the 4- to 8-cell stage in the human (Li et al. 2013). Until EGA, the embryo is transcriptionally quiescent and relies on oocyte mRNAs and proteins for initial development, but must activate its own genome for development to proceed (Hamatani et al.

2004). For the embryo to activate its own genome, it must undergo global demethylation and drastic chromatin reorganisation. This clearance of gamete-specific epigenetic marks is essential for totipotency, and the genome is not extensively remethylated until after implantation (Smith et al. 2012, Guo et al. 2014).

The cleavage stage embryo compacts to form a morula, whereby cell boundaries become nearly indistinguishable and the cells become tightly packed through the formation of tight junctions and filopodia (Fleming et al. 1989). The first differentiation event is the polarisation of the blastomeres into inner and outer layers, resulting in apolar inner cells that become the inner cell

Chapter 1: Introduction mass (ICM), and polar outer cells which are precursors of the trophectoderm (TE) that will go on to form the placenta and yolk sac (Calarco and Epstein 1973, Reeve and Ziomek 1981).

Around the time that the embryo transitions from the oviduct to the uterus, it becomes a blastocyst. TE cells flatten and develop tight seals with junctional complexes to form a transporting epithelium, enabling it to create a specifically regulated internal environment for the development of the ICM (Benos et al. 1985, Bloor et al. 2004). As such, this is a key development from the cleavage stage when all blastomeres were exposed to the external environment. The basolateral membrane of TE cells contains aquaporins (Barcroft et al. 2003) and Na+/K+-ATPases (Watson et al. 1990) that pump sodium into the extracellular space to create an osmotic gradient. This energetically expensive process (Houghton et al. 2003) results in water entering the embryo to create a blastocoel cavity (Watson and Kidder 1988).

In order to implant, the embryo must hatch from the zona pellucida. In vivo, the zona pellucida is degraded by both endometrial and trophectoderm proteases (Gonzales et al. 2001), where implantation serine protease 1 (ISP1, also known as strypsin) is thought to play a vital role

(Perona and Wassarman 1986, O'Sullivan et al. 2001, Sharma et al. 2006). In the absence of endometrial protease activity in vitro, the increasing pressure from the expanding blastocyst, combined with embryo-secreted proteases, facilitates hatching (Leonavicius et al. 2018). After hatching, the process of attachment and implantation begins (Aplin and Ruane 2017).

Metabolism Corresponding to these dramatic changes between fertilisation and implantation are changes in embryo metabolism (Biggers et al. 1967, Gardner and Harvey 2015). The cleavage stage embryo is metabolically similar to the oocyte; its mitochondria are round with few cristae, required for oxidative phosphorylation (OXPHOS) (Lima et al. 2018), and it is relatively quiescent and has low levels of biosynthesis. As such, it has a low requirement for energy, which results in a high ATP:

ADP ratio (Leese et al. 1984). This allosterically inhibits phosphofructokinase, a rate-limiting

Chapter 1: Introduction enzyme in glycolysis, which restricts the early embryo from utilising glucose as an energy source

(Barbehenn et al. 1974). Consequently, the early embryo primarily utilises carboxylic and amino acids, and only low levels of glucose (Leese and Barton 1984, Partridge and Leese 1996, Lane and Gardner 2005a).

After compaction, the embryo becomes more active and rapidly uses ATP for biosynthesis, proliferation and the formation of the blastocoel. Consequently, the ATP: ADP ratio falls, alleviating the allosteric inhibition of phosphofructokinase (PFK), thereby allowing glucose to be metabolised by glycolysis (Barbehenn et al. 1974, Leese and Barton 1984, Houghton et al. 2003).

The preferred energy source then becomes glucose, approximately half of which is metabolised to lactate by aerobic glycolysis (Wales 1969, Gardner and Leese 1990, Houghton et al. 1996), a process specific to embryos, cancers and other rapidly proliferating cells (Warburg 1956). The amino acid requirement of the embryo also changes after compaction; as embryos develop they consume a wider range of amino acids and increase turnover (Partridge and Leese 1996,

Houghton et al. 2002, Booth et al. 2005). Occurring along with the formation of a transporting epithelium and differentiation, these metabolic changes are key developments for the post- compaction embryo.

The metabolisms of the two cell types of the blastocyst are different. TE cells are more metabolically active, consuming more oxygen and producing more ATP than the ICM (Houghton

2006). ICM cells have fewer and spherical mitochondria, while TE cells possess more and elongated mitochondria (Stern et al. 1971, Houghton 2006). ICM cells almost exclusively use aerobic glycolysis to metabolise glucose, whereas TE cells also use oxidative phosphorylation

(Hewitson and Leese 1993). Furthermore, the turnover of amino acids is higher in the TE than the ICM, and the two cell types utilise different amino acid profiles (Houghton 2006). These metabolic differences are thought to be a reflection of the separate roles and activities of the two cell types and are important in differentiation.

Chapter 1: Introduction

It has recently been recognised that metabolism influences epigenetic regulation, a link referred to as metaboloepigenetics (Donohoe and Bultman 2012, Harvey et al. 2016). One-carbon metabolism, which incorporates the folate and methionine cycles is essential for epigenetic regulation because it generates S-adenosyl methionine (SAM), the universal methyl donor for methylation of DNA and histones (Kwong et al. 2010, Xu and Sinclair 2015). Disruption of methionine metabolism or SAM synthesis impairs blastocyst development and differentiation and perturbs methylation (Menezo et al. 1989, Ikeda et al. 2012, Kudo et al. 2015, Sun et al.

2018). Another link between metabolism and methylation is α-Ketoglutarate, a product of the tricarboxylic acid (TCA) cycle, which modulates the activity of demethylases (Tsukada et al. 2006,

Xiao et al. 2012). Acetylation of histones can also be influenced by metabolism of glucose or ketogenic amino acids through the production of acetyl coenzyme A (acetyl-CoA) which is a cofactor for histone acetyltransferases (HAT) (Wellen et al. 2009, Shyh-Chang et al. 2013,

Choudhary et al. 2014, Moussaieff et al. 2015). Through these mechanisms and others, embryo metabolism of amino acids and carbohydrates can influence methylation status of genes and packaging of histones, and therefore gene expression and potentially long-term development

(Gardner and Harvey 2015).

The embryo’s environment in vivo As the embryo moves along the reproductive tract, it experiences gradients of oxygen and nutrients, which correspond to its needs and can influence development (Ng et al. 2018). The human oviduct has higher concentrations of pyruvate and lactate than the uterus, whereas glucose is less concentrated in the oviduct than the uterus (Gardner et al. 1996). Different gradients may exist in the mouse, where the oviduct has a higher glucose concentration than the uterus and there is no difference in lactate or pyruvate (Gardner and Leese 1990, Harris et al. 2005). Amino acid concentrations are also different between the oviduct and the uterus, in particular, the concentrations of non-essential amino acids are generally higher in the oviduct in several species, (Miller and Schultz 1987, Elhassan et al. 2001, Harris et al. 2005),

Chapter 1: Introduction corresponding to the needs of the pre-compaction embryo (Lane and Gardner 1997). Oxygen concentration is also higher in the oviduct compared to the uterus in several species (Fischer and Bavister 1993). In addition to the gradients between the oviduct and the uterus, there are also fluctuations within each part of the tract between ovulation and implantation in oxygen

(Fischer and Bavister 1993, Ottosen et al. 2006), amino acids (Miller and Schultz 1987), pH

(Mather 1975, Nichol et al. 1997, Garcia-Martinez et al. 2018), as well as peaks and rhythmic fluctuations over short time periods (Ng et al. 2018), creating a dynamic environment. These fluctuations and gradients in nutrient concentrations, oxygen and pH can influence embryo development, since the embryo is highly sensitive to its environment (Kwong et al. 2000,

Watkins et al. 2011, Williams et al. 2011, Burkus et al. 2015, Gould et al. 2018).

Oviduct and uterine fluids are extremely complex, consisting of hundreds of different molecules that can influence embryo development (Bhusane et al. 2016, Salamonsen et al. 2016). This includes carbohydrates and amino acids as already discussed, but also many other metabolites

(Lamy et al. 2018, Tribulo et al. 2018) and important molecules such as steroid hormones

(Richardson and Oliphant 1981, Casan et al. 2000), prostaglandins (Nieder and Augustin 1986) and antioxidants (El Mouatassim et al. 2000). There are a wide range of proteins in the secretome of the oviduct and uterus, many of which have only been identified in the last decade due to advances in proteomic technologies (Boomsma et al. 2009, Casado-Vela et al. 2009,

Scotchie et al. 2009, Hannan et al. 2011). Tract-secreted growth factors and cytokines have received particular attention for their potential roles in embryo development and embryo-tract communication (Kaye et al. 1992, Sjoblom et al. 1999, Binder et al. 2014, Robertson et al. 2015), and knock-out mouse models have shown that some tract growth factors and cytokines such as

GM-CSF or LIF are essential for normal pregnancy (Ingman and Jones 2008). Other proteins such as proteases and protease inhibitors (Casslen and Ohlsson 1981) and heat shock proteins

(Lachance et al. 2007) may also be important, and many others are yet to be investigated. More recently, it has been determined that uterine secretions contain exosomes and microRNAs

Chapter 1: Introduction

(miRNAs), which are another avenue of communication between the tract and the embryo

(Vilella et al. 2015, Greening et al. 2016, Lopera-Vasquez et al. 2016). At the same time, the role of the endometrial microbiota in early pregnancy is only just beginning to be understood

(Franasiak and Scott 2015, Moreno et al. 2016). It is clear that the complete makeup of reproductive fluids and each molecule’s role in embryo development is not yet understood.

As the oviduct and uterine environment can affect embryo development, so too can the embryo influence the reproductive tract before attachment and implantation (Maillo et al. 2016). This dialogue is essential to ensure synchrony between the embryo and the endometrium to initiate implantation (Weitlauf 1989). Preimplantation embryos can determine the rate of transport and the timing of implantation (Ortiz et al. 1989, Paria et al. 1993, Wakuda et al. 1999, Ueda et al.

2003, Li et al. 2015), influence vascularisation and the formation of secretory cells (Kolle et al.

2009), and influence the transcriptome, notably including many genes related to immune regulation (Chang et al. 2000, Lee et al. 2002, Beltman et al. 2010, Alminana et al. 2012, Maillo et al. 2015). The presence of embryos in the bovine uterus also changes the protein profile of uterine fluid to be more embryotrophic (Munoz et al. 2012), and protein expression differs depending on embryo sex (Gomez et al. 2013). Furthermore, human in vitro studies demonstrate that endometrial cells respond differently to conditioned media from viable blastocysts compared to non-viable blastocysts, indicating that viable embryos secrete molecules to prepare the endometrium for implantation (Cuman et al. 2013). This communication may be mediated by proteases (Brosens et al. 2014), miRNAs (Cuman et al.

2015, Parks et al. 2018), platelet activating factor (PAF) (Velasquez et al. 1995, Tiemann et al.

1996, Hermoso et al. 2001, Downing et al. 2002) and human chorionic gonadotrophin (hCG)

(Paiva et al. 2011) (Sherwin et al. 2007), and the embryo secretes a wide range of other bioactive molecules that could be part of this embryo-tract dialogue, which in vitro could facilitate embryo-embryo communication (Wydooghe et al. 2017).

Chapter 1: Introduction

Embryo culture in vitro Human embryos fertilised in vitro are cultured for up to six days before transfer to the uterus.

Embryo culture typically involves placing the embryos in a small (10 µl - 1 mL) volume of media in a polystyrene dish, typically covered with mineral oil, inside an incubator to maintain temperature, gas and pH. Most IVF labs maintain their incubators at 37°C with 5 - 6% CO2 to keep the pH of the bicarbonate buffered media at 7.2 – 7.5 (Swain 2012, Hong et al. 2014). Some labs (around 30%) also reduce the oxygen concentration to 5% to mimic the in vivo environment

(Christianson et al. 2014, Wale and Gardner 2016).

Culture media Culture media formulation is among the most important factors determining the success of IVF.

Although embryos can adapt to a range of non-physiological conditions, poorly designed culture media can reduce the ability of embryos to form blastocysts, slow cell division, influence metabolism and epigenetic regulation, and reduce post-implantation viability (Lane and Gardner

2007, Chronopoulou and Harper 2015). Since the birth of Louise Brown, the first IVF baby in

1978, culture media have advanced from simple salt solutions containing glucose, pyruvate, lactate and serum to become more physiological (Leese 1998, Niederberger et al. 2018). Modern culture media vary between manufacturers, but most are now based around an aqueous mix of salts, carbohydrates, vitamins, amino acids, chelators, and albumin (Gardner 2008, Morbeck et al. 2014a, Chronopoulou and Harper 2015). Two of the most important recent additions were amino acids (Gardner and Lane 1993, Devreker et al. 2001) and vitamins (Lane and Gardner

1998), both of which are found in reproductive fluids. Further, sequential or “two-step” media, were designed to reflect the nutrient gradients that occur between the oviduct and uterus and the changing metabolism of the developing embryo (Gardner and Lane 1998). Culture media, while much improved, is still far from a recreation of the in vivo environment.

Although only a few tract-secreted molecules are in human culture media, most, if not all, of the molecules present in the tract have the potential to influence embryo development in vitro.

Chapter 1: Introduction

Some culture media contain one or two additional tract-secreted molecules, one example is the glycosaminoglycan hyaluronan, which is found in high concentrations in oviduct fluid (Carson et al. 1987). Addition of hyaluronan to culture media increases blastocyst hatching, implantation rate and fetal development rate in the mouse and bovine (Furnus et al. 1998, Gardner et al.

1999, Jang et al. 2003), plausibly due to activation of mitogen-activated protein kinase (MAPK) signalling (Marei et al. 2013). Another major component of oviduct fluid added to culture media are immunoglobulins (Aitken 1977), which have been shown to improve mouse (Schneider and

Hayslip 1996, Tanikawa et al. 1999) and human (Weathersbee et al. 1995, Meintjes et al. 2009b) embryo development in vitro. Immunoglobulin supplements also contain unidentified proteins and steroids, and as such their mechanisms of action in embryo culture have not been determined. Some human culture media also contain the hormone insulin, which acts as a growth factor to promote embryo development (Heyner et al. 1989, Harvey and Kaye 1990).

There have been conflicting reports on the mechanism of insulin’s action on embryos of different species (Navarrete Santos et al. 2004, Navarrete Santos et al. 2008, Campbell et al. 2012,

Campbell et al. 2013) and while reports from human studies on blastocyst formation and pregnancy rates have been positive (Fawzy et al. 2016), no randomised controlled trials have been conducted. More recently, media for human embryo culture containing the growth factor granulocyte-macrophage colony-stimulating factor (GM-CSF, also known as CSF2) have been developed. GM-CSF is one of the many growth factors present in the tract and addition of GM-

CSF to embryo culture promotes development by inhibiting cellular stress pathways and apoptosis (Chin et al. 2009). However, this effect may only be apparent under suboptimal culture conditions (Karagenc et al. 2005, Ziebe et al. 2013). There are many other tract-secreted growth factors and cytokines that have an embryotrophic effect in vitro but have not been widely adopted or added to commercial media formulations, such as insulin-like growth factor (IGF)-I

(Harvey and Kaye 1991, Lighten et al. 1998), leukemia inhibitory factor (LIF) (Fry 1992, Dunglison et al. 1996) and heparin-binding EGF-like growth factor (HB-EGF) (Das et al. 1994, Martin et al.

Chapter 1: Introduction

1998). There are two major concerns regarding the addition of growth factors to culture media.

Firstly, growth factors are potent regulators of cell function, and promoting proliferation and preventing apoptosis during the preimplantation stages could have unforeseen long-term consequences. Secondly, growth factors can have complimentary or contradictory actions, hence the relative levels of various growth factors could be important in regulating appropriate embryo development. As such, very few tract-secreted molecules are currently components of embryo culture media. However, embryos themselves secrete growth factors during in vitro culture, although the identities and actions of these growth factors are poorly defined.

Rather than add individual tract-secreted molecules to media, some researchers have co- cultured embryos with oviductal or endometrial cells (Bongso and Fong 1993, Orsi and Reischl

2007, Menezo et al. 2012). This has been trialled with several different somatic cells, including autologous cumulus cells and cell lines such as African Green Monkey Kidney (Vero) cells.

However, these approaches result in highly variable and undefined culture systems, plus the embryos and somatic cells have different requirements in vitro, and there are concerns regarding disease transmission from non-autologous cells. As such coculture is not ideal for human embryo culture, and other methods of supporting embryo development are preferred.

Individual culture of embryos The preimplantation embryo is unusual in that it does not require signalling from neighbouring cells to survive and proliferate (Raff 1992), and therefore, unlike most somatic cells, embryos can be cultured individually. Until recently, most human embryos were cultured in small groups in a drop of medium, but approximately half of IVF clinics worldwide now culture embryos individually (Christianson et al. 2014). Due to technological changes in embryo culture, more patients now receive a single embryo transferred back to the uterus (Sunderam et al. 2018). In the past, doctors transferred multiple embryos to a patient to improve the chances of a pregnancy, but with modern embryology techniques, this practice results in an unacceptably high rate of twin pregnancies because the embryos are now of higher quality (Gardner et al.

Chapter 1: Introduction

2004b). Consequently, a single embryo needs to be selected for transfer out of a group of other morphologically similar embryos. To select the most viable embryo, development is monitored during culture, so that the progress of each embryo can be taken into account when deciding which embryo to transfer, and this requires embryos to be cultured individually. To facilitate monitoring, the use of time-lapse microscopy to track embryos during culture has become increasingly popular in the last five years (Dolinko et al. 2017). These developments have lead to the individual culture of more embryos, but the potential consequences of this change in embryo culture practices are unclear.

Despite the prevalence of individual embryo culture, there is very little research in humans on its use (Reed 2012)(Table 1). Only one study has compared grouped and individual culture of human embryos to the blastocyst stage and found that individual culture resulted in lower development rates on days 4 and 5, and fewer top quality blastocysts (79.2% vs 64.7%) (Ebner et al. 2010). In addition, there was a non-significant decrease in pregnancy and live birth rates from 62.2% to 38.5%. Other studies have compared group and individual culture during cleavage stages only, and while some found that individual culture decreases cell number and development rate (Moessner and Dodson 1995, Rebollar-Lazaro and Matson 2010), others reported no differences (Almagor et al. 1996, Spyropoulou et al. 1999). Most of these studies, with the exception of Almagor et al. (1996), reported no significant change in pregnancy rates.

Only one study has investigated the effect of group culture during the post-compaction period only and reported no difference in development or pregnancy rates (Rijnders and Jansen 1999).

The evidence available suggests that cleavage-stage group culture is beneficial for human embryos, but these studies all used different clinical practices, culture conditions and study designs, so the lack of protocol consistency makes interpretation of the data difficult. In particular, the number of embryos in the group, the duration of group culture and the volume of media are all important variables.

Chapter 1: Introduction

For insight into this issue, we can turn to animal models. Paria and Dey (1990) first demonstrated that mouse embryos cultured in groups were more likely to form blastocysts and had more cells than individually cultured embryos. Since then, many research groups have confirmed in multiple species that individual culture results in inferior preimplantation development, including changes in allocation to the ICM and TE, and increases in apoptosis (Lane and Gardner

1992, Gardner et al. 1994, Keefer et al. 1994, Brison and Schultz 1997, Isobe 2014), at least in part through the action of p53 (Chandrakanthan et al. 2006). Embryos from monoovulatory species, which would not normally encounter another embryo in vivo, also benefit from group culture (O'Doherty et al. 1997, Larson and Kubisch 1999, Fujita et al. 2006, Gopichandran and

Leese 2006). Only a handful of studies have compared the post-implantation development of individually or group-cultured embryos, and all have observed only a trend for decreased implantation or live birth rates (Lane and Gardner 1992, Kato and Tsunoda 1994, Isobe 2014).

Despite the abundance of data regarding the effect of group culture on blastocyst development, there is no data available on possible changes to embryo physiology, epigenetics, or the long- term development and health of offspring as a result of group or individual culture.

Chapter 1: Introduction

Table 1. Group culture vs individual culture of human embryos. *Treatments conducted in series. EBSS: Earle’s balanced salt solution; HSA: human serum albumin; SSR: synthetic serum replacement.

Moessner and Almagor et al. Spyropoulou et al. Rijnders and Jansen Rebollar-Lazaro and Ebner et al. (2010) Dodson (1995) (1996) (1999) (1999) Matson (2010)* Results Group culture No effect of group No difference in No difference in Group culture Group culture resulted in higher culture on cell cleavage rate, cell blastocyst rate, resulted in more resulted in more cell number, no numbers, numbers, implantation rate or useable blastocysts compacting effect on morphology score or morphological pregnancy rate. for women <35, but embryos on Day 3, morphology grade. development rates, score, or not >35. No more blastocysts on Number of embryos but resulted in implantation or difference in Day 5, and more top per group correlated higher pregnancy pregnancy rate. implantation or quality blastocysts. to cell number. rate. pregnancy rate Trend for higher pregnancy and live birth rate. Individual culture 1 embryo in 1 mL 1 embryo in 700 µL 1 embryo in 20 µL 1 embryo in 5 µl or 1 embryo in 15 µL 1 embryo in 30 µL 160 µl Group culture 2-5 embryos in 1 mL 3-6 embryos in 700 3-5 embryos in 20 µL 2-4 embryos in 160 >3 embryos in 15 µL 3-5 embryos in 30 µL µL µl or 10-20 µl (5 µl/embryo) Culture period Day 1-2 Day 1-2 Day 1-2 All embryos cultured Embryo cultured in Day 1-5 in groups Day 1-3, treatments Day 1-3, then assigned to then all cultured treatments Day 3-5 individually Day 3- 5/6 Number of patients 55 91 159 25 760 72 Oxygen 20% 20% 20% 20% Not specified Not specified Culture medium Modified Ham’s F10 Ham's F10 + 15% EBSS + SSR + HSA Mix of EBSS and Cook Cleavage MediCult + 15% serum serum Ham’s F10 + 9% Medium and EmbryoAssist and pasteurised plasma Blastocyst Medium BlastAssist

Chapter 1: Introduction

Culturing embryos in groups promotes development due to the microenvironment they create during culture. This was demonstrated by two studies where embryos were adhered to the culture dish and as the distance between embryos increased, and therefore the dilution of the embryo-conditioned microenvironment, there was a decrease in blastocyst rate and cell number and altered metabolism (Stokes et al. 2005, Gopichandran and Leese 2006). Further, collecting embryo-conditioned culture media, then using it to culture new embryos can improve development (Stoddart et al. 1996, Fujita et al. 2006). The beneficial effects of this embryo- conditioned microenvironment are increased if embryos are of high quality or more developmentally advanced (Spindler and Wildt 2002, Spindler et al. 2006). Concentrating the microenvironment by reducing the embryo:media volume ratio, known as embryo density, can improve the development of both individual and grouped embryos (Wiley et al. 1986, Paria and

Dey 1990, Lane and Gardner 1992, Ferry et al. 1994). However, the group culture microenvironment is not always beneficial, as poor quality embryos or unfertilised oocytes can have a detrimental effect on their neighbours (Salahuddin et al. 1995, Tao et al. 2013). The embryo’s in vitro microenvironment consists of a wide variety of molecules that can influence preimplantation development, including paracrine signalling molecules, discussed in detail below. Identifying the embryo-secreted molecules responsible for promoting development during group culture could lead to the development of a better culture medium for individual embryo culture.

Microwell dishes Time-lapse incubators require embryos to be cultured in specialised microwell dishes instead of conventional culture, and these microwells can improve the development of individually cultured embryos. Culture in microwell dishes can improve the blastocyst rate, cell numbers, and ICM size of mouse (Vajta et al. 2008, Dai et al. 2012), bovine (Pereira et al. 2005), porcine

(Vajta et al. 2008) and human (Vajta et al. 2008, Hashimoto et al. 2012) embryos compared to conventional individual culture. Importantly, the effect on embryo development is dependent

Chapter 1: Introduction on the shape and size of the microwells (Taka et al. 2005, Feltrin et al. 2006, Hoelker et al. 2010,

Feltrin 2015) and the composition of the culture media (Ieda et al. 2018). The benefit of these dishes may be due to the reduced media volume surrounding the embryo which concentrates the embryo-conditioned microenvironment, as demonstrated by culture with only one microwell (Vajta et al. 2000, Dai et al. 2012). Alternatively, there may be paracrine communication between embryos, although there are several reports supporting (Dai et al.

2012, Lehner et al. 2017, Ieda et al. 2018) or contradicting (Vajta et al. 2000, Pereira et al. 2005,

Sugimura et al. 2013, Kang et al. 2015) this hypothesis, probably due to differences in microwell size and distance. Microwell dishes, therefore, provide a promising alternative for culture of individual embryos.

Oxygen One of the most important determinants of embryo development in vitro is the concentration of oxygen in the incubator (Wale and Gardner 2016, Bolnick et al. 2017). Although a concentration of around 2-8% oxygen is more physiological (Mastroianni and Jones 1965, Fischer and Bavister 1993), it is estimated that around 75% of IVF clinics worldwide culture embryos under atmospheric oxygen (Christianson et al. 2014). Compared to culture under 5% oxygen, this non-physiological excess of oxygen has a wide range of detrimental effects on embryo development, including slower cleavage divisions (Wale and Gardner 2010, Kirkegaard et al.

2013), lower blastocyst rates and fewer cells per embryo (Whitten 1971, Tervit et al. 1972, Quinn and Harlow 1978, Dumoulin et al. 1999), more apoptosis (Van Soom et al. 2002, Yuan et al.

2003), aneuploidy (Bean et al. 2002), and DNA damage (Takahashi et al. 2000, Kitagawa et al.

2004). Embryos cultured in 5% and 20% oxygen also exhibit differences in gene expression

(Harvey et al. 2004, Kind et al. 2005, Rinaudo et al. 2006), protein expression (Katz-Jaffe et al.

2005), protein secretion (Kubisch and Johnson 2007), and metabolism (Khurana and Wales 1989,

Wale and Gardner 2012). These changes are likely to be associated with the differences observed in histone remodelling and methylation (Gaspar et al. 2015, Li et al. 2016), which

Chapter 1: Introduction indicate that the effects of oxygen during the preimplantation culture period may be programming later development. Indeed, culture under atmospheric oxygen perturbs postimplantation development, resulting in lower implantation, pregnancy, and live birth rates in humans (Catt and Henman 2000, Meintjes et al. 2009a, Waldenstrom et al. 2009, Kovacic et al. 2010, Gomes Sobrinho et al. 2011), and higher miscarriage rates in mice (Karagenc et al.

2004), compared with culture in 5% oxygen. Atmospheric oxygen is a well-characterised culture stress, and combined with its clinical relevance, this makes it a useful model for stress in vitro.

Oxygen can influence embryo development through several mechanisms. Exposure to atmospheric oxygen can cause an increase in intracellular reactive oxygen species (ROS) in embryos (Goto et al. 1993, Kitagawa et al. 2004). ROS are important intracellular signalling molecules, but an imbalance in ROS can cause not only dysregulation of signalling pathways but also damage to lipids, proteins and DNA (Guerin et al. 2001, Harvey et al. 2002) and altered epigenetic marks (Menezo et al. 2016). On the other hand, a low oxygen environment can stabilise hypoxia-inducible factors (HIFs), which are transcription factors with a wide range of target genes, including glucose transporters and metabolic enzymes such as lactate dehydrogenase (Semenza et al. 1994, Harvey et al. 2004, Kind et al. 2005, Redel et al. 2012). HIF-

1α is not activated at 5% oxygen in the blastocyst (Harvey et al. 2004, Yoon et al. 2013), but other HIFs may be active at this concentration. HIF-2α responds to 5% oxygen in other cell types

(Holmquist-Mengelbier et al. 2006), and is activated in mouse blastocysts at 3% oxygen (Ma et al. 2017), and bovine blastocysts at 2% oxygen (Harvey et al. 2004). Through these mechanisms, high or low oxygen concentrations can regulate intracellular signalling pathways, metabolism, and epigenetic regulation of gene expression in the embryo.

The embryo’s in vitro microenvironment Although the in vitro culture environment is seemingly controlled and static, with defined media components and constant temperature and gas, the embryos themselves create a dynamic microenvironment (Gardner 2008). They consume nutrients from the media and produce a wide

Chapter 1: Introduction variety of metabolites and other bioactive molecules including nucleic acids, lipids, metabolites, exosomes and proteins including growth factors and cytokines. Among those molecules that have been identified, there is considerable overlap with molecules present in oviduct or endometrial fluids, and as such, culturing embryos together may partially compensate for the absence of paracrine signals from the tract. Many of the embryo-secreted molecules identified to date have been characterised for their roles in signalling to the tract or as biomarkers of embryo viability, and their potential actions on other embryos have not been investigated.

Below is a description of several components of the embryo’s in vitro microenvironment, with an emphasis on molecules that are known to influence embryo physiology, development or viability.

Metabolites Embryos consume and produce metabolites during culture, creating gradients of these molecules which can influence embryo metabolism. Metabolism of carbohydrates is dependent on their concentration and the concentration of other metabolites; for example, Gardner and

Leese (1988, 1990) found that by changing the concentrations of pyruvate, lactate and glucose to more physiological levels, the glycolysis rate of mouse embryos also became more physiological. Furthermore, amino acid utilisation (Lamb and Leese 1994) and transport (Van

Winkle et al. 2006, Tan et al. 2011) are dependent on the presence or concentration of other amino acids. Amino acid metabolism and carbohydrate metabolism are also linked, for example, aspartate is the rate-limiting factor in the malate-aspartate shuttle which regenerates cytosolic

NAD+ required for the metabolism of glucose to pyruvate, and the absence of aspartate in the medium prevents the embryo from utilising lactate as an energy source (Lane and Gardner

2005a, Mitchell et al. 2009). Likewise, the absence of pyruvate in the medium changes the embryo’s utilisation of amino acids (Orsi and Leese 2004). These examples demonstrate how the nutritional environment of the embryo determines its physiology, and thus it is important to provide physiological levels of these nutrients in the artificial environment of in vitro culture.

Chapter 1: Introduction

As well as carbohydrates and amino acids, embryos alter the concentration of other metabolites during culture, such as oxygen (Mills and Brinster 1967, Trimarchi et al. 2000), carbon dioxide

(Brinster 1967), fatty acids (Hillman and Flynn 1980, Yamada et al. 2012) and lipids (Braga et al.

2015, Borges et al. 2016), gradients of which may influence metabolism. Recent metabolic profiling by mass spectrometry has now also provided data on turnover of a range of novel or under-researched metabolites (Sanchez-Ribas et al. 2012, Yamada et al. 2012, Bellver et al. 2015,

Gomez et al. 2016, Nomm et al. 2018), all of which contribute to the embryo microenvironment in vitro and could influence embryo development.

Measurement of metabolites in culture medium can be used as an indicator of viability (Renard et al. 1980). For example, higher pyruvate consumption by cleavage stage embryos is associated with human blastocyst development (Hardy et al. 1989b), and at the blastocyst stage, high glucose consumption together with a low glycolytic rate is indicative of post-transfer development and live birth in the mouse and human (Gardner and Leese 1987, Lane and

Gardner 1996, Gardner et al. 2011). Similar to carbohydrate metabolism, amino acid metabolism has been correlated with morphokinetics, blastocyst development, DNA damage, aneuploidy and pregnancy rate in the human, mouse, and other species (Houghton et al. 2002, Brison et al.

2004, Booth et al. 2007, Seli et al. 2008, Sturmey et al. 2009, Picton et al. 2010, Lee et al. 2015,

D'Souza et al. 2016, Souza et al. 2018). However, there has been little consistency between these studies which aim to characterise amino acids as indicators of viability. This may be due in part to species differences, the stage of the embryo during testing, and culture conditions, all of which affect embryo metabolism (Sturmey et al. 2008, Wale and Gardner 2012).

Beyond production of ATP and biosynthesis, metabolites also have other functions within the embryo. For example, pyruvate is an antioxidant and as such can protect embryos from damage from excessive ROS (Andrae et al. 1985). Lactate, often considered just a waste product of glycolysis, is an important signalling molecule and pH regulator (Gardner 2015). Amino acids

Chapter 1: Introduction have many roles in culture, acting as osmolytes (Van Winkle et al. 1990, Lawitts and Biggers

1992, Dumoulin et al. 1997, Steeves and Baltz 2005), antioxidants and chelators (Lindenbaum

1973, Suzuki et al. 2007) and intracellular pH buffers (Edwards et al. 1998). Furthermore, amino acids can regulate the activity of key cell signalling pathways such as mechanistic target of rapamycin (mTOR) (Van Winkle et al. 2006). These are just a few examples of the ways metabolite gradients during culture can influence embryo development beyond regulating metabolism.

Immunosuppressive factors In the 1980s, it was demonstrated that embryos produced unidentified immunosuppressive factors (Smart et al. 1981, Daya and Clark 1986). These molecules facilitate embryo-tract communication, but in vitro can also act as autocrine or paracrine signalling molecules between embryos. The first embryo-secreted immunosuppressive molecule to be thoroughly characterised was the phospholipid PAF (O'Neill 1985, Collier et al. 1988). PAF is secreted by both the tract and the embryo from the zygote stage and is an embryotrophic autocrine/paracrine signalling molecule which acts through the phosphoinositide-3-kinase

(PI3K) signalling pathway (O'Neill 2005, O'Neill et al. 2015). Another immunosuppressive factor is Preimplantation Factor (PIF), which is a peptide secreted by mouse and human embryos

(Roussev et al. 1996, Barnea et al. 1999, Stamatkin et al. 2011a) and may function as a growth- promoting autocrine/paracrine factor in vitro (Stamatkin et al. 2011a, Stamatkin et al. 2011b,

Barnea et al. 2014, Goodale et al. 2017). These immunosuppressive molecules may therefore have important roles in promoting embryo development in vitro.

Growth factors and cytokines In the 1990s, growth factors and cytokines were identified in embryo-conditioned medium. The first cytokines detected were interleukins (IL)-1 (Zolti et al. 1991, Austgulen et al. 1995, Baranao et al. 1997), IL-2 (Orvieto et al. 1997), IL-6 (Zolti et al. 1991, Austgulen et al. 1995, Yu et al. 2012) and IL-10 (Ozornek et al. 1995, Criscuoli et al. 2005), along with tumour necrosis factor (TNF)

Chapter 1: Introduction

(Witkin et al. 1991, Zolti et al. 1991) and interferon gamma (IFNγ) (Ozornek et al. 1995, Ozornek et al. 1997). Among the first growth factors reported were platelet-derived growth factor (PDGF)

(Svalander et al. 1991), insulin-like growth factors (IGF)-I (Inzunza et al. 2010) and IGF-II

(Hemmings et al. 1992, Winger et al. 1997) and transforming growth factors (TGF)-α and TGF-β

(Hemmings et al. 1992, Austgulen et al. 1995). Subsequently, many more cytokines and growth factors have been identified in embryo-conditioned media using protein arrays (Dominguez et al. 2008, Dominguez et al. 2015) or multiplex ELISAs (Borgatti et al. 2008, Lindgren et al. 2018), and the protein or mRNA of many others has been detected in the embryo and are probably also secreted (Sharkey et al. 1995, Thouas et al. 2015). Some of these embryo-secreted growth factors and cytokines have been added to embryo culture and have been found to promote development and therefore are likely candidates for embryo-embryo communication. Examples include IGF-I (Harvey and Kaye 1991, Lighten et al. 1998), IGF-2 (Harvey and Kaye 1992), platelet- derived growth factor (PDGF) (Larson et al. 1992), TGF-α (Paria and Dey 1990), and IL-6 (Shen et al. 2009).

Other proteins To identify novel proteins of interest, the embryo secretome has been investigated using untargeted proteomics. However, few proteins previously identified as secreted by embryos have been detected by these methods (Beardsley et al. 2010, Cortezzi et al. 2011, Foresta et al.

2016). Some novel embryo-secreted proteins have been identified as potential viability markers, such as ubiquitin (Katz-Jaffe et al. 2006), corticotrophin-releasing hormone (CRH) (Katz-Jaffe et al. 2010), lipocalin-1 (McReynolds et al. 2011) and apolipoprotein A1 (ApoA1) (Mains et al. 2011) but to date there has been little consistency between reports aiming to characterise the embryo secretome using proteomics, and few of the proteins identified by proteomics have been investigated in detail or their role in the embryo-trophic microenvironment determined.

Chapter 1: Introduction

Nucleic acids and microvesicles In the last 5 years, embryo-secreted exosomes (Saadeldin et al. 2014), miRNAs (Kropp et al.

2014, Rosenbluth et al. 2014), mRNAs (Kropp and Khatib 2015a), mitochondrial DNA (mtDNA)

(Stigliani et al. 2013) and nuclear DNA (Hammond et al. 2017) have been identified in embryo culture media. While cell-free DNA has received a great deal of interest for its potential application in preimplantation genetic testing (PGT) (Feichtinger et al. 2017, Liu et al. 2017, Yang et al. 2017, Capalbo et al. 2018, Kuznyetsov et al. 2018, Vera-Rodriguez et al. 2018), it also has the potential to influence the activity of neighbouring embryos (Konecna et al. 2018). Both miRNAs and exosomes (which may also contain miRNAs) have the potential to be important for embryo-maternal signalling (Cuman et al. 2015, Vilella et al. 2015, Lopera-Vasquez et al. 2016,

Giacomini et al. 2017, Gross et al. 2017, Lv et al. 2018, Parks et al. 2018) and may also be significant in embryo-embryo signalling in vitro. Embryos can take up exosomes from culture media (Saadeldin et al. 2014) and embryo-secreted exosomes can improve bovine blastocyst development and live birth rate (Qu et al. 2017). Also, embryo-secreted miR-24 influences bovine embryo development and gene expression (Kropp and Khatib 2015b). As such, these exosome and nucleic acids could be important autocrine/paracrine signalling molecules in vitro.

Preimplantation environment influences postnatal development The preimplantation embryo is sensitive to changes in its environment, which can determine its growth and influence fetal development and beyond into adulthood. This concept is known as the Developmental Origins of Health and Disease (DOHaD) hypothesis, first proposed by David

Barker (Barker et al. 1989, Feuer and Rinaudo 2016). Chronic diseases can occur when there is a mismatch between the early environment and the adult environment. This has been demonstrated in vivo in rodents, where short-term changes to maternal diet, restraint stress, or infection during preimplantation development can influence postnatal growth, behaviour, immune response, and cardiovascular health (Kwong et al. 2000, Watkins et al. 2011, Williams et al. 2011, Burkus et al. 2015, Gould et al. 2018). Similarly, alterations to the in vitro culture

Chapter 1: Introduction environment can have lasting consequences. For example, a series of experiments compared culture of mouse embryos in “poor” conditions (atmospheric oxygen and simple medium) with

“good” conditions (5% oxygen and potassium simplex optimized medium (KSOM) with amino acids) and found sex-specific differences in postnatal growth trajectory, glucose tolerance, fat composition and insulin secretion (Donjacour et al. 2014, Feuer et al. 2014). Another study found that changing the medium protein source from bovine serum albumin (BSA) to fetal calf serum

(FCS) delayed postnatal neuromotor development and altered body composition and growth rates, and altered behaviour and memory in mice (Fernandez-Gonzalez et al. 2004). This lasting effect of environment on development can occur from a relatively short exposure. Culturing pronucleate mouse embryos in culture media with altered carbohydrate composition or pH for only 15 h resulted in changes in birth weight and postnatal growth trajectory (Banrezes et al.

2011), and mouse blastocysts cultured without amino acids for only 6 h reduced fetal weight

(Lane and Gardner 1998). The effects of changing the preimplantation environment can even carry through the next two generations in mice (Mahsoudi et al. 2007). These long-term consequences of short-term exposure to altered environmental conditions may be due to epigenetic regulation, since the embryo undergoes crucial epigenetic programming events during the preimplantation development. Indeed, changing the preimplantation environment can affect preimplantation DNA and histone methylation (Mann et al. 2004, Gaspar et al. 2015,

Li et al. 2016, Salvaing et al. 2016) and expression of imprinted genes (Doherty et al. 2000,

Fernandez-Gonzalez et al. 2004, Mann et al. 2004, Schwarzer et al. 2012), and induce different patterns of DNA methylation in the placenta (Mann et al. 2004, Ghosh et al. 2017) and fetus

(Khosla et al. 2001). Since environment can influence the metabolic activity of the embryo

(Gardner and Harvey 2015), metabolomics provides a plausible mechanism to explain how the preimplantation environment can induce epigenetic changes and thereby influence long-term health. It is clear that the culture environment of the preimplantation embryo is key in

Chapter 1: Introduction determining not only blastocyst development, but long-term health, and efforts must be made to optimise it.

Multiple stresses The preimplantation embryo is adaptable and will survive in a range of conditions, however, conditions outside the optimal range will cause an embryo stress. The consequence of this stress during the preimplantation period has the potential to cause long-term harm (Feuer and

Rinaudo 2016). A stressor can be any stimulus that triggers a response from the embryo to restore homeostasis or repair and protect itself, or, conversely, the absence of a stimulus required for normal development. A response to a stressor can be observed as upregulation of specific stress response proteins or a change in gene expression or cellular function such as altered metabolism, which results in slower cell divisions, apoptosis, or loss of viability (Feuer and Rinaudo 2012, Puscheck et al. 2015).

Susceptibility to stress can be stage-specific; in general, the pre-compaction embryo is more sensitive due to the lack of a transporting epithelium and regulatory transport systems (Gardner and Lane 2005, Lane and Gardner 2005b). Stress experienced during the precompaction stages is often manifest at the blastocyst stage, and embryos may not recover even when the stress is removed post-compaction (Wale and Gardner 2010).

When an embryo is subject to one source of stress in vitro, it may become more susceptible to additional stress from other sources (Awonuga et al. 2013). For example, mouse blastocysts can alleviate the stress of ammonium in the culture medium by transamination to glutamine and alanine, but not when they are exposed to the additional stress of 20% oxygen (Wale and

Gardner 2013). Individual in vitro culture may be regarded as culture stress because individually cultured embryos are less likely to form a blastocyst, have fewer cells, more apoptosis, and altered allocation to the ICM (Paria and Dey 1990, Lane and Gardner 1992, Brison and Schultz

1997). This implies that embryos cultured individually may be less able to survive exposure to

Chapter 1: Introduction additional stresses. This is supported by evidence from Hughes et al. (2010), who found that embryos cultured in groups were more resilient to hydroperoxide from the mineral oil overlay than embryos cultured individually. Importantly, embryos are not only subject to the stresses of culture (Wale and Gardner 2016), but the genetic background, infertility and health of both parents can determine embryo quality (Finger et al. 2015, McPherson et al. 2015, McPherson et al. 2018) and the ability of embryos to survive in vitro stress (Sakatani et al. 2017).

Summary IVF is a common treatment for infertility, resulting in thousands of babies born each year.

However, due to success rates of around 21% per cycle, many couples require multiple cycles of treatment to conceive a child, and many others remain childless. Further, there are concerns regarding the short- and long-term health of IVF children. Both success rates and health outcomes may be improved by optimisation of embryo culture conditions. Advances in embryo culture technologies have prompted a move towards individual culture, but this may be detrimental for preimplantation and postimplantation development. The consequences of this practice need to be investigated further, particularly in the wider context of changing other variables within a culture system, resulting in added stress for the embryo. Further, the in vitro microenvironment of the embryo should be understood, for its potential in providing biomarkers of embryo health, but also with the aim of understanding how the embryo- conditioned environment regulates development.

Hypotheses and Aims Hypothesis: The microenvironment created by the preimplantation embryo in vitro regulates its development and viability, and its ability to cope with external stressors.

Aims:

- Investigate the influence of culture conditions on development of individual preimplantation embryos in vitro.

- Characterise the impact of combining two stressful culture conditions (individual culture and atmospheric oxygen) on preimplantation embryo development, health, and developmental potential.

Chapter 1: Introduction

- Identify embryo-secreted factors and investigate their role in embyrotrophic signalling.

Chapter 2: Materials and methods

Chapter 2: Methods

Chemicals and reagents Unless otherwise stated, all chemicals and reagents were obtained from Sigma-Aldrich, St Louis,

USA.

Animals This study was carried out in accordance with the Australian code of practice for the care and use of animals for scientific purposes. Ethical approval was obtained from the University of

Melbourne Animal Ethics Committee (#1513740.1 and #1413159.3) prior to experimentation.

All efforts were made to minimise the suffering of animals and to reduce the number of animals required. F1 hybrid (C57BL/6 x CBA) mice were bred by the Florey Institute of Neuroscience and

Mental Health animal facility and maintained in a standard animal research facility in the School of BioSciences 4 at the University of Melbourne. Mice were housed in individually ventilated cages (Optimice, Animal Care Systems, Centennial, CO, USA) with a 12 h light–dark photoperiod

(6 am - 6 pm) with controlled temperature and food and water available ad libitum.

C57Bl/6 x CBA F1 mice were selected because they are an established model strain for the study of embryo culture. They are known to respond well to hormone stimulation and therefore produce more embryos per mouse than some other strains. In addition, their embryos grow well in culture, and therefore fewer are needed.

Quality control and quality assurance in the embryology laboratory All chemicals and plastics were prescreened in a mouse embryo assay prior to use (Gardner et al. 2005). Pronucleate oocytes were cultured in simple G1 medium without amino acids, and after 24 h were moved to simple G1 without amino acids or albumin. On the fifth day of culture, blastocyst development was assessed and cell number determined by staining with bizbenzamide. If >80% reached the expanded blastocyst stage and the mean cell number was

>50, the component tested was considered suitable for use.

Chapter 2: Methods

The temperature and gas levels inside the incubators were monitored at least fortnightly.

Temperature was measured using a thermometer with a thermocouple (Traceable, Webster, TX,

USA). Oxygen concentration was measured with an O2 meter (Oxor III, Bacharach, New

Kensington, PA, USA) and carbon dioxide with a CO2 meter (GM70, Vaisala, Helsinki, Finland).

Generation and collection of embryos Ovarian stimulation and mating To reduce the number of mice required, exogenous hormones were used to stimulate the ovaries of female mice. Fertilised embryos were generated by superovulation of four week old

F1 hybrid females with intraperitoneal injections of 5 international units (IU) pregnant mare serum gonadotropin (PMSG, Folligon; Intervet, Bendigo East, Vic, Australia) at the mid-point of the light phase, followed 48 h later by 5 IU human chorionic gonadotropin (hCG) (Chorulon;

Intervet). Superovulated females were then mated with males of the same strain overnight.

Embryo collection Pronucleate oocytes were collected 22 h after hCG injection (Gardner et al. 2004a). Mice were killed by cervical dislocation and placed ventral side up. The abdomen was disinfected with 80% ethanol then an incision was made in the ventral midline to expose the peritoneum, which was cut with fine scissors. The visceral organs were moved to the side to expose the reproductive tract. The utero-tubal junction was held with fine forceps and the uterus pulled uterus taught, then connective tissue was removed from the uterus using fine scissors. The oviduct was dissected and placed in a 35 mm petri dish (Falcon Easy-Grip; Corning Life Sciences, Tewksbury,

MA, USA) containing 2 mL warmed G-MOPS PLUS handling medium (Vitrolife, Göteborg,

Sweden). The oviduct was transferred to a 60 mm centre well organ culture dish (Falcon) containing 400 µL G-MOPS PLUS under an SMZ 1500 dissection microscope (Nikon Instruments,

Melville, NY, USA) with a heated stage (Tokai Hit, Shizuoka, Japan). The swollen ampulla was torn using fine forceps, releasing the cumulus mass. The oviduct was discarded and 400 µL hyaluronidase was added (Type IV-S, Sigma), resulting in a final concentration of 550 IU/ml

Chapter 2: Methods hyaluronidase (bovine testes type IV-S; Sigma-Aldrich, St Louis, MO, USA). As soon as the cumulus cells were removed, pronucleate oocytes were moved to a fresh drop of G-MOPS PLUS by pipetting with a hand-pulled Pasteur pipette made from borosilicate glass, with a diameter slightly larger than the embryos. Embryos were washed three times in G-MOPS PLUS and once in pre-incubated G1 culture medium. Embryos were pooled, and if at least one pronucleus was observed, they were allocated to culture treatments.

Embryo culture Culture media G1 and G2 culture media were prepared in the laboratory as described previously (Gardner and

Lane 2007, 2014), with specific modifications. To reflect the current commercial formulation of

G media, choline chloride, folic acid, inositol, nicotinamide and taurine were omitted from G2.

Media were supplemented with 2.5 mg/ml recombinant albumin (G-MM, Vitrolife) instead of human serum albumin, to eliminate the potential effects of contaminants inherent in serum albumin (Bar-Or et al. 2005, Dyrlund et al. 2014, Morbeck et al. 2014b). The composition of all culture media is shown in Table 1.

For metabolic experiments, alanyl-glutamine was substituted with L-glutamine for the purposes of measuring amino acid utilisation, and the concentration of glucose was reduced to 0.5 mM in

G2 medium to facilitate measurement of glucose uptake.

For embryo handling during collection, a modified version of G1 was used, supplemented with the buffer MOPS, 3-(N-Morpholino) propanesulfonic acid (Good 1966); this was purchased from

Vitrolife (G-MOPS PLUS).

Culture media containing the cytokine interleukin 6 (IL-6) was prepared by adding 1 µL of 100

µg/mL recombinant mouse IL-6 (Life Technologies, Thermo Fischer Scientific, Scoresby, Vic,

Australia) or recombinant human IL-6 (R&D Systems, Minneapolis, MN, USA) to 999 µL G1 or G2 media, resulting in 100 ng/mL IL-6 in the media. Serial dilutions 1:10 were then made, and the

Chapter 2: Methods media was immediately used to create culture dishes, which were equilibrated in the incubator for at least 4 h before use.

Media preparation Media were made from concentrated stock solutions, which were prepared by weighing each component on an analytical balance (Quintix 124-1S, Sartorius Lab Instruments, Goettingen,

Germany) and dissolving them in Milli-Q water. The essential amino acids stock was purchased from Corning Life Sciences (MEM Cellgro; Corning Life Sciences, Tewksbury, MA, USA). The composition of the stocks is shown in Appendix A. Stocks were filtered through a 0.2 µM low protein-binding filter and stored at 4⁰C. Media and stocks were prepared in a laminar flow hood.

To prepare the media, Milli-Q water was added to a 14 ml round bottom tube (Falcon) using a sterile serological pipette. The stock solutions were then added using an Eppendorf Research pipette. The pH of G2 medium was adjusted to 8.0 +/- 0.05 using 2.0 M sodium hydroxide

(NaOH). The pH of G1 was tested (Orion VersaStar, ThermoFischer) and was discarded and remade if it was not 7.8 +/- 0.05. The osmolality of the media was tested (Model 3320

Osmometer, Advanced Instruments, Norwood, MA, USA), G1 was 273 ± 10 mOsmol/kg and G2

270 ± 10 mOsmol/kg. Media was filtered through a 0.2 µM filter and stored at 4⁰C for up to 4 weeks.

Chapter 2: Methods

Table 1. Composition of embryo culture media Component G1 G2 Metabolic G1 Metabolic G2

Sodium chloride (NaCl) 90.08 mM 90.08 mM 90.08 mM 90.08 mM

Potassium chloride (KCl) 5.5 mM 5.5 mM 5.5 mM 5.5 mM

Sodium phosphate 0.25 mM 0.25 mM 0.25 mM 0.25 mM (NaH2PO4) Magnesium sulfate 1 mM 1 mM 1 mM 1 mM heptahydrate (MgSO4.7H20) Sodium bicarbonate 25 mM 25 mM 25 mM 25 mM (NaHCO3) Recombinant albumin (G- 2.5 mg/ml 2.5 mg/ml 2.5 mg/ml 2.5 mg/ml MM) Hyaluronan 0.125 mg/ml 0.125 mg/ml 0.125 mg/ml 0.125 mg/ml

Gentamycin 0.01 mg/ml 0.01 mg/ml 0.01 mg/ml 0.01 mg/ml

Calcium chloride (CaCl3) 1.8 mM 1.8 mM 1.8 mM 1.8 mM

Glucose 0.5 mM 3.15 mM 0.5 mM 0.5 mM

Sodium L-lactic acid 10.5 mM 5.87 mM 10.5 mM 5.87 mM

Sodium pyruvate 0.32 mM 0.1 mM 0.32 mM 0.1 mM

Ethylenediaminetetraacetic 0.01 mM 0.01 mM acid (EDTA) Alanyl-glutamine 0.5 mM 1 mM

Alanine 0.1 mM 0.1 mM 0.1 mM 0.1 mM

Aspartic acid 0.1 mM 0.1 mM 0.1 mM 0.1 mM

Asparagine 0.1 mM 0.1 mM 0.1 mM 0.1 mM

Glutamic acid 0.1 mM 0.1 mM 0.1 mM 0.1 mM

Glycine 0.1 mM 0.1 mM 0.1 mM 0.1 mM

Proline 0.1 mM 0.1 mM 0.1 mM 0.1 mM

Serine 0.1 mM 0.1 mM 0.1 mM 0.1 mM

Taurine 0.1 mM 0.1 mM

Arginine 0.3 mM 0.3 mM

Chapter 2: Methods

Cystine 0.05 mM 0.05 mM

Histidine 0.1 mM 0.1 mM

Isoleucine 0.2 mM 0.2 mM

Leucine 0.2 mM 0.2 mM

Lysine 0.2 mM 0.2 mM

Methionine 0.05 mM 0.05 mM

Phenylalanine 0.01 mM 0.01 mM

Threonine 0.2 mM 0.2 mM

Tryptophan 0.25 mM 0.25 mM

Tyrosine 0.1 mM 0.1 mM

Valine 0.2 mM 0.2 mM

Pantothenate 0.0042 mM 0.0042 mM

Pyridoxal 0.0049 mM 0.0049 mM

Thiamine 0.0030 mM 0.0030 mM

Riboflavin 0.0003 mM 0.0003 mM

Glutamine 0.5 mM 0.5 mM

Chapter 2: Methods

Preparation of culture dishes Standard culture dishes Unless otherwise specified, embryo cultures were performed in 35 mm or 60 mm Petri dishes

(Falcon Easy-Grip; Corning Life Sciences, Tewksbury, MA, USA) under paraffin oil (Ovoil;

Vitrolife). Drops of media were made under oil using an eVol positive displacement pipette (SGE

Analytical Science, Ringwood, Vic, Australia) in order to accurately deliver the small volumes employed and prevent evaporation of media during dish preparation (accurate to ± 1.0%).

Microwell dishes Embryos were cultured in the same incubator in 4 different dish types. Control embryos were cultured individually in 2 µl drops in 35 mm dishes as described above. Their development was compared to embryos in 3 commercially available microwell dishes designed for the Primo

Vision and EmbryoScope time-lapse systems (Vitrolife) (Figure 1). Microwell dishes were prepared according to the manufacturer’s instructions. When the medium was changed after 48 h culture, the manufacturers recommend aspirating 90% of the media and replacing it with fresh pre-incubated media, but to maintain consistency with the 2 µl drop controls the entire dish was changed. The microwell dishes are made from polystyrene, tested for embryotoxicity, and have conical-shaped microwells.

The Primo Vision culture dishes are available in a 9-well or 16-well format. The microwells in the

9-well dish are 0.4 mm diameter, 0.27 mm deep, with a volume of 0.11 µl. The microwells in the

16-well dish are 0.35 mm diameter, 0.27 mm deep, with 0.09 µl volume. A single drop of media covers all the microwells: 40 µl in the 9-well dish and 70 µl in the 16-well dish (4.4 µl per embryo).

An additional 20 µl wash drop was also made. Half of the media drop was laid down first, then covered with 3.5 ml oil. The dish was tapped on the bench to remove air bubbles then media was added to the final volume. Dishes were equilibrated in the incubator at least 4 h, and any remaining bubbles were removed by prodding with a micropipette before loading the embryos into the microwells. All the microwells of each dish were used for embryo culture (i.e. 9 or 16 embryos per dish).

Chapter 2: Methods

The EmbryoSlide culture dish, designed for the EmbryoScope time-lapse system, has 12 separate wells. At the bottom of each well is a microwell, 0.2 mm in diameter with a volume of approximately 0.02 µl. 25 µl media was placed in each well, plus the extra wells for washing, and overlaid with 1.5 ml oil. Air bubbles were removed as described above.

Figure 1. Diagram of microwells (A) Well-of-the-well (WOW) format (Primo Vision) and (B) single microwell (EmbryoScope) format. Not to scale.

Chapter 2: Methods

Culture protocol Standard culture protocol Unless otherwise specified, embryos were cultured in G1 medium for 48 h, then transferred to

G2 medium for a further 24 h or 48 h in a humidified multi-gas incubator at 37°C (MCO-5M;

Sanyo Electric, Osaka, Japan). For reduced oxygen experiments the incubator atmosphere was

5% O2, 6% CO2 and 89% N2, and for 20% oxygen experiments the incubator atmosphere was 6%

CO2 in air. To maintain embryo density between treatments, individual embryos were cultured in 2 µl drops of media, and groups of 10 embryos cultured in 20 µl drops of media.

To ensure that using two drop volumes did not affect osmolality, dishes with 2 µl and 20 µl drops of media were prepared and incubated for 48 h as described. The osmolality after incubation was measured using a Model 3320 Osmometer (Advanced Instruments). This machine requires

20 µl minimum sample volume, so 10 x 2 µl drops were pooled to create one sample.

Culture protocol for metabolic experiments For metabolic experiments, individual embryos were cultured in 1 µl metabolic medium and groups of 10 embryos in 10 µl. Additional control drops were incubated without embryos.

Embryos were cultured in G1 medium for 48 h, then in G2 medium for a further 24 h, and then in a new drop of G2 for a further 24 h. Embryos that did not appear to be developmentally arrested or degenerating were selected for metabolic analysis. To collect conditioned media without contamination from the oil overlay, the media drop was removed from the culture dish with a 20 µL pipette and expelled onto the inside of the lid of the culture dish. The oil then ran off the drop of media, and 7 µl of media was collected using a fresh pipette tip. Individual 1 µl drops were pooled into groups of 10 and 10 µl drops were collected individually. Media was collected into 1.5 mL Eppendorf tubes and snap frozen in liquid nitrogen and stored at -80⁰C.

The same sample was used for amino acid, glucose and pyruvate measurements.

Chapter 2: Methods

Culture protocol to determine the effect of embryo-conditioned media on embryo development in vitro Embryo-conditioned media was produced by culturing 10 embryos in 20 µl media. After 48 h culture, grouped embryos were moved from G1 to G2 media, and that 20 µl drop of conditioned

G1 was used to culture a single pronucleate oocyte. After a further 48 h, the grouped embryos were removed from G2 and the nuclei stained. The single embryos were then moved from conditioned G1 to conditioned G2. Control embryos were cultured individually in media that had been incubated without embryos. Embryos were stained at 118 h post-hCG.

Culture protocol to determine the effect of single culture during precompaction or postcompaction stages Embryos were cultured in groups of 10 in 20 µl or individually in 2 µl under 5% oxygen. When the embryos were moved from G1 to G2 on day 3 of culture, half of the group-cultured embryos were changed to single culture, and half of the singly-cultured embryos were moved to group culture. This resulted in four treatments: Single, Single/Group, Group/Single, and Group. ICM and TE were stained at 118 h post-hCG.

Culture protocol to determine the effect of embryo density and oxygen on single embryo development Single embryos were cultured in 20 µl or 2 µl drops of media, under 5% or 20% oxygen, resulting in four treatments: 5% 20 µl, 5% 2 µl, 20% 20 µl, and 20% 2 µl. ICM and TE were stained at 118 h post-hCG.

Culture protocol for IL-6 dose response During the IL-6 dose-response experiments, individual embryos were cultured in 2 µl drops of media (with or without IL-6, as described above) under 5% oxygen.

Assessment of preimplantation embryo development Morphological assessment Embryos were removed from the incubator and development was assessed on the morning of days 3, 4 and 5 of culture (70, 94 and 118 h post-hCG) using a dissecting microscope with a heated stage, unless a time-lapse incubator was used. Developmental stages were defined as

Chapter 2: Methods follows: ‘compacting’: the loss of membrane definition between blastomeres; ‘early blastocyst’: the presence of a blastocoel cavity less than half the volume of the embryo; ‘blastocyst’: the presence of a cavity occupying at least half the volume of the embryo; ‘expanded’: the increase in volume of the blastocyst and thinning of the zona; ‘hatching’: the appearance of cells outside the zona; and ‘fully hatched’: the complete evacuation of the embryo from the zona (Gardner and Lane 2014) (Figure 2).

Figure 2. Developmental stages of the preimplantation mouse embryo From top left to bottom right, pronucleate oocyte, 2-cell embryo, 4-cell embryo, morula, blastocyst, blastocyst, hatching blastocyst. Images from MCOK-5M[RC] imaging incubator. Scale bar 50 µm.

Chapter 2: Methods

Morphokinetics Embryo development during culture was monitored using a multi-gas cell imaging incubator fitted with an inverted microscope and turntable (MCOK-5M[RC]; Sanyo Electric) (Figure 3) as described previously (Wale and Gardner 2010, Lee et al. 2015). The embryo culture dishes rest on a motorised stage, while images are taken through the fixed Olympus microscope with a 10X objective. The light source is a 0.1W white LED. This style of time-lapse incubator is unique in that is allows assessment of embryo morphokinetics while embryos are cultured in different dish types, drops sizes or even in groups. Images were obtained every 15 min during culture and the time of cleavage events recorded. Cleavage times were defined as the first time point when new blastomeres were separated by cell membranes. Notation for cleavage events is based on those used by Meseguer and colleagues (Meseguer et al. 2011): cleavage time to 2 cells is represented as t2, and so on for 3 to 8 cells. Syn-t2 is the duration from syngamy to 2 cells; cc2 is the duration of the second cell cycle of the embryo, i.e. the interval between t2 and t3, likewise cc3 is the third cell cycle, i.e. the interval between t4 and t5; s2 is the duration of the second synchrony, i.e. the interval between t3 and t4, likewise s3 is the duration of the third synchrony, i.e. the interval between t5 and t8. Notation of post-compaction events is as described in the embryo culture section. Cleavage times are expressed as h post-hCG injection, or as h post-t2 (to reduce the potential influence of variation in mating and fertilisation times). Time-lapse experiments in either 5% oxygen or 20% oxygen were conducted in series to compare individual and group culture at each oxygen concentration.

Chapter 2: Methods

Figure 3. Interior of MCOK-5M[RC] time-lapse incubator. Image shows 35 mm culture dishes on the turntable. The metal cylinder at the rear of the incubator above the turntable is the LED light source. The camera is out of view, directly beneath the dish under the light source.

Nuclear stain To determine the number of cells present, embryos were incubated in 0.1 mg/ml bisBenzimide

(Hoescht 33258, Sigma) in G-MOPS and 10% ethanol for 30 min on a heated stage at 37⁰C.

Blastocysts were then washed briefly in G-MOPS PLUS and mounted in glycerol on a glass microscope slide. Images were captured on an Eclipse TS100 inverted fluorescent microscope with a DS-Fi1 camera and Digital Sight control unit (Nikon Instruments) at x200 magnification

(Figure 4). Cells were counted using ImageJ (1.49v, National Institutes of Health, USA).

Chapter 2: Methods

Figure 4. A blastocyst after nuclear staining with bisBenzamide Scale bar 0.1 mm

Differential stain Allocation of cells in the blastocyst to the ICM or TE was assessed using a differential staining protocol (Hardy et al. 1989a). All procedures were performed at 37°C, and blastocysts were washed in G-MOPS PLUS between all steps except the last. Simple G1 (sG1) medium without

NEAAs, alanyl-glutamine, taurine or HSA, containing 4 mg/ml polyvinylpyrrolidone (PVP; Sigma-

Aldrich) (sG1+PVP) was used to dilute (2,4,6-trinitrobenzene sulfonic acid) TNBS and anti-DNP

(2,4-Dinitrophenol) produced in rabbit.

Blastocysts were incubated in 0.5% pronase (Sigma-Aldrich) until the zona was no longer visible, then in 0.5% TNBS for 10 min. Blastocysts were then transferred to 10% anti-DNP antibody produced in rabbit for 10 min, and then into guinea pig serum (IMVS, Adelaide, SA, Australia) diluted 50% in 0.02 mg/ml propidium iodide in G-MOPS until blebbing of the cell membranes was observed. The final step was incubation in 0.1 mg/ml bisBenzimide (Hoescht 33258) in G-

MOPS and 10% ethanol for 30 min. Blastocysts were then washed briefly and mounted in

Chapter 2: Methods glycerol on a glass microscope slide. Propidium iodide is unable to penetrate tight junctions between cells to reach the ICM and as such stains only TE cells red (~620 nm). BisBenzamide stains all cells of the blastocyst blue (~470 nm). Images were captured on an Eclipse TS100 inverted fluorescent microscope with a DS-Fi1 camera and Digital Sight control unit (Nikon

Instruments). Cells of the TE appeared pink and the ICM appeared blue (Figure 5). Cells were counted manually using ImageJ (1.49v, National Institutes of Health, USA).

Figure 5. Blastocyst stained using differential staining technique Cells of the TE appear pink and the ICM appears blue. Scale bar 0.1 mm.

Assessment of peri-implantation and postimplantation development Blastocyst outgrowths At 124 h post hCG, blastocyst attachment and outgrowth potential were assessed in vitro

(Hannan et al. 2011), optimised from an earlier protocol (Cole et al. 1965). Flat-bottomed 96- well tissue culture dishes (Falcon, Corning Life Sciences) were rinsed with sterile phosphate- buffered saline (PBS) then coated with a solution of 10 mg/ml fibronectin (BD BioSciences)

Chapter 2: Methods overnight at 4ᵒC. Coated wells were rinsed with sterile PBS and incubated for 2 h at room temperature with 4 mg/ml BSA (MP Biomedicals, Seven Hills, NSW, Australia) in PBS. Wells were rinsed again with PBS, then filled with 150 µl of G2 with HSA and 5% FCS (Invitrogen, Thermo

Fisher Scientific, Scoresby, Vic, Australia), overlaid with 120 µl of paraffin oil (Ovoil) and the plate equilibrated at 6% CO2, 5% O2 and 89%N2 at 37ᵒC for at least 4 h. Embryos were selected for outgrowth assays if they had reached the blastocyst stage or beyond. One blastocyst was placed into each well, and outgrowths were imaged at 189 and 213 h post-hCG, using an inverted microscope with a heated stage (Ti-U eclipse; Nikon Instruments) with a CoolSNAP HQ camera

(Photometrics, Tucson, AZ, USA). Images were obtained using NIS Elements BR 3.00, SP7

Laboratory Imagining software (Nikon Instruments) and outgrowth area was measured using

Image J software (Figure 6).

Figure 6. Blastocyst outgrowths The image on the right has a red line indicating the outline drawn around the image of the outgrowth to calculate the area. Scale bar 0.1 mm.

Chapter 2: Methods

Embryo transfer experiments Surgical procedures All surgical instruments were washed with detergent and soaked in distilled water, then packaged and autoclaved. The surgeon wore a clean laboratory coat and a surgical mask, hands washed with chlorohexidine and gloves worn. Coarse hair was removed from the surgical site with clippers (PG-6020, Remington, DeForest, WI, USA). Fine hair was removed with the application of 1% sodium hydroxide cream (Nair Sensitive, Church & Dwight, Ewing, NJ, USA) for

1 min then washed with water. The skin was then cleaned using a scrub of chlorohexidine then a wash of chlorohexidine to remove clippings and organic debris. Surgical procedures were performed using a dissecting microscope (Typ 355110, Wild Heerbrugg, Switzerland) with a heated plate set at 37⁰C (Ratek Instruments, Boronia, VIC, Australia) and a cold light source

(Intralux 150H, Volpi, Auburn, NY, USA). The microscope stage was cleaned with 70% ethanol and covered with a drape. A second drape was placed next to the microscope to arrange the surgical equipment on. A third drape covered the animal. After prepping the mouse the surgeon then scrubbed their hands and put on clean gloves. Between animals, instruments were soaked in 70% ethanol and sterilised with heated glass beads (Germinator 500, CellPoint Scientific,

Gaithersburg, MD, USA).

Anaesthesia Anaesthesia was induced by isofluorane gas (Veterinary Companies of Australia, Kings Park,

NSW, Australia) administered using the Sleep Safe Small Animal Anaesthetic Machine and a face mask (Miden Medical, Tullamarine, Vic, Australia). The mouse first breathed 100% oxygen at 2 litres per minute for 30 sec before the isofluorane was introduced and increased over the following 30 sec from 0% to 4%. After the mouse became anaesthetised (approximately 1 min), the isofluorane was reduced to 1-2.5% to maintain anaesthesia during surgery (approximately

15 – 30 min). Lack of reflex reaction to pinching of the tail or feet indicated anaesthesia of sufficient depth to commence surgery. If a response was noted the procedure was halted and the percentage of isofluorane increased by 0.5% to a maximum of 2.5% and the surgery not

Chapter 2: Methods recommenced until lack of reflex reaction to tail or feet pinching was observed. To end anaesthesia, the isofluorane was reduced to 0% and the mouse was removed from the respirator to the recovery chamber and allowed to breathe normal air.

Analgesia To relieve post-operative pain, after the mouse was anaesthetised a single dose of

Buprenorphine (0.05 mg/kg) (Temgesic; Reckitt Benckiser, Slough, Berkshire, UK) was administered subcutaneously in 0.1 mL with a 25 gauge needle into the scruff of the neck. After surgery but while the mouse was still anaesthetised, a single dose of Carprofen (5 mg/kg)

(Carprieve; Norbrook Laboratories, Tullamarine, Vic, Australia) was administered subcutaneously in 0.1 mL with a 25 gauge needle into the scruff of the neck. To aid recovery, a subcutaneous injection of sterile saline was also administered (maximum of 0.5 mL).

Post-operative care Following surgery, the mouse was placed in the recovery chamber, a cardboard box lined with a paper towel half on and half off a warm plate set at 37⁰C. When the mouse had partially recovered and was able to walk normally, it was placed into its normal environment. Animals were housed individually post-surgery to avoid fighting and gnawing the surgical site. Mice were examined twice daily for the first 72 hours following surgery to ensure that the surgical site was healing and the mice showed no signs of poor recovery, and their recovery was recorded on the

Animal Monitoring Sheet.

Vasectomy surgery Male mice were vasectomised to mate with females and induce a pseudopregnancy (Gardner et al. 2004a). During anaesthesia, the mouse was placed on his back and the scrotal area prepared for surgery as described above. A small incision in the scrotal sac was made, then each testis was then exposed in turn and the vas deferens excised using a cautery pen to cauterise the wound and prevent bleeding (Bovie Medical Corporation, Clearwater, FL, USA). The wound was then

Chapter 2: Methods closed with a suture. The males were housed individually for at least one month after surgery to allow full recovery before mating with a female.

Embryo transfer surgery Adult female mice (6 - 12 weeks old) were housed with vasectomised males and considered pseudopregnant when a copulation plug was observed in the morning (day 0.5 of pregnancy).

On day 3.5 of pregnancy, mice underwent embryo transfer surgery (Hogan et al. 1986). A small

(1 cm) dorsal incision was made below the last rib. The underlining connective tissue was then dissected away gently and an incision made into the peritoneum directly above the fat pad. The uterine horn was exposed and a small hole made in the lumen with a 35 gauge needle and the embryos (5 per horn) inserted into the lumen with a polished glass pipette. The reproductive tract was eased back into the body cavity using blunt forceps, avoiding contact with the rest of the uterus and the ovary. This was repeated for the other uterine horn, again transferring 5 embryos, so there were 10 embryos in total in the tract. Once all the embryos have been transferred the skin wound was sealed with sterile surgical clips (MikRon AUTOCLIP; Clay Adams,

BD).

For synchronicity between embryo development and the pseudopregnant recipient, embryos were transferred on day 4 of culture (94-96 h post-hCG) and were selected for transfer if they had reached at least the blastocyst stage. Prior to transfer, embryos were incubated for less than

1 h in G2 media with hyaluronan and recombinant HSA (EmbryoGlue, Vitrolife), since the addition of hyaluronan to the transfer medium improves the implantation rate and fetal development rate (Gardner et al. 1999). Group-cultured embryos were transferred into one horn of each recipient, and single-cultured embryos into the other, alternating left and right for each treatment, and each recipient received embryos from only one oxygen concentration.

Fetal and placental dissection Embryo transfer recipients were killed on embryonic day 15 by cervical dislocation. They were placed on their back, their abdomen sterilized with 80% ethanol, then an incision was made

Chapter 2: Methods along the ventral midline. The reproductive tract removed and placed in a 90 mm petri dish

(Vitrolife) on ice. The number of fetuses and resorptions were counted. The uterine wall was cut open, the individual fetuses and placentas removed from the extraembryonic membrane, and the umbilical cords removed. Fetuses and placentas were weighed on an analytical balance

(Extend ED124S, Sartorius, Goettingen Germany) and measured using callipers, and the morphology of the fetal ear, skin, eye and limbs were assessed to determine developmental age

(Table 2) (Wahlsten and Wainwright 1977, Lane and Gardner 1994). Fetuses were sexed by removing the gonads and examining their morphology under an SMZ 1500 microscope.

Table 2. The morphological scale used to assess fetal development Adapted from (Lane and Gardner 1994).

Age (days) Ear features Eye features Limb features Skin features

13 Distinct meatus Round. Cornea Footplate Large head and pinna barely evident. indented. Hand follicles protrusion Faint ring of smooth pigment 14 Pinna growing Round. Faint Foot digits Follicles on out and forward cornea. Circle of separated. Hand abdomen pigment digits webbed 15 Distinct pinna Almond-shaped. Digits separated Follicles on head flap, almost over Distinct cornea. and splayed and abdomen meatus Dark pigment

Placenta histology Placentas were fixed in 4% paraformaldehyde at 4⁰C for 20 h, cut in half, then fixed for a further

6 h. To remove the paraformaldehyde, placentas were then kept in PBS for 24 h, which was replaced by fresh PBS for a further 24 h. Placentas were then stored in 70% ethanol until all tissues had been collected. Fixed placentas were dehydrated, cleared and infiltrated with wax in a Sakura Tissue-Tek VIP6 (Table 3) before they were embedded in paraffin wax and 5 μm thick sections were cut on a microtome onto a microscope slide.

Chapter 2: Methods

Table 3. Sakura Tissue-Tek VIP6 Processing Schedule (short cycle) Solution Duration

70% Ethanol 15 min

90% Ethanol 15 min

100% Ethanol 30 min

Xylene 30 min

Wax 30 min

Wax 15 min

Wax was removed from sections using xylene then stained with a Masson trichrome stain (Table

4) (Masson 1929, Lamar Jones et al. 2008). This protocol stains nuclei blue/black, collagen green, and cytoplasm, muscle and red blood cells red. Sections were then dehydrated in ethanol, and coverslips applied. Embedding, sectioning and staining were performed by the Biomedical

Sciences Histology Facility at the University of Melbourne. Morphology was examined at 20x magnification on a BX51 microscope with a DP70 camera and DP controller software v3.1.267

(Olympus Australia, Notting Hill, Victoria). Four sections of each placenta were examined. Areas of the junctional zone and labyrinth were measured using Image J software (Figure 7).

Chapter 2: Methods

Table 4. Masson Trichrome stain protocol Solution Duration and Composition of solution

temperature

Bouin’s fixative 60 min at 60⁰C 75 mL saturated aqueous picric acid, 25 mL

40% formaldehyde, 5 mL glacial acetic acid.

Weigert’s Iron 2 min 1 part Solution A (5 g haemotoxylin in 500 ml

Haematoxylin absolute ethanol), and 1 part Solution B (20

ml 30% aqueous ferric chloride, 5 ml

hydrochloric acid, 500 ml distilled water). Mix

Solutions A and B immediately before use.

Cytoplasmic (Plasma) 5 min 2 parts 1% Ponceau 2R and 1 part 1% Acid

Stain fuchsin in 1% acetic acid.

1% Phosphomolybdic acid 3 min

2% Light Green in 1% 5 min

acetic acid

1% Acetic acid 1 min

Chapter 2: Methods

Figure 7. Day 15 placenta stained with Masson trichrome X20. Regions of the placenta are outlined and labelled; Dec: decidua, JZ: junctional zone, Lab: labyrinth, CP: chorionic plate.

Analysis of embryo-conditioned media Glucose and pyruvate measurements Glucose and pyruvate concentrations in the conditioned media were analysed by microfluorescence assays based on enzymatic reactions involving the consumption or production of NAD(P)H (Leese and Barton 1984). Enzymatic cocktails were prepared as previously described (Gardner and Leese 1990).

Glucose cocktail contained 0.42 mM Dithiothreitol (DTT), 3.08 mM MgSO4, 0.42 mM ATP, 1.24 mM NADP+ (Roche), 14.17 U/ml hexokinase and 7.08 U/ml glucose-6-phosphate dehydrogenase in EPPS buffer, pH 8.0. Glucose added to this cocktail creates the following reaction:

ℎ푒푥표푘𝑖푛푎푠푒 𝑔푙푢푐표푠푒 + 퐴푇푃 → 𝑔푙푢푐표푠푒 6 푝ℎ표푠푝ℎ푎푡푒 + 퐴퐷푃

𝑔푙푢푐표푠푒 6 푝ℎ표푠푝ℎ푎푡푒 푑푒ℎ푦푟푑푟표𝑔푒푛푎푠푒 𝑔푙푢푐표푠푒 6 푝ℎ표푠푝ℎ푎푡푒 + 푁퐴퐷푃+→ 6 푝ℎ표푠푝ℎ표𝑔푙푢푐표푛푎푡푒

+ 푁퐴퐷푃퐻 + 퐻⁺

Chapter 2: Methods

To create a standard curve, G2 medium was made containing 0.6 mM glucose with no pyruvate or lactate, then serial dilutions were made using the same media without glucose, to create the following concentrations of glucose: 0.6, 0.3, 0.15, 0.075, 0.0375 and 0 mM.

The pyruvate cocktail contained 0.12 mM NADH and 27.1 U/ml lactate dehydrogenase in EPPS buffer, pH 8.0. Pyruvate added to this cocktail creates the following reaction:

푙푎푐푡푎푡푒 푑푒ℎ푦푟푑푟표𝑔푒푛푎푠푒 푝푦푟푢푣푎푡푒 + 푁퐴퐷퐻 + 퐻⁺ → 푙푎푐푡푎푡푒 + 푁퐴퐷⁺

To create a standard curve, G1 media samples were made containing 0.4 mM pyruvate with no glucose or lactate, then serial dilutions were made using the same media without pyruvate, to create the following concentrations of pyruvate: 0.4, 0.2, 0.1, 0.05, 0.025 and 0 mM.

1 µl of conditioned medium was added to 4 µl cocktail and incubated for 20 min at room temperature in the dark. When exposed to UV light, NADPH and NADH fluoresce at 460 nm and this fluorescence was quantified as an indirect measure of glucose and pyruvate respectively, using a NanoDrop 3300 fluorospectrometer (ThermoFisher Scientific, Waltham, MA, USA) as previously described (Choi et al. 2016). These values were converted to mM values using a standard curve. To determine the consumption or production of glucose or pyruvate, the sample measurement was subtracted from the measurement of control media incubated without embryos. Metabolite turnover was expressed as per embryo per hour.

Amino acid measurements Amino acid analysis was performed using a triple-quadrupole mass spectrometer (LC-QqQMS) as previously described (Wale and Gardner 2013, Lee et al. 2015). Frozen 1 µl culture media samples were vacuum dried then resuspended in 10 µl MilliQ water. Alongside these, 10 µl aliquots of each amino acid standard were then prepared. To all the standards and samples, 70

µl borate buffer (pH 8.8) was then added and mixed by vortex for 20 sec followed by centrifugation (1 min). 20 µl of the derivatization-labelling reagent 6-aminoquinolyl-N- hydroxysuccinimidyl carbamate (Aqc, 10 mM) was then added, vortexed immediately for 20 sec,

Chapter 2: Methods and then warmed on a heating block (Thermomixer; Eppendorf) with shaking (1000 rpm) for 10 min at 55⁰C. The final solution was then allowed to cool to ambient temperature before centrifugation (1 min), followed by liquid chromatography-mass spectrometry (LC-MS) analysis using an Agilent 1200 LC-system coupled to an Agilent 6420 ESI-QqQ-MS. Norleucine (25 µM) was used as an internal standard in borate buffer containing sodium borate (200 mM), the antioxidant ascorbic acid (1 mM) and the reducing agent tris(2-carboxyethyl)phosphine (10 mM). Concentrations of amines were quantified by comparison to a standard curve and the amino acid turnover was calculated by comparing results to control media incubated without embryos. Mass spectrometry was performed by Metabolomics Australia at the School of

BioSciences, University of Melbourne.

Statistical analysis For all tests, differences were considered statistically significant when P<0.05. Unless specified, all analyses were performed in SPSS Statistics v22 (IBM, Armonk, NY, USA). The homogeneity of variance was assessed by visualisation of the residuals on a scatterplot, and if the data were considered skewed then it was transformed by either a square root or logarithmic transformation, depending on which was most appropriate for the data.

Preimplantation development Individual culture and oxygen experiments In chapter 4, developmental stage of embryos was analysed by Fischers exact test. In chapter 5, this was analysed by a Generalised linear model with Poisson regression and log-linear link function with oxygen, grouping and replicate as factors. Cell numbers were compared by

General Linear Model Univariate analysis with oxygen and grouping as factors and replicate as a random factor. This was followed by the same test with treatment as a factor and replicate as a random factor, then multiple comparisons were performed with Bonferroni post-hoc analysis.

For morphokinetic data, experiments in 5% and 20% oxygen were performed in series, and so

Chapter 2: Methods were analysed separately by General Linear Model Univariate with grouping as a factor and replicate as a random factor.

Embryo density & oxygen Developmental stage of embryos was analysed using a Generalised Linear Model with binomial distribution and logit link function, using JMP 13.0.0. Cell numbers were compared using a

General Linear Model Univariate with drop size and oxygen as factors and replicate as a random factor.

Precompaction vs post-compaction Developmental stage of embryos was analysed using a Generalised Linear Model with binomial distribution and logit link function, using JMP 13.0.0. Cell numbers were compared using a

General Linear Model Univariate with treatment as the factor and replicate as a random factor.

Microwell experiments Cell numbers and morphokinetic data in each dish type were compared by General Linear Model

Univariate analysis with dish type as a factor and replicate as a random factor, then the least significant difference (LSD) test was used to compare each treatment with the control.

Conditioned media experiment Developmental stage of embryos was analysed using a Generalised Linear Model with binomial distribution and logit link function, using JMP 13.0.0 (SAS, Cary, NC, USA). Cell numbers were compared using a General Linear Model Univariate, with treatment (grouped, single or single + conditioned media) as the factor and replicate as a random factor.

IL-6 dose response Developmental stage of embryos was compared to the control by Yates’ Corrected Chi-Square.

Comparison of cell numbers was conducted by General Linear Model Univariate analysis with dose as a factor and replicate as a random factor. Multiple comparisons were performed with the least significant difference (LSD) test to compare each dose with the control.

Chapter 2: Methods

Metabolic data Metabolic data were compared by General Linear Model Univariate analysis with oxygen and grouping as factors and replicate as a random factor. This was followed by the same test with treatment as a factor and replicate as a random factor. Multiple comparisons were performed with Bonferroni post-hoc analysis.

Postimplantation development Outgrowth and fetal and placental data were analysed by comparing grouped or individually cultured embryos within the same oxygen concentration. For outgrowths, a General Linear

Model Univariate analysis was used with grouping as a factor and replicate as a random factor.

For fetal and placental data, a General Linear Model Univariate analysis was used with grouping as a factor and litter size as a covariate. Implantation rate and fetal development rate per recipient was analysed by Wilcoxon matched-pair signed-rank test and non-pregnant recipients were excluded. Fetal sex was analysed by a Mann-Whitney test.

Osmolality in 2 µl and 20 µl culture drops. The osmolality of incubated drops of media (G1 and G2) was subtracted from the osmolality of non-incubated media to find the difference that resulted from incubation. This value was compared for 2ul and 20ul drops using General Linear Model Univariate analysis with drop size, oxygen, media type and replicate as factors.

Chapter 3: In vitro culture of individual mouse preimplantation embryos: the role of embryo density, microwells, oxygen, timing and conditioned media

Chapter 3: Individual culture

Supplementary data

Supplementary Figure 1. Correlation between cell number of group cultured embryos used to make conditioned media, and cell number of single embryos cultured in embryo-conditioned media n = 56-57, 3 independent biological replicates.

Chapter 3: Individual culture

Supplementary Table 1. The effect of precompaction or post-compaction single embryo culture Development assessed on days 3, 4 and 5 of culture (70, 94 and 118 h post-hCG). Five independent biological replicates. No significant differences between treatments.

Group Group/Single Single/Group Single n 100 90 110 120 Day 3 3-8 cells (%) 5 2 9 7 Compacting/morula (%) 95 98 91 93 Day 4 Degenerate (%) 0 0 1 1 Compacting/morula (%) 3 2 2 3 Early blastocyst (%) 2 7 7 5 Blastocyst (%) 8 12 21 18 Expanded (%) 24 14 30 24 Hatching (%) 63 64 39 49 Total blastocysts (%) 95 90 90 91 Day 5 Degenerate (%) 0 0 3 3 Expanded (%) 2 8 4 6 Hatching (%) 88 88 83 88 Fully hatched (%) 9 3 9 3 Total blastocysts (%) 99 99 96 97

Chapter 3: Individual culture

Supplementary Table 2. The effect of embryo density and oxygen on singly cultured embryo development Development assessed on days 3, 4 and 5 of culture (70, 94 and 118 h post-hCG). Four independent biological replicates. Different letters represent significant differences within a row.

5% oxygen 20% oxygen P 2 µL 20 µL 2 µL 20 µL Oxygen Density Interaction n 101 98 92 92 Day 3 3-8 cells (%) 9 10 16 7 Compacting or morula 90 90 84 93 (%) Day 4 Degenerate (%) 1 0 0 1 Compacting or morula 3a 7a 24b 6a <0.01 NS <0.01 (%)

Early blastocyst (%) 10 15 16 18 Blastocyst (%) 29a 24a 30a 46b <0.05 NS <0.05

Expanded (%) 23 20 18 17 a a b b Hatching (%) 34 34 11 11 <0.0001 NS NS

Total blastocysts (%) 85a 78a 60b 74a <0.01 NS <0.05 Day 5 Degenerate (%) 3 2 9 8 a b b b Blastocyst (%) 0 5 4 8 <0.05 <0.05 NS a a b b Expanded (%) 16 8 41 34 <0.0001 NS NS a a b b Hatching (%) 77 78 42 50 <0.0001 NS NS

Fully hatched (%) 3 6 2 0

Total blastocysts (%) 96 98 90 91

Chapter 3: Individual culture

Supplementary Table 3. Development of single embryos in embryo-conditioned media Development assessed on days 3, 4 and 5 of culture (70, 94 and 118 h post-hCG). No significant differences between treatments. Three independent biological replicates.

Grouped Single Control media Conditioned media n 56 57 56 Day 3 3-8 cell (%) 3 0 0 Compacting/morula (%) 96 100 100 Day 4 Degenerate (%) 0 2 0 Compacting/morula (%) 2 4 0 Early blastocyst (%) 7 4 5 Blastocyst (%) 18 15 5 Expanded blastocyst (%) 23 25 21 Hatching (%) 51 51 68 Total blastocysts (%) 92 90 94 Day 5 Degenerate (%) 1 5 0 Blastocyst (%) 2 5 4 Expanded blastocyst (%) 6 11 2 Hatching (%) 74 60 66 Fully hatched (%) 17 18 29 Total blastocysts (%) 98 95 100

Chapter 4: Combined effects of individual culture and atmospheric oxygen on preimplantation mouse embryos in vitro

Chapter 4: Individual culture and oxygen effects preimplantation development

Supplementary Data Supplementary Table 1. Culture of embryos in atmospheric or reduced oxygen, in groups of 10 or individually. Numbers in columns represent the % of embryos at each developmental stage. N= 180-250 embryos per treatment, 11 replicates. Approximately half of the embryos were removed from culture on day 4 for staining, resulting in fewer embryos on day 5. Grouped 5% oxygen (5G), Single 5% oxygen (5S), Grouped 20% oxygen (20G), Single 20% oxygen (20S).

5% O2 grouped 5% O2 single 20% O2 grouped 20% O2 single P Day 3 n 190 182 248 205 Degenerate, 1-cell or 2-cells 0 2 1 3 a a ab b 3-8 cells 3 5 6 11 5S-20S <0.05, 5G-20S <0.01 a a a b Compacting or morula 97 93 93 86 5G-20S <0.01, others <0.05 Day 4 n 190 182 248 205 Degenerate, 1-cell or 2-cells 0 2 1 2 a a b b 3-8 cells 0 0 10 6 <0.001 a a c b Compacting or morula 2 5 12 21 5S-20G <0.05, others <0.001 5G-20G 5S-20G <0.01, others a a b b Early blastocyst 4 4 13 19 <0.001 Blastocyst 17 19 20 25 ab a bc c Expanded 25 32 19 13 5S-20S <0.001, others <0.01 5S-20G <0.05, 5G-5S <0.01, others a b c d Hatching 51 37 27 14 <0.001 Day 5 n 111 99 168 122 a ab a b Degenerate, 1-cell or 2-cells 1 2 1 7 5G-20S <0.05, 20G-20S <0.01 Compacting or morula 0 0 1 4 a ab ab b Early blastocyst 0 1 1 5 <0.05 a a b b Blastocyst 1 0 21 27 <0.001 a b c c Expanded 4 21 40 43 5S-20G <0.01, others <0.001 a b c d Hatching 80 56 36 14 <0.001 a a b b Fully hatched 14 20 1 0 <0.001

Chapter 5: Individual culture and atmospheric oxygen during culture affect mouse preimplantation embryo metabolism and postimplantation development

Chapter 5: Individual culture and oxygen affect metabolism and postimplantation development Abstract Culture of preimplantation embryos under reduced oxygen or in groups improves development in multiple species. Compared to embryos cultured under 5% oxygen in groups, mouse embryos cultured under atmospheric oxygen or individually develop at a slower rate, resulting in reduced blastocyst rate and smaller inner cell masses. Furthermore, the detrimental effects of combining atmospheric oxygen and individual culture are cumulative. The aim of this study was to determine if single embryo culture under atmospheric or reduced oxygen alters preimplantation metabolism and postimplantation development compared to culture in groups. Mouse embryos were cultured under 5% or 20% oxygen, individually or in groups of 10. Concentrations of pyruvate, glucose and amino acids in spent media were analysed after 48, 72 and 96 h of culture.

Blastocysts were assessed by outgrowth assay or transferred to pseudopregnant recipients, and fetal and placental weight, length and morphology were assessed. Compared to group culture, individually cultured blastocysts had lower net consumption of glucose and aspartate and higher glutamate production. Atmospheric oxygen reduced uptake of glucose and aspartate and increased production of glutamate and ornithine. Combining 20% oxygen and single culture resulted in further metabolic changes: decreased leucine, methionine and threonine consumption. Under 5% oxygen, individual culture decreased placental labyrinth area but had no other effects on fetal and placental development or outgrowth size. Under 20% oxygen, however, individual culture reduced outgrowth size and fetal and placental weight compared to group cultured embryos. Preimplantation metabolism of glucose and amino acids is altered by both oxygen and individual culture, and fetal weight is reduced by individual culture under atmospheric oxygen but not 5% oxygen. This study raises concerns regarding the increasing prevalence of single embryo culture in human IVF and adds to the existing evidence regarding the detrimental effects of atmospheric oxygen during embryo culture. Furthermore, these data demonstrate the cumulative nature of stress during embryo culture and highlight the importance of optimising each element of the culture system.

Chapter 5: Individual culture and oxygen affect metabolism and postimplantation development Introduction Many factors determine the success of in vitro fertilisation (IVF), from patient characteristics such as age and aetiology of infertility, to laboratory conditions such as media formulation, pH and temperature. All these variables need to be optimised to improve the chances of a successful pregnancy and a healthy baby. Suboptimal embryo culture induces stress in the embryo, causing fewer embryos to form blastocysts and reducing the inner cell mass (ICM), thereby reducing pregnancy rates and potentially compromising the long-term health of babies conceived (Feuer and Rinaudo 2016, Gardner and Kelley 2017). Two key laboratory variables documented to perturb embryo development are individual embryo culture (Paria and Dey

1990, Ebner et al. 2010) and atmospheric oxygen (Bontekoe et al. 2012, Kirkegaard et al. 2013), both of which are commonly used in human IVF.

Culture of multiple embryos together in groups, rather than individually, improves the development of mouse, bovine and sheep embryos, with group culture resulting in faster cleavage divisions, more embryos developing to the blastocyst stage, more cells per blastocyst and in the ICM, and fewer apoptotic cells (Paria and Dey 1990, Lane and Gardner 1992, Ferry et al. 1994, Gardner et al. 1994, Brison and Schultz 1997, Kelley and Gardner 2016). Evidence suggests that there is also a benefit of group culture for human embryos, with reports of higher cell numbers, blastocyst rates and pregnancy rates following group culture (Moessner and

Dodson 1995, Almagor et al. 1996, Ebner et al. 2010, Rebollar-Lazaro and Matson 2010). Group culture also reduces susceptibility to other sources of stress during culture, examples of which include atmospheric oxygen (Kelley and Gardner 2016), and peroxides from oil (Hughes et al.

2010, Ainsworth et al. 2017). Some of the beneficial effects of group culture can be replicated by culturing individual embryos in embryo-conditioned medium (Stoddart et al. 1996, Kelley and

Gardner 2017) and it has been proposed that embryo-secreted paracrine molecules, such as growth factors (Paria and Dey 1990, Brison and Schultz 1997) or platelet-activating factor (PAF)

(O'Neill 2005) are responsible for this effect. It is likely that these embryo-secreted molecules

Chapter 5: Individual culture and oxygen affect metabolism and postimplantation development substitute for tract-secreted factors that the embryo would be exposed to in vivo (Robertson et al. 2015, Wydooghe et al. 2017).

An estimated 55% of clinics worldwide routinely culture human embryos individually rather than in groups (Christianson et al. 2014), yet surprisingly little is known about the potential consequences of this practice beyond blastocyst formation and cell numbers, which are fundamental but unsophisticated measures of viability. Embryo metabolism is an important indicator of embryonic health and viability, but the potential effects of individual or group culture on this key parameter have not yet been investigated. Further, there have been few attempts to determine if individual culture is detrimental to fetal and placental development.

Animal and human studies that have investigated post-implantation outcomes following individual embryo culture to blastocyst stage have reported only a trend for decreased implantation rates or live birth rates (Lane and Gardner 1992, Kato and Tsunoda 1994, Ebner et al. 2010, Isobe 2014). Moreover, there has been no investigation of other fetal, placental or postnatal outcomes in any species following individual embryo culture.

In all species tested, including humans, reduced oxygen is beneficial for embryo culture, resulting in faster cleavage divisions (Wale and Gardner 2010, Kirkegaard et al. 2013), higher blastocyst formation and cell numbers (Whitten 1971, Tervit et al. 1972, Dumoulin et al. 1999), fewer apoptotic cells (Van Soom et al. 2002, Yuan et al. 2003), less frequent aneuploidy (Bean et al. 2002), and less DNA damage (Takahashi et al. 2000, Kitagawa et al. 2004). A possible mechanism for these outcomes may be a decrease in intracellular reactive oxygen species (ROS) compared to culture in atmospheric oxygen (Goto et al. 1993, Kitagawa et al. 2004). There is also evidence of differences between embryos cultured in 5% and 20% oxygen in gene expression (Harvey et al. 2004, Kind et al. 2005, Mantikou et al. 2013), histone remodelling and methylation (Gaspar et al. 2015, Li et al. 2016), as well as changes in the proteome, secretome and metabolism (Khurana and Wales 1989, Katz-Jaffe et al. 2005, Kubisch and Johnson 2007,

Chapter 5: Individual culture and oxygen affect metabolism and postimplantation development

Wale and Gardner 2012). Changes to preimplantation embryo development are also observed post-transfer, as reduced oxygen during culture results in higher implantation, pregnancy, and live birth rates in humans (Catt and Henman 2000, Meintjes et al. 2009a, Waldenstrom et al.

2009, Gomes Sobrinho et al. 2011), and lower miscarriage rates in mice (Karagenc et al. 2004), compared with culture in atmospheric oxygen. In spite of the evidence that atmospheric oxygen is detrimental to embryo development and that a concentration of 2-8% is more physiological

(Mastroianni and Jones 1965, Fischer and Bavister 1993), there is no global consensus on the use of reduced oxygen for embryo culture (Gardner 2016, Nastri et al. 2016), and it remains common practice in human IVF to use atmospheric oxygen in incubators. Christianson et al.

(2014) found that of 265 clinics in 71 countries surveyed, only 24% used reduced oxygen for all embryo culture. Importantly, embryos cultured in atmospheric oxygen are also more susceptible to a second stress during culture, such as ammonium (Wale and Gardner 2013) or simple medium lacking amino acids (Feuer et al. 2016). It has recently been shown that combining individual culture and atmospheric oxygen is detrimental to cleavage timings, blastocyst formation, and blastocyst cell numbers (Kelley and Gardner 2016). Consequently, it was the aim of this study to further investigate the effect of individual and group culture on preimplantation metabolism and postimplantation fetal and placental development, and also the role of oxygen concentration in determining these outcomes.

Chapter 5: Individual culture and oxygen affect metabolism and postimplantation development Materials & Methods Experimental design Embryos were cultured in groups of 10 or individually, in either physiologic (5%) or atmospheric

(20%) oxygen, resulting in 4 treatment groups. The embryo:media volume ratio was maintained between treatments. In the metabolic experiments, conditioned media was collected for analysis at three time points, and embryos were stained to determine the number of cells. In experiment 2, embryos were transferred to a recipient mouse or used in an in vitro outgrowth assay to assess postimplantation developmental capacity.

Animals F1 hybrid (C57BL/6 X CBA) mice were maintained in a standard animal research facility in individually ventilated cages (Optimice, Animal Care Systems, Centennial, CO, USA) with a 12 h light–dark photoperiod (6 am - 6 pm) and controlled temperature, with food and water available ad libitum.

Fertilised embryos were generated by superovulation of four week old females with intraperitoneal injections of 5 international units (IU) pregnant mare serum gonadotropin

(PMSG, Folligon; Intervet, Bendigo East, Vic, Australia) at the mid-point of the light phase, followed 48 h later by 5 IU human chorionic gonadotropin (hCG) (Chorulon; Intervet), and mated with males of the same strain overnight. All experiments we approved by The University of

Melbourne Animal Ethics Committee.

Embryo culture Pronucleate oocytes were collected 22 h after hCG injection in G-MOPS PLUS handling medium, containing 5 mg/ml human serum albumin (HSA) (Vitrolife, Göteborg, Sweden) as described previously (Gardner and Lane 2007, 2014). Embryos were incubated in G-MOPS PLUS containing

550 IU/ml hyaluronidase (bovine testes type IV-S; Sigma-Aldrich, St Louis, MO, USA) until cumulus cells were removed, then washed three times in G-MOPS PLUS and once in pre- incubated G1 culture medium. Embryos were pooled, then allocated to treatments.

Chapter 5: Individual culture and oxygen affect metabolism and postimplantation development

G1/G2 media were prepared in the laboratory as described previously (Gardner and Lane 2007,

2014), with modifications. Media were supplemented with recombinant albumin (2.5 mg/ml; G-

MM, Vitrolife), hyaluronan (0.125 mg/ml; Vitrolife) and gentomycin (10 µg/mL; Sigma). In addition, choline chloride, folic acid, inositol, nicotinamide and taurine were omitted from G2.

All additional media chemicals were supplied by Sigma-Aldrich, except essential amino acids

(MEM Cellgro; Corning Life Sciences, Tewksbury, MA, USA). In metabolic experiments, alanyl- glutamine was substituted with L-glutamine and glucose was reduced to 0.5 mM to facilitate measurement of utilisation by embryos.

Embryo cultures were performed in 35 mm Petri dishes (Falcon Easy-Grip; Corning Life Sciences,

Tewksbury, MA, USA) under paraffin oil (Ovoil; Vitrolife) in a humidified multi-gas incubator at

37°C (MCO-5M; Sanyo Electric, Osaka, Japan). For reduced oxygen experiments the incubator atmosphere was 5% O2, 6% CO2 and 89% N2, for 20% oxygen experiments the incubator atmosphere was 6% CO2 in air. Drops of media were made directly under oil using an eVol positive displacement pipette (SGE Analytical Science, Ringwood, Vic, Australia) to accurately deliver small volumes (accurate to ± 1.0%) and prevent evaporation of media during dish preparation. All chemicals and plastics were pre-screened in a mouse embryo assay prior to use

(Gardner et al. 2005). Embryo manipulations were performed on an SMZ 1500 microscope

(Nikon Instruments, Melville, NY, USA) with a heated stage (Tokai Hit, Shizuoka, Japan).

In the metabolic experiments, individual embryos were cultured in 1 µl metabolic medium and groups of 10 embryos in 10 µl. Additional control drops were incubated without embryos.

Embryos were cultured in G1 medium for 48 h, then in G2 medium for a further 24 h, and then in a new drop of G2 for a further 24 h. Conditioned media was collected, 10 µl drops were snap frozen individually, and 1 µl drops were pooled in groups of 10 before freezing and stored at -

80⁰C. The same sample was used for amino acid, glucose and pyruvate measurement.

Chapter 5: Individual culture and oxygen affect metabolism and postimplantation development

For all other experiments, 2 µl culture medium per embryo was used, with embryos cultured in

G1 medium for 48 h, then transferred to G2 medium for 48 h.

Nuclear staining At 118 h post-hCG, embryos in metabolic experiments were stained to determine the number of cells present. Embryos were incubated in 0.1 mg/ml bisBenzimide (Hoescht 33258, Sigma) in

G-MOPS and 10% ethanol for 30 min. Blastocysts were then washed briefly and mounted in glycerol on a glass microscope slide. Images were captured on an Eclipse TS100 inverted fluorescent microscope with a DS-Fi1 camera and Digital Sight control unit (Nikon Instruments).

Cells were counted using ImageJ (1.49v, National Institutes of Health, USA).

Glucose and pyruvate measurements Glucose and pyruvate concentrations in conditioned media were analysed by microfluorescence assays. Enzymatic cocktails were prepared as previously described (Gardner and Leese 1990).

Glucose cocktail contained 0.42 mM DTT, 3.08 mM MgSO4, 0.42 mM ATP, 1.24 mM NADP

(Roche), 14.17 U/ml hexokinase and 7.08 U/ml G6PDH in EPPS buffer. The pyruvate cocktail contained 0.12 mM NADH and 235 U/ml lactate dehydrogenase in EPPS buffer.

One µl of conditioned medium was added to 4 µl cocktail and incubated for 20 min at room temperature in the dark. The fluorescence of NADPH or NADH was quantified as an indirect measure of glucose and pyruvate respectively, using a NanoDrop 3300 fluorospectrometer

(ThermoFisher Scientific, Waltham, MA, USA) as previously described (Choi et al. 2016). These values were converted to mM values using a standard curve and the sample measurement was subtracted from the measurement of control media incubated without embryos.

Chapter 5: Individual culture and oxygen affect metabolism and postimplantation development each amino acid standard were prepared. To all standards and samples, 70 µl borate buffer (pH

8.8) was added, followed by 20 µl of the derivatization-labelling reagent 6-aminoquinolyl-N- hydroxysuccinimidyl carbamate (Aqc, 10 mM). Samples were warmed on a heating block

(Thermomixer; Eppendorf) with shaking (1000 rpm) for 10 min at 55⁰C. Liquid chromatography- mass spectrometry (LC-MS) was performed using an Agilent 1200 LC-system coupled to an

Agilent 6420 ESI-QqQ-MS (Agilent Technologies, Santa Clara, CA, USA). Norleucine (25 µM) was used as an internal standard in borate buffer containing sodium borate (200 mM), the antioxidant ascorbic acid (1 mM) and the reducing agent tris(2-carboxyethyl)phosphine (10 mM). Concentrations of amines were quantified by comparison to the standard curve and amino acid turnover was calculated by subtracting results from control media incubated without embryos.

Blastocyst outgrowth Blastocyst attachment and outgrowth potential were assessed in vitro at 124 h post hCG

(Hannan et al. 2011). Flat-bottomed 96-well tissue culture dishes (Falcon, Corning Life Sciences) were coated with a solution of 10 mg/ml fibronectin (BD BioSciences) overnight at 4ᵒC. Coated wells were rinsed with PBS, then incubated for 2 h at room temperature with 4 mg/ml bovine serum albumin (BSA) (MP Biomedicals, Seven Hills, NSW, Australia), rinsed, then filled with 150

µl of G2 medium supplemented with 5% HSA and 5% fetal calf serum (FCS) (Invitrogen, Thermo

Fisher Scientific, Scoresby, Vic, Australia), overlaid with 120 µl of paraffin oil (Ovoil) and the plate equilibrated at 6% CO2, 5% O2 and 89% N2 at 37ᵒC for at least 4 h. Embryos were selected for outgrowth assays if they had reached the blastocyst stage or beyond. One blastocyst was placed into each well, and outgrowths were imaged at 189 and 213 h post-hCG, using an inverted microscope with a heated stage (Ti-U eclipse; Nikon Instruments) at ×10 magnification with a

CoolSNAP HQ camera (Photometrics, Tucson, AZ, USA). Images were obtained using NIS

Elements BR 3.00 SP7 Laboratory Imagining software (Nikon Instruments) and outgrowth area was measured using ImageJ.

Chapter 5: Individual culture and oxygen affect metabolism and postimplantation development

Embryo transfer Adult female mice (6 - 12 weeks old) were housed with vasectomised males and considered pseudopregnant when a copulation plug was observed in the morning (day 0.5 of pregnancy).

On day 3.5 of pregnancy, mice were anaesthetised with isofluorane gas (Veterinary Companies of Australia, Kings Park, NSW, Australia) and analgesia was given as subcutaneous injections of buprenorphine (0.05 mg/kg) (Temgesic; Reckitt Benckiser, Slough, Berkshire, UK) and carprofen

(5 mg/kg) (Carprieve; Norbrook Laboratories, Tullamarine, Vic, Australia). A dorsal incision was made, each uterine horn isolated, and 5 embryos were transferred to each uterine horn using a polished glass pipette containing EmbryoGlue (Vitrolife). The wound was sealed with surgical clips (MikRon AUTOCLIP; Clay Adams, BD).

Embryos were transferred on day 4 of culture (94-96 h post-hCG) and were selected for transfer if they had reached at least the blastocyst stage. Five group-cultured embryos were transferred into one horn of each recipient, and 5 single-cultured embryos into the other, alternating left and right, and each recipient received embryos from only one oxygen concentration. Embryo transfer recipients were sacrificed on day 14.5 to determine the number of fetuses and resorptions present. Fetuses and placentas were weighed and measured, and fetal development assessed (Wahlsten and Wainwright 1977).

Placenta histology Placentas were fixed in 4% paraformaldehyde at 4°C for 20 h, bisected sagittally, then fixed for a further 6 h. Fixed placentas were dehydrated in a series of alcohols and then embedded in paraffin wax. Microtome sections (5 μm) were stained with a Masson trichrome stain as previously described (Roberts et al. 2003, Cuffe et al. 2011). Sections were examined at 20x magnification on a BX51 microscope with a DP70 camera and DP controller software v3.1.267

(Olympus Australia, Notting Hill, Victoria). Six placentas per treatment were randomly selected for analysis and four sections of each placenta were examined. Areas of the junctional zone and labyrinth were measured using ImageJ (Roberts et al. 2003).

Chapter 5: Individual culture and oxygen affect metabolism and postimplantation development

Statistical analysis For all tests, differences were considered statistically significant when P<0.05. All analyses were performed in SPSS Statistics v22 (IBM, Armonk, NY, USA). The homogeneity of variance was assessed by visualisation of the residuals on a scatterplot, and if the data were considered skewed then it was transformed. Developmental stage of embryos was analysed by Generalised linear model with Poisson regression and log-linear link function with replicate as a factor.

Metabolic data and cell numbers were compared by General Linear Model Univariate analysis with replicate as a random factor. Since the number of cells per embryo was likely to directly influence the metabolic turnover, metabolic data was also analysed using cell number as a covariate. Pairwise comparisons were performed with Bonferroni post hoc analysis.

Implantation rate and fetal development rate per recipient was analysed by Wilcoxon matched- pair signed-rank test and non-pregnant recipients were excluded. Fetal sex was analysed by a

Mann-Whitney test. General Linear Model Univariate analysis was used for fetal and placental data with litter size as a covariate. Three unviable fetuses (one from 20% oxygen single culture and two from 5% oxygen group culture) were counted as miscarriages and were not included in the analysis of fetal & placental parameters.

Chapter 5: Individual culture and oxygen affect metabolism and postimplantation development Results Blastocyst rates and cell numbers are reduced by individual culture and atmospheric oxygen Embryos were cultured in 5% or 20% oxygen, in groups of 10 or individually, and embryos were selected for metabolic analysis that did not appear to be developmentally arrested or degenerating. Even with this selection, developmental differences were observed between treatments (Figure 1A).

On day 3, there was a significant positive effect of grouping (P<0.01) on the percentage of embryos to reach the compacting or morula stage, but there was no effect of oxygen and no significant interaction, and a Bonferroni post hoc analysis showed no significant pairwise differences between treatments.

On day 4 of culture, significantly fewer embryos in 20% oxygen single culture had reached the blastocyst stage or beyond (70.1 ± 2.8%) than all other treatments (5% oxygen grouped 97.1 ±

3.4%, 5% oxygen single 90.1 ± 3.3%, 20% oxygen grouped 88.1 ± 3.2%; P<0.001). This was largely due to more morula and early blastocysts in 20% oxygen single culture, and more hatching blastocysts in the other treatments (Supplementary Table 1). The effects of grouping (P<0.001) and oxygen (P<0.001) were both significant, as was the interaction (P<0.05).

A similar pattern was observed on day 5 of culture, as fewer embryos in 20% oxygen single culture were hatching or fully hatched than in any other treatment (33.9 ± 4.2%; P<0.001), and there were also fewer hatching or fully hatched blastocysts in 20% oxygen group culture than

5% oxygen group culture (69.9 ± 6.1% vs 96.8 ± 7.2%; P<0.05), although this was not different to

5% oxygen single culture (72.9 ± 6.2%). The effects of grouping (P<0.001), oxygen (P<0.001), and the interaction were all significant (P<0.05).

Chapter 5: Individual culture and oxygen affect metabolism and postimplantation development

Figure 1. Developmental stage of embryos in metabolism assays Embryos were cultured under 5% or 20% oxygen, in groups of 10 embryos or individually. Boxes represent 25-75th percentile, whiskers represent 5-95th percentile, line represents median, ‘+’ represents mean. n = 10 groups of 10 embryos per treatment selected for metabolic analysis. Spent medium from 10 individually cultured embryos was pooled for metabolic analysis, and this graph shows development per pool of 10 individually cultured embryos or 10 group cultured embryos. Different superscripts indicate significant differences. (A) Graph shows % of embryos per group of 10 that reached the designated developmental stage or beyond on days 3, 4 and 5 of culture (70, 94 and 118 h post-hCG). Day 3: grouping <0.01, oxygen NS, interaction NS. Day 4: grouping <0.001, oxygen <0.001, interaction NS; pairwise P<0.001 between all treatments. Day 5: grouping <0.001, oxygen <0.001, interaction <0.05; pairwise 5G vs 20G <0.05, 20S vs all <0.001. (B) Graph shows mean number of cells per embryo within each group of 10 embryos on day 5 (118 h post-hCG). Oxygen P<0.001, grouping P<0.001, interaction NS. 5G vs 5S P<0.05, 5G vs 20G P<0.01, others P<0.001.

100

Chapter 5: Individual culture and oxygen affect metabolism and postimplantation development

The number of cells per embryo within each group of 10 embryos was lower after culture in 20% oxygen single culture than all other treatments (42.2 ± 4.1; P<0.001), less than half that of 5% oxygen group culture (110.9 ± 4.1) (Figure 1B). Embryos from 5% oxygen group culture also had more cells than either 5% oxygen single culture (78.3 ± 4.1; P<0.05) and 20% oxygen group culture (76.1 ± 4.1; P<0.01). There was a significant effect of oxygen (<0.001) and grouping

(<0.001) on the number of cells, with no significant interaction.

Glucose metabolism is decreased by individual culture and atmospheric oxygen On day 3, there was a significant effect of oxygen (P<0.05) on glucose consumption, with embryos cultured in 20% oxygen consuming less glucose than those in 5% oxygen. The effect of grouping and the interaction between grouping and oxygen were not significant (Figure 2).

Posthoc analysis showed no pairwise differences between treatments (5% oxygen grouped 0.81

± 0.17, 5% oxygen single 0.49 ± 0.17, 20% oxygen grouped 0.22 ± 0.17, 20% oxygen single 0.32

± 0.17 pmol/embryo/h).

On day 4, the significant effect of oxygen had disappeared and been replaced by a significant effect of grouping (P<0.05), as embryos cultured individually consumed less glucose than those in groups (Figure 2). The interaction between oxygen and grouping was not significant. There were no pairwise differences between treatments (5% oxygen grouped 1.17 ± 0.40, 5% oxygen single 0.06 ± 0.40, 20% oxygen grouped 0.86 ± 0.42, 20% oxygen single 0.39 ± 0.40 pmol/embryo/h).

On day 5, both oxygen and grouping influenced glucose consumption (P<0.001), but the interaction was not significant (Figure 2). Embryos in 5% oxygen group culture consumed more glucose (6.21 ± 0.38 pmol/embryo/h) than embryos in 5% oxygen single culture (4.49 ± 0.38 pmol/embryo/h, P<0.05) or 20% oxygen group culture (4.00 ± 0.38 pmol/embryo/h; P<0.01), while embryos in 20% oxygen single culture consumed less glucose than all other treatments

(1.69 ± 0.38 pmol/embryo/h; P<0.001).

101

Chapter 5: Individual culture and oxygen affect metabolism and postimplantation development

Figure 2. Glucose consumption per embryo on Days 3, 4 and 5 of culture Embryos were cultured under 5% or 20% oxygen, in groups of 10 embryos or individually. Media was collected on days 3, 4 and 5 (70, 94 and 118 h post-hCG) to determine glucose consumption. Boxes represent 25-75th percentile, whiskers represent 5-95th percentile, line represents median, ‘+’ represents mean. n = 10 groups of 10 embryos per treatment. Graph shows glucose consumed per embryo over 48 h (day 3) or 24 h (days 4 and 5). Asterisks indicate significant effect of factors in model, without significant differences in pairwise comparisons. Different letters indicate significant differences between treatments. Day 3: oxygen P<0.05, grouping NS, interaction NS. Day 4: oxygen NS, grouping P<0.05, interaction NS. Day 5: Oxygen P<0.001, grouping P<0.001, interaction NS; 5G vs 5S P<0.05, 5G vs 20G P<0.01, others P<0.001.

When the number of cells per embryo was included in the analysis as a covariate, there was a significant interaction between oxygen and grouping (P<0.05), such that embryos in 20% oxygen single culture consumed less than all other treatments (Table 1).

There was no effect of treatment on the concentration of glucose in control drops without embryos (data not shown).

Pyruvate metabolism is not affected by individual culture or atmospheric oxygen There was no effect of treatment on the consumption of pyruvate per embryo on day 3 (5% oxygen grouped 2.65 ± 0.19, 5% oxygen single 1.97 ± 0.19, 20% oxygen grouped 2.30 ± 0.19, 20%

102

Chapter 5: Individual culture and oxygen affect metabolism and postimplantation development oxygen single 2.55 ± 0.19 pmol/embryo/h). There was no significant turnover of pyruvate on days 4 or 5 (data not shown).

Amino acid metabolism is altered by individual culture and atmospheric oxygen Media from the final 24 h of culture was collected and analysed for the 20 proteinogenic amino acids plus ornithine. There was a non-significant trend for embryos cultured in groups in 5% oxygen to consume more of the 20 amino acids (not including ornithine) than embryos cultured individually in 20% oxygen (Figure 3), however there was no difference between treatments in total amino acid production or total amino acid turnover (total production + total consumption per embryo) of amino acids.

Aspartate consumption per embryo was higher in embryos cultured in groups at 5% oxygen (-

2.30 ± 0.7 pmol/embryo/h; P<0.001) than embryos cultured individually in 5% oxygen (-1.61 ±

0.07 pmol/embryo/h) or in groups in 20% oxygen (-1.82 ± 0.07 pmol/embryo/h) (Figure 4).

Aspartate consumption was lowest in single culture in 20% oxygen compared to all other treatments (-1.20 ± 0.07 pmol/embryo/h; P<0.001). There was a significant effect of oxygen

(<0.001) and grouping (<0.001), but the interaction was not significant. When the number of cells was included as a covariate, grouped embryos consumed significantly more aspartate per embryo than singly cultured embryos but the effect of oxygen was not significant (Table 1).

Glutamate was consumed by embryos cultured in 5% oxygen in groups (-0.158 ± 0.94 pmol/embryo/h) but was produced by embryos in all other treatments (5% oxygen single 0.338

± 0.94; P<0.01, 20% oxygen grouped 0.465 ± 0.94; <0.001, 20% oxygen single 0.557 ± 0.10 pmol/embryo/h; P<0.001) (Figure 4). There was a significant effect of oxygen (<0.001), and grouping (P<0.01), and the interaction was significant (<0.05). When cell number was included in the analysis as a covariate, embryos cultured in groups under 5% oxygen still had significantly less glutamate production than all other treatments (Table 1).

103

Chapter 5: Individual culture and oxygen affect metabolism and postimplantation development

Figure 3. Overall amino acid utilization on day 5 Embryos were cultured under 5% or 20% oxygen, in groups of 10 embryos or individually. Media was collected on day 5 (118 h post-hCG) to determine utilization of 20 amino acids (ornithine not included) over 24 h from day 4 to 5 of culture. n = 10 groups of 10 embryos per treatment. Boxes represent 25-75th percentile, whiskers represent 5-95th percentile, line represents median, ‘+’ represents mean. n = 10 groups of 10 embryos per treatment. Graph shows total pmol of amino acid consumption, production, turnover (total production + total consumption) per embryo per hour.

Table 1: Metabolism Embryos were cultured under 5% or 20% oxygen, in groups of 10 embryos or individually. Media was collected on day 5 (118 h post-hCG) to determine utilization of 20 amino acids plus ornithine and glucose over 24 h from day 4 to 5 of culture. n = 10 groups of 10 embryos per treatment. Table shows mean ± sem of metabolite utilization per embryo per hour, calculated with cell number as a covariate. Negative numbers indicate net uptake and positive numbers indicate net production. Total consumption, productions and turnover of amino acids do not include ornithine.

104

Chapter 5: Individual culture and oxygen affect metabolism and postimplantation development

Two-way analysis 5% oxygen 5% oxygen 20% oxygen 20% oxygen grouped single grouped single Oxygen Grouping Interaction Alanine 0.085 ± 0.173 0.279 ± 0.095 0.324 ± 0.094 0.588 ± 0.176 Arginine 0.372 ± 0.650 -0.422 ± 0.355 -0.089 ± 0.355 -0.428 ± 0.660 Asparagine -0.232 ± 0.099 -0.124 ± 0.054 -0.105 ± 0.054 -0.41 ± 0.101 Aspartate -2.004 ± 0.101 -1.597 ± 0.055 -1.826 ± 0.55 -1.501 ± 0.102 <0.001 Cysteine -0.239 ± 0.132 0.033 ± 0.072 0.043 ± 0.072 0.121 ± 0.134 Glutamate -0.044 ± 0.459 0.339 ± 0.095 0.459 ± 0.095 0.437 ± 0.180 <0.05 Glutamine -0.602 ± 1.119 0.246 ± 0.611 0.799 ± 0.610 1.419 ± 1.136 Glycine -1.085 ± 0.337 -0.913 ± 0.185 -0.398 ± 0.194 -0.486 ± 0.348 Histidine 0.189 ± 0.289 0.212 ± 0.158 0.324 ± 0.157 0.267 ± 0.293 Isoleucine -0.851 ± 0.346 -0.352 ± 0.189 -0.498 ± 0.189 -0.308 ± 0.351 Leucine -0.758 ± 0.259 -0.368 ± 0.142 -0.309 ± 0.141 -0.041 ± 0.263 Lysine -0.544 ± 0.305 -0.203 ± 0.167 -0.223 ± 0.166 0.087 ± 0.310 Methionine 0.155 ± 0.071 -0.002 ± 0.039 -0.015 ± 0.039 0.064 ± 0.072 Phenylalanine -0.160 ± 0.129 0.038 ± 0.071 0.018 ± 0.071 0.149 ± 0.131 Proline -0.355 ± 0.146 -0.048 ± 0.080 -0.138 ± 0.079 0.139 ± 0.148 <0.05 Serine -0.067 ± 0.149 -0.012 ± 0.081 -0.038 ± 0.081 0.155 ± 0.151 Threonine -0.695 ± 0.261 -0.120 ± 0.143 -0.325 ± 0.143 0.143 ± 0.265 <0.05 Tryptophan -0.047 ± 0.037 -0.002 ± 0.020 -0.019 ± 0.020 0.004 ± 0.038 Tyrosine -0.190 ± 0.127 0.011 ± 0.070 0.007 ± 0.069 0.087 ± 0.129 Valine -0.601 ± 0.238 -0.163 ± 0.130 -0.209 ± 0.130 0.016 ± 0.241 Total consumed -10.985 ± 2.553 -7.714 ± 1.395 -6.486 ± 1.393 -4.270 ± 2.592 Total produced 3.048 ± 1.615 4.588 ± 0.897 4.044 ± 0.941 5.423 ± 1.734 Total turnover 14.333 ± 1.874 12.144 ± 1.040 11.406 ± 1.092 8.916 ± 2.012 Ornithine 0.358 ± 0.050 0.392 ± 0.027 0.504 ± 0.027 0.580 ± 0.050 <0.01 Glucose -4.667 ± 0.836 -4.833 ± 0.467 -4.500 ± 0.504 -2.708 ± 0.831 <0.05

105

Chapter 5: Individual culture and oxygen affect metabolism and postimplantation development

Leucine was consumed most by embryos cultured in groups in 5% oxygen (-0.69 ± 0.14 pmol/embryo/h), and least by single embryos in 20% oxygen (-0.12 ± 0.14 pmol/embryo/h;

P<0.05) (Figure 4). There was a significant effect of oxygen (P<0.05), but not grouping, and the interaction was not significant. This pattern reflects the number of cells per sample, and when cell number was used as a covariate, there was no significant effect of treatment (Table 1).

Like leucine, methionine consumption was higher in embryos cultured in 5% oxygen in groups (-

0.14 ± 0.04 pmol/embryo/h) than single embryos in 20% oxygen which were producing methionine instead (0.5 ± 0.4 pmol/embryo/h; P<0.05) (Figure 4). There was a significant effect of oxygen (<0.05), and grouping (P<0.05), but no significant interaction. This pattern reflects the number of cells per sample, and when the number of cells was accounted for in the analysis, there was no effect of treatment (Table 1).

Grouped embryos appeared to consume more proline than individually cultured embryos, but this was not significant (Figure 4) until differences in cell number between treatments were accounted for (Table 1).

Threonine was consumed more by embryos cultured in 5% oxygen in groups (-0.59 ± 0.14 pmol/embryo/h) than embryos in 20% oxygen single culture (0.04 ± 0.14 pmol/embryo/h;

P<0.05) (Figure 4). There was a significant effect of grouping (P<0.01), but not oxygen, and the interaction was not significant. When cell number was included as a covariate, grouped embryos still consumed more threonine per embryo than singly cultured embryos (Table 1).

106

Chapter 5: Individual culture and oxygen affect metabolism and postimplantation development

Figure 4. Amino acid consumption/production Embryos were cultured under 5% or 20% oxygen, in groups of 10 embryos or individually. Media was collected on day 5 (118 h post-hCG) to determine utilization of 20 amino acids plus ornithine over 24 h from day 4 to 5 of culture. n = 10 groups of 10 embryos per treatment. Boxes represent 25-75th percentile, whiskers represent 5-95th percentile, line represents median, ‘+’ represents mean. Ala: alanine; Arg: arginine; Asn: asparagine; Asp: aspartate; Cys: cysteine; Gln: glutamine; Glu: glutamate; Gly: glycine; His: histidine; Ile: isoleucine; Leu: leucine; Lys: lysine; Met: methionine; Orn: ornithine; Phe: phenylalanine; Pro: proline; Ser: serine; Thr: threonine; Trp: tryptophan; Tyr: tyrosine; Val: valine. Amino acid utilization per embryo per hour. Different letters indicate significant differences between treatments. Asp: 5S vs 20S <0.01, others <0.001. Glu: 5G vs 5S <0.01, others <0.001. Leu: 5G vs 20S P<0.05. Met: 5G vs 20S P<0.05. Orn: 5S vs 20G P<0.05, 5G vs 20G P<0.01, others P<0.001. Thr: 5G vs 20S P<0.05.

107

Chapter 5: Individual culture and oxygen affect metabolism and postimplantation development

Like leucine, methionine and threonine, several amino acids including isoleucine, lysine, phenylalanine, tyrosine and valine appeared to be consumed more by embryos cultured at 5% oxygen in groups than other treatments, but these differences were not significant (Figure 4A).

There was no observable effect of the culture conditions on the utilisation of alanine, arginine, asparagine, cysteine, glutamine, glycine, histidine, serine, or tryptophan.

Ornithine was produced in all samples (Figure 4), but embryos cultured in 5% oxygen (grouped

0.37 ± 0.03, single 0.39 ± 0.03 pmol/embryo/h) produced less than those in 20% oxygen

(grouped 0.50 ± 0.03; P<0.05, single 0.57 ± 0.03 pmol/embryo/h; P<0.001). The effect of oxygen was significant (<0.001), but grouping and the interaction were not. This was still the case when cell number was included as a covariate (Table 1).

Postimplantation development is similar following individual or grouped culture under 5% oxygen Embryos cultured in groups or individually under 5% were removed from culture at 124 h post- hCG to determine their ability to attach and initiate implantation in vitro, or removed from culture at 94 h post-hCG and transferred to pseudopregnant recipients. All the embryos formed outgrowths (n=44-49), and there was no significant difference in outgrowth area formed at 189 h post-hCG (0.143 ± 0.010 vs 0.157 ± 0.009 mm2) or 213 h post-hCG (0.171 ± 0.014 vs 0.190 ±

0.014 mm2). The rate of growth was not affected by the preimplantation culture conditions.

After transfer to recipient mice, there were no differences in implantation rate, fetal development rate or sex ratio (Table 2).

108

Chapter 5: Individual culture and oxygen affect metabolism and postimplantation development

Figure 5. Fetal and placental development Embryos were cultured under 5% oxygen (A) or 20% oxygen (B), in groups of 10 embryos or individually, then transferred into pseudopregnant recipients. Fetal and placental development was assessed on embryonic day 15. Boxes represent 25-75th percentile, whiskers represent 5- 95th percentile, line represents median, ‘+’ represents mean. * indicates significantly different to embryos cultured in groups at the same oxygen concentration; *P<0.05, *** P<0.001

109

Chapter 5: Individual culture and oxygen affect metabolism and postimplantation development

Table 2. Postimplantation development after culture in 5% oxygen Embryos were cultured under 5% oxygen in groups of 10 embryos or individually, then transferred into synchronous pseudopregnant recipients on day 4 of culture 94-96 h post-hCG. Fetal and placental development was assessed on embryonic day 15. Non-pregnant recipients were excluded. Placentas were stained with Masson’s Trichrome to differentiate junctional zone and labyrinth (n=6). * indicates significantly different to embryo cultured in groups; *P<0.05.

Grouped Single Recipients (n) 13 13

Implantations n (%) 3.0 ± 0.5 (61.2 ± 10.4) 2.8 ± 0.4 (55.4 ± 7.9) Fetuses n (% of transferred) 2.0 ± 0.5 (40.8 ± 9.6) 1.5 ± 0.3 (29.2 ± 6.2) Fetuses n (% of implanted) 2.0 ± 0.5 (57.0 ± 10.8) 1.5 ± 0.3 (55.6 ± 10.6) Fetal sex - % male 41.1 ± 10.7 68.3 ± 12.8

Fetus n 28 19 Weight (mg) 205 ± 7 208 ± 8 Crown-rump length (mm) 11.0 ± 0.1 11.1 ± 0.1 Weight : length 1.87 ± 0.05 1.86 ± 0.06 Skin (days) 14.5 ± 0.12 14.68 ± 0.14 Limbs (days) 14.82 ± 0.10 14.67 ± 0.12 Ear (days) 14.86 ± 0.07 14.88 ± 0.09 Eye (days) 14.33 ± 0.14 14.26 ± 0.17 Development (days) 14.64 ± 0.07 14.64 ± 0.09

Placenta Weight (mg) 105 ± 4 108 ± 5 Diameter (mm) 8.05 ± 0.15 8.06 ± 0.17 Depth (mm) 2.60 ± 0.15 2.43 ± 0.18 Area (mm2) 10.37 ± 0.60 8.95 ± 0.60 Labyrinth (mm2) 4.42 ± 0.28 3.44 ± 0.28* Junctional zone (mm2) 3.23 ± 0.31 3.08 ± 0.31 Labyrinth : Junctional zone 1.45 ± 0.14 1.17 ± 0.14 Labyrinth : Total area 0.43 ± 0.02 0.38 ± 0.02 Junctional zone : Total area 0.31 ± 0.02 0.35 ± 0.02 Labyrinth + Junctional zone : Total area 0.73 ± 0.02 0.74 ± 0.02

Fetal : placental weight 2.04 ± 0.09 1.99 ± 0.10

110

Chapter 5: Individual culture and oxygen affect metabolism and postimplantation development

Fetal and placental development was not affected by single culture, as determined by weight, length and fetal development score (Figure 5A). The area of the labyrinth in the placenta was significantly smaller after individual culture (P<0.05), but the junctional zone and total area were not affected.

Postimplantation development is perturbed following individual culture under 20% oxygen Most of the embryos cultured in groups or individually under 20% oxygen formed outgrowths

(20% oxygen grouped 98% ± 14%, 20% oxygen single 89% ± 14%; n=45-48). Individually cultured embryos formed smaller outgrowths than group-cultured embryos at 189 h post-hCG (0.173 ±

0.013 vs 0.214 ± 0.013 mm2; P<0.05) and 213 h post-hCG (0.212 ± 0.019 vs 0.272 ± 0.018 mm2;

P<0.05). The rate of growth was not affected by the preimplantation culture conditions. After transfer to recipient mice, there were no differences in implantation rate, fetal development rate or sex ratio (Table 3). Individually cultured embryos developed into fetuses that were 11% lighter than fetuses from group cultured embryos (P<0.001; Figure 5B). The fetuses also had a shorter crown-rump length (P<0.001), and their weight:length ratio was smaller (P<0.01), indicating that the fetuses were skinnier (Table 3). Despite this, the fetuses were not developmentally delayed as determined by the morphology of selected features. The placentas were also 15% lighter (P<0.05), although no difference in diameter or depth was observed, or any difference in the area of the placental labyrinth or junctional zone. The fetal:placental weight ratio was not affected by individual culture.

111

Chapter 5: Individual culture and oxygen affect metabolism and postimplantation development

Table 3. Postimplantation development after culture in 20% oxygen Embryos were cultured under 20% oxygen in groups of 10 embryos or individually, then transferred into synchronous pseudopregnant recipients on day 4 of culture 94-96 h post-hCG. Fetal and placental development was assessed on embryonic day 15. Non-pregnant recipients were excluded. Placentas were stained with Masson’s Trichrome to differentiate junctional zone and labyrinth (n=6). * indicates significantly different to embryos cultured in groups; *P<0.05, **P<0.01, P<0.001

Grouped Single Recipients (n) 12 12

Implantations n (%) 3.4 ± 0.4 (71.7 ± 7.6) 3.0 ± 0.4 (60.0 ± 8.2) Fetuses n (% of transferred) 1.8 ± 0.5 (37.8 ± 9.0) 1.6 ± 0.4 (31.7 ± 7.6) Fetuses n (% of implanted) 1.8 ± 0.5 (49.6 ± 12.2) 1.6 ± 0.4 (48.5 ± 10.6) Fetal sex - % male 58.3 ± 13.4 50.0 ± 12.1

Fetus n 22 20 Weight (mg) 217 ± 5 192 ± 5*** Crown-rump length (mm) 11.3 ± 0.1 10.8 ± 0.1*** Weight : length 1.91 ± 0.04 1.77 ± 0.04** Skin (days) 14.73 ± 0.08 14.63 ± 0.09 Limbs (days) 14.98 ± 0.04 14.89 ± 0.05 Ear (days) 14.82 ± 0.08 14.84 ± 0.09 Eye (days) 14.47 ± 0.16 14.14 ± 0.17 Development (days) 14.74 ± 0.06 14.64 ± 0.06

Placenta Weight (mg) 112 ± 5 95 ± 5* Diameter (mm) 8.05 ± 0.16 7.57 ± 0.17 Depth (mm) 2.40 ± 0.12 2.47 ± 0.17 Area (mm2) 9.83 ± 0.89 8.87 ± 0.89 Labyrinth (mm2) 3.92 ± 0.43 3.03 ± 0.43 Junctional zone (mm2) 3.12 ± 0.45 3.02 ± 0.45 Labyrinth : Junctional zone 1.41 ± 0.31 1.26 ± 0.31 Labyrinth : Total area 0.41 ± 0.05 0.34 ± 0.05 Junctional zone : Total area 0.32 ± 0.04 0.34 ± 0.04 Labyrinth + Junctional zone : Total area 0.73 ± 0.03 0.68 ± 0.03

Fetal : placental weight 2.06 ± 0.10 2.06 ± 0.11

112

Chapter 5: Individual culture and oxygen affect metabolism and postimplantation development Discussion This study confirms and expands upon previous findings that a combination of atmospheric oxygen and individual culture is detrimental to preimplantation embryo development (Kelley and Gardner 2016, 2017). Differences between group-cultured and individually-cultured embryos are caused by changes embryos make to their microenvironment in vitro (Stokes et al.

2005, Gopichandran and Leese 2006). These include altering the metabolite composition of the media, and secretion of embryotrophic signalling molecules (Thouas et al. 2015, Wydooghe et al. 2017), which can influence embryo physiology and development. Some of molecules identified in the embryo secretome are also present in the oviduct or uterine lumen, and as such their production in vitro may partially compensate for the lack of signals from the endometrium

(Robertson et al. 2015, Salamonsen et al. 2016).

In humans, embryos would normally be alone in the tract, in contrast to mice, which are polyovulatory. It is not clear if preimplantation mouse embryos interact in vivo, but group culture is beneficial for both polyovulatory and monoovulatory species, demonstrated by experiments with bovine embryos (O'Doherty et al. 1997, Larson and Kubisch 1999, Fujita et al.

2006, Gopichandran and Leese 2006), and interestingly there is also an inter-species benefit of group culture (Spindler and Wildt 2001, Stilley et al. 2003, Spindler et al. 2006). Considering the overlap between embryo-secreted and tract-secreted molecules, it is possible that the benefit of group culture is not due to embryo-specific secreted molecules, but rather a more generic effect of paracrine signals from other cells.

Despite morphologically normal embryos being selected for metabolic analysis on day 5 of culture, development rates and cell numbers were reduced by culturing individually or in atmospheric oxygen. A combination of both individual culture and atmospheric oxygen had an additive effect, resulting in blastocysts with less than half the number of cells of blastocysts cultured in 5% oxygen in groups, which is consistent with previous findings (Kelley and Gardner

113

Chapter 5: Individual culture and oxygen affect metabolism and postimplantation development

2016, 2017). Fast development in vitro and high blastocyst cell number is an indicator of viability in the mouse (Lane and Gardner 1997, Lee et al. 2015), which corresponds to the results from the embryo transfer experiments.

Group culture influences metabolism by changing the microenvironment There are multiple mechanisms by which group culture could influence embryo metabolism.

During in vitro culture, embryos take up and secrete molecules in the media, creating gradients of these molecules around them (Thouas et al. 2015, Wydooghe et al. 2017). A single embryo in a relatively large volume of media will create a smaller gradient than a group of embryos

(Matsuura 2014, Ieda et al. 2018), therefore, embryos cultured in groups experience a different microenvironment than embryos cultured individually, and molecules in this microenvironment can directly regulate metabolism. For example, embryo-secreted growth factors and cytokines

(such as IGF-I (Inzunza et al. 2010)) can stabilise hypoxia-inducible factor (HIF)-1α or activate mammalian target of rapamycin complex 1 (mTORC1), both of which are potent regulators of metabolism (Wullschleger et al. 2006, Yoon et al. 2013). Embryo-secreted phospholipid platelet- activating factor (PAF) (Ryan et al. 1990) and miRNAs (Rosenbluth et al. 2014, Zhang et al. 2017) also regulate metabolic pathways. The concentration of metabolites in the medium can also influence metabolism; for example, a high concentration of lactate, which is produced by blastocysts at a high rate, reduces pyruvate uptake by the blastocyst and changes its metabolic fate (Lane and Gardner 2000, Gardner 2015). The concentration of amino acids can also influence their uptake (Lamb and Leese 1994), in part due to competition for transporters (Van

Winkle et al. 2006, Tan et al. 2011). Embryos also create an oxygen gradient during culture

(Trimarchi et al. 2000), and thus grouped embryos probably experience a lower oxygen concentration, which can influence metabolism (Khurana and Wales 1989, Wale and Gardner

2012). Furthermore, blastocysts cultured in groups also have larger ICMs (Kelley and Gardner

2016), and since the ICM and TE have different metabolic patterns (Gopichandran and Leese

2003, Houghton 2006), this may also explain some of the observed differences in metabolism.

114

Chapter 5: Individual culture and oxygen affect metabolism and postimplantation development

The tumour suppressor protein p53 appears to be responsible, in part, for the lower blastocyst rate of individually cultured embryos (Chandrakanthan et al. 2006) and may also cause increased apoptosis in the ICM (Brison and Schultz 1997, Ganeshan et al. 2017). Embryo-secreted molecules may suppress p53 when embryos are cultured in groups (O'Neill et al. 2015), but p53 can also be activated by dysregulation of metabolic pathways (Vousden and Prives 2009).

Furthermore, in addition to p53’s classical roles in DNA repair and cell cycle arrest, it regulates glucose metabolism (Vousden and Prives 2009). As such, the observed changes in metabolism following individual culture may indicate metabolic stress and therefore activate p53, or the observed changes in metabolism may be caused by activated p53 induced by changes in paracrine signalling.

Oxygen influences metabolism through HIFs and ROS Oxygen can affect embryo metabolism through several different mechanisms. Low oxygen environments can activate HIF-2α in blastocysts (Harvey et al. 2004, Ma et al. 2017), which moderates transcription of glucose transporters and metabolic enzymes such as lactate dehydrogenase, causing a shift towards glycolysis (Semenza et al. 1994, Harvey et al. 2004, Kind et al. 2005, Redel et al. 2012). On the other hand, a high oxygen environment causes an increase in ROS (Goto et al. 1993, Kitagawa et al. 2004), which can dysregulate many important signalling pathways and transcription factors which can alter metabolism and other cell functions (Harvey et al. 2002, Yoshida et al. 2011, Zhang et al. 2016). Excess ROS can also damage DNA and oxidate lipids and proteins, which stimulates repair mechanisms, slows cell division and ultimately changes metabolic requirements (Guerin et al. 2001, Harvey et al. 2002). Through these mechanisms, high or low oxygen in vitro can influence embryo metabolism.

Glucose and pyruvate metabolism is altered by group culture and oxygen At the blastocyst stage, the embryo’s main energy source is glucose (Gardner and Leese 1986), and high glucose consumption together with a low glycolytic rate is indicative of post-transfer development and live birth (Gardner and Leese 1987, Lane and Gardner 1996, Gardner et al.

115

Chapter 5: Individual culture and oxygen affect metabolism and postimplantation development

2011). In this study, blastocyst glucose consumption on days 4-5 was highest in embryos cultured in 5% oxygen in groups, indicating that they were the most viable. Combining individual culture with 20% oxygen resulted in glucose uptake that was around one-third of the uptake of embryos cultured in 5% oxygen in groups. Since these blastocysts had different numbers of cells, cell number was included in the analysis as a covariate, and embryos cultured individually under

20% oxygen still consumed less glucose per embryo compared to all other treatments, indicating that blastocysts cultured under different these conditions develop different physiologies. The lower glucose uptake could also be a result of developmental retardation, since formation and expansion of the blastocoel and degradation of the zona both require high glucose consumption

(reviewed by (Gardner and Harvey 2015)) and 34% of the blastocysts cultured individually under

20% oxygen had not expanded, and 66% had not hatched.

The lower glucose uptake under atmospheric oxygen is consistent with previous reports

(Khurana and Wales 1989, Wale and Gardner 2012). Further, Wale and Gardner (2012) found that the glycolytic rate of blastocysts cultured under atmospheric oxygen was higher, indicating a reduction in the oxidation of glucose under atmospheric oxygen.

Glucose has cellular functions beyond the production of ATP, including biosynthesis of nucleic acids and complex sugars, and glycolysis is also a source of acetyl Co-A, which is a cofactor for histone acetylation (Moussaieff et al. 2015). Changes in glucose metabolism, therefore, can influence the epigenetic regulation of gene expression, a phenomenon referred to as metaboloepigenetics (Harvey et al. 2016). These epigenetic changes allow the embryo to adapt to its in vitro environment, but the epigenetic marks may persist and ultimately cause long-term harm (Fleming et al. 2015). As such, altered glucose metabolism may be either the cause or consequence of reduced viability in embryos cultured individually under atmospheric oxygen.

Precompaction embryos utilise pyruvate as their main source of carbohydrates (Biggers et al.

1967, Leese and Barton 1984), and higher pyruvate consumption by cleavage stage embryos is

116

Chapter 5: Individual culture and oxygen affect metabolism and postimplantation development associated with human blastocyst development (Hardy et al. 1989b). Pyruvate uptake during the first 48 h of culture was not significantly affected by either oxygen or group culture, although it tended to be higher during culture under 5% oxygen compared to 20% oxygen, which is consistent with earlier data (Wale and Gardner 2012). In addition to being a source of energy, pyruvate is also an antioxidant (Andrae et al. 1985), so embryos in atmospheric oxygen may increase pyruvate uptake to counteract increased ROS.

Amino acid metabolism is altered by group culture and oxygen Like carbohydrate metabolism, amino acid metabolism is indicative of embryo viability and has been correlated with morphokinetics, blastocyst development, DNA damage, aneuploidy and pregnancy rate in the human, mouse, and other species (Houghton et al. 2002, Brison et al. 2004,

Booth et al. 2007, Seli et al. 2008, Sturmey et al. 2009, Picton et al. 2010, Lee et al. 2015, D'Souza et al. 2016, Souza et al. 2018). While there was no significant effect of culture conditions on total net turnover, the metabolism of several specific amino acids was altered.

Both group culture and 5% oxygen increased aspartate consumption, such that blastocysts in cultured in groups under 5% oxygen consumed the most, and embryos in 20% oxygen single culture consumed the least. This is consistent with reports that fast-cleaving embryos consume more aspartate and are more viable (Lee et al. 2015). Aspartate is a rate-limiting factor in the malate-aspartate shuttle, which regenerates cytosolic NAD+ required for the metabolism of glucose to pyruvate (Lane and Gardner 2005a, Mitchell et al. 2009). A decrease in glucose metabolism, as observed during individual culture in 20% oxygen, could reduce the requirement for aspartate for the MAS or conversely, a disrupted MAS would decrease glucose consumption

(Mitchell et al. 2009). As such, the observed changes in aspartate metabolism are indicative of changes in cellular activity in individually cultured embryos.

Embryos cultured under 5% oxygen in groups mostly consumed glutamate, however, embryos cultured individually or under 20% oxygen produced glutamate instead, most from the

117

Chapter 5: Individual culture and oxygen affect metabolism and postimplantation development transamination of other amino acids (Orsi and Leese 2004, Wale and Gardner 2013). This switch in glutamate metabolism restricts the embryo’s ability to transaminate ammonium (Wale and

Gardner 2012, 2013), indicating that embryos cultured individually or under 20% oxygen are more vulnerable to the toxicity of ammonium, which is generated during culture by the breakdown of amino acids. Significantly, human embryos that produce glutamate are less likely to implant (Seli et al. 2008), and slow-cleaving mouse and bovine embryos produce more glutamate than fast-cleaving embryos (Lee et al. 2015, Milazzotto et al. 2016), which supports the observation that embryos cultured under 5% oxygen in groups are more viable than embryos cultured individually or under 20% oxygen.

A combination of 20% oxygen and individual culture resulted in less uptake of methionine, leucine and threonine compared to 5% oxygen group culture, and approximately half of embryos switched to production of these amino acids. When cell number was included in the analysis, only the change in threonine consumption due to individual culture was statistically significant, indicating that leucine and methionine consumption may be closely correlated to the number of cells per blastocyst.

Leucine is utilised in the biosynthesis of cholesterol, an essential component of cell membranes

(Van Winkle et al. 1990), which may contribute to the correlation between cell number and leucine uptake. Leucine is also an important activator of mTORC1, which regulates many cell functions (Van Winkle et al. 2006), and as such, changes in leucine uptake may influence embryo physiology via mTOR signalling. Since leucine is an essential amino acid, its release into the media must derive from protein breakdown, indicating that embryos cultured individually in

20% oxygen may increase proteolysis. This is a clear difference in cellular activity between blastocysts cultured under 5% oxygen in groups or under 20% oxygen individually.

Disruption of methionine metabolism impairs blastocyst development, differentiation and methylation (Menezo et al. 1989, Ikeda et al. 2012, Kudo et al. 2015). Methionine is converted

118

Chapter 5: Individual culture and oxygen affect metabolism and postimplantation development to S-Adenosyl methionine (SAM), which is the universal methyl donor for methylation and is required for polyamine synthesis. Without SAM synthesis, embryos cannot make the transition from morula to blastocyst (Sun et al. 2018). More cell division may cause increased demand for

SAM and therefore methionine to methylate newly synthesised DNA, lipids and proteins, which may explain the correlation between cell number and methionine uptake. Perturbations in methionine utilisation could also alter the heritable epigenetic landscape of embryos.

Threonine can be metabolised by the folate cycle (Kwong et al. 2010), which generates acetyl

CoA, which can be involved in histone acetylation as discussed above (Moussaieff et al. 2015).

Mouse ESCs require threonine to maintain high levels of SAM, H3K4me3 and a pluripotent state

(Wang et al. 2009, Shyh-Chang et al. 2013), and while the metabolic fate of threonine in blastocysts has not been determined, changes to threonine metabolism could influence epigenetic regulation, causing long-term consequences as a result of individual culture.

Proline consumption was not different between the treatments, however, when cell number was included in the analysis, grouped embryos consumed more proline than individually cultured embryos. Proline can be reversibly converted to glutamate or ornithine, or it can be used by the embryo as an osmolyte (Dawson and Baltz 1997, Anas et al. 2007). In ESCs, proline metabolism regulates differentiation (Tan et al. 2011), activates mTOR (Washington et al. 2010), and modulates histone methylation (Comes et al. 2013). Proline transporters are differentially expressed in the ICM and TE (Tan et al. 2016), and while the role of proline in blastocyst differentiation has not been investigated, the larger ICM in group-cultured embryos (Kelley and

Gardner 2016) may be responsible for the difference in proline consumption and it may have consequences for epigenetic regulation of gene expression.

The only non-proteinogenic amino acid analysed, ornithine is a precursor for polyamine synthesis. Mice lacking ornithine decarboxylase (ODC), which converts ornithine to putrescine, develop blastocysts that die peri-implantation due to widespread apoptosis in the ICM

119

Chapter 5: Individual culture and oxygen affect metabolism and postimplantation development

(Pendeville et al. 2001). Polyamines have multiple roles within cells, including ROS scavenging

(Ha et al. 1998), stabilising chromatin (Snyder 1989), and DNA synthesis (Zwierzchowski et al.

1986), and are required for pluripotency in ESCs (Zhao et al. 2012). Ornithine can also be reversibly converted to glutamate or proline, the significance of which have been discussed above. Net production of ornithine into culture media was observed in all embryos, and this was higher under 20% oxygen, as previously observed (Wale and Gardner 2013). This may be caused by an increase in synthesis from arginine without a corresponding increase in polyamine synthesis, as suggested by a non-significant increase in arginine consumption under 20% oxygen.

This data indicates that culture under atmospheric oxygen may perturb polyamine synthesis, providing another potential mechanism for the lower viability of these embryos.

Post-implantation development is perturbed by individual culture Peri-implantation viability as assessed by outgrowth assay was not affected by individual culture under 5% oxygen, but under 20% oxygen, individual culture resulted in smaller outgrowths than embryos that had been cultured in groups, indicating they were less viable. This was supported by results from in vivo embryo transfer experiments. Individual culture under 5% oxygen had minimal observed effects on post-implantation development, whereas under atmospheric oxygen foetuses and placentas were smaller after individual culture.

Previous investigations into the post-implantation development of embryos cultured individually have reported decreases in implantation and live birth rates compared to group culture, but these differences have not been statistically significant. Two studies in the mouse reported non-significant decreases in implantation rate (38% vs 28%) and fetal development rate (28% vs 21%) (Lane and Gardner 1992) or percentage of live fetuses (26% to 15%) (Kato and

Tsunoda 1994) following individual culture under atmospheric oxygen. In the bovine, Isobe

(2014) also found a non-significant increase in stillbirth rate (25% vs 14%) and lower live birth rate (75% v 86%) after individual culture under 5% oxygen. Consistent with the results from animal studies, Ebner et al. (2010) found that individual culture of human embryos reduced live

120

Chapter 5: Individual culture and oxygen affect metabolism and postimplantation development births from 62% to 39% compared to group-cultured embryos, and again this difference was not statistically significant. Taken together with the results from the current study, it appears that there may be subtle post-implantation effects of individual culture. Given that altering the preimplantation environment can influence postnatal and adult growth, metabolism and neuromotor development (Fernandez-Gonzalez et al. 2004, Donjacour et al. 2014), studies into the health of these offspring and their postnatal development is warranted even if the decreases in live birth rate and fetal weight are not statistically significant. Furthermore, given the metabolic changes observed at the blastocyst stage, the epigenetic status of these fetuses and placentas may be influenced by the preimplantation environment, as has been observed following culture in media lacking amino acids (Mann et al. 2004). Such heritable changes can have consequences for adult health (Fleming et al. 2015), but these effects are not necessarily observable through measures of fetal and placental size.

Placental function is essential for the health of the fetus, and preimplantation environment can influence placental growth, epigenetics and gene expression (Mann et al. 2004, Delle Piane et al. 2010, de Waal et al. 2014, de Waal et al. 2015, Ghosh et al. 2017). In the current study, individual culture under 20% oxygen caused a decrease in placental weight, although the fetal:placental weight ratio was not affected, indicating slower but uniform growth. The placental labyrinth contains the villi where nutrients pass from maternal to fetal blood, and the size of the labyrinth determines the ability of the placenta to support fetal development (Coan et al. 2008). Labyrinth area was smaller after individual culture under both oxygen concentrations, although due to the small number of samples and the high degree of variability under 20% oxygen, this was not statistically significant. This data, therefore, indicate that individual culture may reduce the efficiency of the placenta.

Although the effects of oxygen on fetal and placental development were not assessed in this study, other investigators have shown that culture 5% oxygen results in fewer miscarriages in

121

Chapter 5: Individual culture and oxygen affect metabolism and postimplantation development the mouse (Karagenc et al. 2004), and higher implantation, pregnancy and live birth rates in the human (Catt and Henman 2000, Meintjes et al. 2009a, Waldenstrom et al. 2009, Gomes

Sobrinho et al. 2011, Bontekoe et al. 2012).

Two culture stresses are worse than one The two culture stresses of atmospheric oxygen and individual culture have an additive detrimental effect on the development and metabolism of preimplantation embryos. Combining the two stresses had consequences that were either exaggerated compared to one stress or caused further perturbations that were not observed when the embryo was only exposed to one stress. This may be because the further the culture conditions become from optimal, the more resources an embryo uses to adapt and survive, or there is an accumulation of trauma, and the manifestations of stress become more exaggerated. There are many other examples of the additive nature of these culture stresses, for example, embryos cultured in atmospheric oxygen are more sensitive to the stresses of ammonium, or media lacking amino acids (Wale and

Gardner 2013, Feuer et al. 2016). Similarly, individually cultured mouse embryos are more sensitive to peroxides in oil than group cultured embryos (Hughes et al. 2010).

This concept of cumulative culture stresses is highly relevant for human embryo culture. If embryos are routinely cultured individually or in atmospheric oxygen, they may be more vulnerable to a second or third stress introduced to the culture system, such as a batch of poor quality oil, or a drop in laboratory air quality. There are many potential sources of stress to the embryo or the gametes during ART (Gardner and Kelley 2017) and these data highlight the importance of optimising every aspect of the culture system to minimise stress. Importantly, patient factors such as age, obesity, aetiology of infertility, and response to ovarian stimulation also contribute to gamete quality and subsequent embryo development, and patient factors from both parents can interact to result in a cumulative effect on embryo development (Finger et al. 2015, McPherson et al. 2015, McPherson et al. 2018).

122

Chapter 5: Individual culture and oxygen affect metabolism and postimplantation development

This cumulative and interacting nature of culture stresses may also help to explain some of the variation in the scientific literature between clinics, as the culture environment determines how embryos respond to a variable of interest, depending on the level of stress experienced. For example, culturing embryos individually can exaggerate the beneficial effect of adding growth factors or antioxidants to culture media (Paria and Dey 1990, Truong et al. 2015) or exaggerate the detrimental effect of additional stress such as peroxides in oil (Hughes et al. 2010). The same paradigm applies to oxygen; for example, labs using atmospheric oxygen report adverse perinatal outcomes following blastocyst culture, whereas labs using reduced oxygen have reported no detrimental effects (Gardner 2016).

In conclusion, embryos cultured individually exhibit not only slower cleavage divisions, fewer cells, smaller ICMs, and more apoptosis compared to embryos cultured in groups (Brison and

Schultz 1997, Kelley and Gardner 2016), they also have different physiologies, demonstrated by the net turnover of glucose and amino acids. These differences are exacerbated by culture under

20% oxygen, which itself induces many changes to metabolism and development (Wale and

Gardner 2016). The observed changes in preimplantation metabolism following individual culture or exposure to atmospheric oxygen may influence epigenetic control of gene expression

(Harvey et al. 2016), which could persist and result in altered fetal and postnatal development

(Fleming et al. 2015). Indeed, the detrimental effects of individual culture on fetal and placental growth was observed following culture under 20% oxygen, but not 5% oxygen, although more subtle changes may be present. This study, therefore, raises concerns regarding the prevalence of individual culture in human IVF and reinforces the importance of optimisation of every component of the embryo culture system due to the additive effect of culture stresses.

123

Chapter 5: Individual culture and oxygen affect metabolism and postimplantation development

Supplementary data Supplementary table 1. Developmental stage of embryos in metabolism assays Embryos were cultured under 5% or 20% oxygen, in groups of 10 embryos or individually. n = 10 groups of 10 embryos per treatment selected for metabolic analysis. Table shows mean ± SEM of % embryos per group of 10 at each developmental stage on days 3, 4 and 5 of culture (70, 94 and 118 h post-hCG). Different superscripts indicate significant differences between treatments by post-hoc analysis.

5% oxygen 5% oxygen 20% oxygen 20% oxygen grouped single grouped single Grouping Oxygen Interaction 3-8 cells (%) 7.0 ± 2.9 22.0 ± 5.3 9.0 ± 3.4 17.0 ± 4.6 <0.01 NS NS Day 3 Compacting (%) 93.4 ± 4.4 78.3 ± 4.0 91.4 ± 4.3 83.4 ± 4.1 <0.01 NS NS Compacting (%) 0.8 ± 0.9a 4.4 ± 2.0a 2.6 ± 1.5a 15.8 ± 4.1b <0.01 <0.05 NS Early blastocyst (%) 2.0 ± 1.2a 5.0 ± 2.0ab 7.0 ± 2.3ab 12.0 ± 3.1b NS <0.05 NS Day 4 Blastocyst (%) 1.0 ± 3.1 2.0 ± 4.4 1.0 ± 3.1 2.3 ± 4.9 <0.01 NS NS Expanded (%) 15.2 ± 4.1 20.7 ± 4.9 16.0 ± 4.2 14.4 ± 4.0 NS NS NS Hatching (%) 66.0 ± 7.5a 41.0 ± 5.9bc 57.0 ± 6.9ab 25.0 ± 4.6c <0.001 <0.05 NS Early blastocyst (%) 0.0 ± 0.0 3.0 ± 1.5 1.0 ± 1.0 6.0 ± 4.0 NS NS NS Blastocyst (%) 0.8 ± 0.7a 5.4 ± 2.1ab 6.1 ± 2.3ab 16.0 ± 4.2b <0.01 <0.01 NS Day 5 Expanded (%) 2.8 ± 1.5a 14.1 ± 3.4b 18.8 ± 3.9bc 32.0 ± 5.3c <0.001 <0.001 NS Hatching (%) 94.4 ± 7.0a 70.6 ± 6.1b 64.6 ± 5.8bc 32.8 ± 4.1c <0.001 <0.001 <0.05 Fully hatched (%) 2.0 ± 1.6 2.0 ± 1.6 5.0 ± 2.5 1.0 ± 1.1 NS NS NS

124

Chapter 6: Addition of interleukin-6 to mouse embryo culture increases blastocyst cell number and influences the inner cell mass to trophectoderm ratio

Chapter 6: Interleukin-6

126

Chapter 6: Interleukin-6

127

Chapter 6: Interleukin-6

128

Chapter 6: Interleukin-6

129

Chapter 6: Interleukin-6

130

Chapter 6: Interleukin-6

131

Chapter 6: Interleukin-6

132

Chapter 7: General discussion

Chapter 7: General Discussion

Human IVF clinics have historically cultured embryos in groups, but a need to select the single best embryo for transfer combined with advances in embryo monitoring has led many clinics to culture embryos individually. This seemingly innocuous change has been conducted with very little investigation into its potential consequences, but the studies presented in this thesis demonstrate that culturing embryos individually can influence both preimplantation and postimplantation development in the mouse model. Furthermore, and perhaps of the most significance, not only can embryo density alter embryo development, it determines how the embryos respond to other stresses during culture.

In chapter 3, grouped and individual culture of mouse embryos was compared, and the impact of altering selected culture conditions was investigated with the aim of improving outcomes for individually cultured embryos. Firstly, under 5% oxygen, individual culture for either the precompaction or post-compaction half of the culture period resulted in fewer cells per blastocyst and smaller ICMs compared to embryos cultured in groups throughout, indicating that development was impaired by individual culture during either the pre-compaction or post- compaction stage. However, there may be some benefit of culturing in groups for only half of the culture compared to continuous individual culture, indicated by more trophectoderm cell numbers, which may be beneficial for implantation and the establishment of the placenta. These findings are relevant to human IVF because some clinics (estimated to be around 5 - 16%

(Christianson et al. 2014)) culture embryos individually for half of the culture period. Evidence from this study and other animal models suggests while this practice is preferable to continuous individual culture, it may still compromise embryo development compared to group culture

(Wright et al. 1978, O'Neill 1998, Stokes et al. 2005), and the limited evidence available from clinical studies supports this (Rebollar-Lazaro and Matson 2010).

Secondly, the interaction between oxygen concentration and embryo density was investigated.

Under 5% oxygen, individually cultured embryos were more likely to form hatching blastocysts

134

Chapter 7: General Discussion and had more cells in both the TE and ICM compared to culture under 20% oxygen. Interestingly, reducing the embryo density by decreasing the media volume was only beneficial under 5% oxygen, not 20% oxygen, as shown by an increase in cell numbers in the TE. No increase in cell number was observed in the ICM, unlike the effect of increasing embryo density by adding embryos rather than reducing media volume. Possibly the optimal embryo density under 20% oxygen is higher than 1:2 µL and a range of media volumes would need to be tested to observe an effect of changing embryo density at this oxygen concentration. An interaction between oxygen concentration and embryo density is consistent with work in the bovine (Nagao et al.

1998) and goes some way to explaining why human studies on optimal embryo density have produced conflicting results (De Munck et al. 2015, Minasi et al. 2015). This interaction is plausibly due to differences in the microenvironment of embryos cultured under 20% and 5% oxygen. Embryos cultured under 20% oxygen typically have fewer cells and plausibly produce fewer embryotrophic molecules including growth factors and cytokines, thereby requiring a smaller media volume to create an effective concentration. Alternatively, since embryos in atmospheric oxygen are less viable they may secrete more embryotoxic or stress-inducing molecules (Salahuddin et al. 1995, Spindler and Wildt 2002, Tao et al. 2013) such as TNF-α (Zolti et al. 1991, Pampfer et al. 1994, Kurzawa et al. 2001) and therefore decreasing the media volume does not promote development. Oxygen also regulates metabolism, resulting in different concentrations of metabolites in the media under 5% or 20% oxygen (Khurana and Wales 1989,

Wale and Gardner 2012), which can influence embryo physiology (Gardner and Leese 1988,

Lamb and Leese 1994, Lane and Gardner 2000, Orsi and Leese 2004, Lane and Gardner 2005a).

Embryos also consume oxygen, creating a gradient around themselves (Trimarchi et al. 2000), so it’s possible that a low oxygen environment combined with high embryo density results in an optimal oxygen microenvironment, one even lower than the gaseous 5%. Many IVF clinics do not consider embryo density to be an important variable in determining culture outcome (Reed

2012), but these new data suggest that embryo density should be optimised like all other

135

Chapter 7: General Discussion elements of the culture system. With this in mind, optimal embryo density is likely to differ between clinics, due to differences in oxygen concentration and media formulations (Gardner et al. 1994, Kato and Tsunoda 1994).

Microwell dishes have recently been introduced into IVF labs to facilitate the monitoring of embryos by time-lapse microscopy. The volume of media in a microwell is around 100 times less than conventional culture, and the high embryo density may allow individual embryos to establish an embryotrophic environment, and thus improve development compared to conventional individual culture (Vajta et al. 2000, Matsuura 2014). Studies on the benefits of microwells to date have all been carried out using custom-built dishes and the efficacy of commercially available microwell dishes have not previously been reported. In this experiment, embryo culture in three different commercially available microwell dishes increased the number of cells in the ICM compared to conventional single culture. A larger ICM is indicative of greater viability (Lane and Gardner 1997) and a greater ICM:TE ratio is more similar to group-cultured embryos, which supports the hypothesis that microwells create a microenvironment similar to group culture. However, culture in different dishes resulted in different cleavage times, indicating that the size and shape of the microwell influences embryo development, consistent with previous data (Hoelker 2010). The EmbryoSlide dishes appeared to perform better than those from the PrimoVision, but since mouse embryos are smaller and less active than human embryos, the optimum size and shape of microwells for human embryos will likely be different.

This experiment was not designed to determine if there is communication between embryos cultured in groups in microwell dishes, but the data suggest that there may be little benefit to culture in a dish with multiple microwells under a single drop of media (PrimoVision) compared to a single microwell (EmbryoSlide). However, other differences between the dishes mean that this is not conclusive. Attempts to determine if microwell dishes allow paracrine communication between embryos have produced conflicting results (Vajta et al. 2000, Pereira et al. 2005, Dai et al. 2012, Sugimura et al. 2013, Kang et al. 2015, Lehner et al. 2017, Ieda et al. 2018), but clearly

136

Chapter 7: General Discussion this is dependent on the size and spacing of the microwells, and there have been no reports on the possibility of paracrine communication in commercial microwell dishes. Given the results using animal models, more extensive human studies into the possible benefits of microwells are warranted. Specifically, the ideal shape and size of microwells for human embryo culture should be established, and the potential benefit of multiple microwells under a single drop of medium determined.

Finally, Chapter 3 explored the effect of culturing individual embryos in embryo-conditioned media to determine if embryo-secreted molecules present in the media would promote development under these conditions. Individual embryos cultured in embryo-conditioned media had more cells at the blastocyst stage and a higher rate of hatching. This outcome is consistent with similar studies in the mouse (Stoddart et al. 1996) and bovine (Fujita et al. 2006) under more stressful culture conditions, and confirms that culture of individual pronucleate mouse embryos in embryo-conditioned media can substitute for culture in groups, at least in regard to blastocyst formation, cell number and hatching. The factors responsible for this growth- promoting effect are incompletely defined, but once identified they could inform the development of future culture media formulations.

Data presented in this thesis highlight the importance of the embryo’s microenvironment during culture and raise questions regarding the significance of changing or refreshing the media during blastocyst culture. The use of time-lapse incubators for embryo monitoring allow undisturbed culture because the embryos do not need to be removed from the incubator for assessment, however, there is concern that not renewing the medium will allow build-up of waste products

(particularly ammonium) and deplete nutrients (Gardner and Lane 1993, Lane and Gardner

1994). Also, unlike sequential media, undisturbed culture does not mimic the changing nutrient profile of oviduct and uterine fluids and accommodate the changing needs of the embryo

(Gardner and Lane 1998). Human studies to date have found no effect of refreshing the medium

137

Chapter 7: General Discussion on day 3 (with the same culture medium) on morphokinetics, blastocyst development, implantation or pregnancy rates compared with continuous culture (Macklon et al. 2002, Costa-

Borges et al. 2016, Pena et al. 2018). This indicates that one disruption of the microenvironment is not stressful enough to have obvious effects on development, and on the other hand, removing waste products and replenishing nutrients has no obvious benefit, at least under the culture conditions used. This does not take into account changing the nutritional composition of the media. Studies comparing one-step and sequential media are generally difficult to interpret due to the number of variables involved, but two studies comparing one-step and sequential media from the same manufacturer found no differences in blastocyst rate, implantation rate or pregnancy rate (Ciray et al. 2012, Hardarson et al. 2015). In the future, development of microfluidic systems may be able to incorporate the benefits of both approaches (Urbanski et al. 2008, Swain et al. 2013), but at the moment it is not clear if disturbing the microenvironment to refresh or change the medium is detrimental or beneficial.

The aims of chapters 4 and 5 were to expand upon the findings of chapter 3 by investigating the interaction between group culture and oxygen concentration (Figure 1). In keeping with a large body of evidence on the detrimental effects of atmospheric oxygen during preimplantation culture (reviewed by (Bontekoe et al. 2012, Wale and Gardner 2016)), embryos cultured in 5% oxygen (grouped or individually) had faster cleavages, higher blastocyst rates, and more cells per blastocyst. Furthermore, oxygen concentration affected blastocyst metabolism, with atmospheric oxygen causing a reduction in the net uptake of aspartate and glucose, and an increase in the production of ornithine, consistent with previous studies (Khurana and Wales

1989, Wale and Gardner 2012). Culture of embryos individually under either oxygen concentration resulted in slower cleavage divisions, fewer blastocysts hatching, fewer cells per blastocyst and a reduction in %ICM compared to embryos cultured in groups. Individually cultured blastocysts also had lower net glucose and aspartate uptake and a reduction in net uptake of proline and threonine. It is interesting that some of the observed effects of

138

Chapter 7: General Discussion atmospheric oxygen and individual culture were the same, so that embryos cultured individually under 20% oxygen were doubly affected. For example, the cell number of blastocysts on day 5 of culture under was reduced from 134.1 ± 3.4 under 5% oxygen in groups to 104.5 ± 3.2 by changing to individual culture, or 73.4 ± 2.2 by changing to atmospheric oxygen. By changing both conditions, the cell number was further reduced to 57.0 ± 2.8, a 58% decrease compared to the control. This same additive effect of the two stressful culture conditions was also observed in cleavage timings, day 5 hatching rate, and glucose, aspartate, leucine, methionine and threonine uptakes. On the other hand, some outcomes revealed an interacting effect of oxygen and individual culture. Net production of glutamate, which is negatively associated with embryo viability (Seli et al. 2008, Lee et al. 2015, Milazzotto et al. 2016), was observed under all culture conditions except the control (5% oxygen group culture), indicating perhaps that below a certain level of stress the embryo can use glutamate to transaminate ammonium, but under stressful conditions glutamate is taken up instead (Wale and Gardner 2013). Another example of interaction is glucose metabolism; when cell number was included in the analysis embryos cultured individually under 20% oxygen consumed significantly less glucose than embryos in all other treatments, indicating that the cells in these blastocysts were probably less active than under other culture conditions. Likewise, some of the morphokinetic delays were only observed during individual culture in 20% oxygen, such as the 6 h delay in cavitation and 4 h delay in hatching compared to group culture under the same oxygen concentration. Compared to culture in groups under 5% oxygen, culture of embryos individually or under atmospheric oxygen was detrimental and altered metabolism. However, combining the two detrimental conditions resulted in earlier and longer delays to development, fewer and smaller blastocysts, and more exaggerated changes to metabolism.

139

Chapter 7: General Discussion

Figure 1. The effects of individual culture and 20% oxygen on mouse blastocysts compared to culture under 5% oxygen in groups The yellow circle represents individually cultured blastocysts compared to group cultured blastocysts (in either atmospheric or reduced oxygen). The pink circle represents blastocysts cultured under 20% oxygen compared to 5% oxygen (in either group culture or individual culture). The orange overlap in the middle represents the blastocysts cultured individually under 20% oxygen compared to blastocysts culture either under 5% oxygen individually, or 20% oxygen in groups. Some of the observed phenotypes are caused by either individual culture or oxygen and are not influenced by the other stressor, e.g. reduction in %ICM is observed after individual culture compared to group culture, regardless of oxygen concentration. Other phenotypes, indicated by double arrows, are additive effects of the two stressors, e.g. cell numbers are lower after individual culture compared to group culture, and lower after culture under 20% oxygen compared to 5% oxygen, thus combining individual culture and 20% oxygen results in a further reduction in cell numbers. Other effects were observed only when individual culture and 20% oxygen were combined, e.g. changes to leucine and methionine metabolism. The results from this thesis are in blue and other researchers’ finding in green, based on which it is likely that embryos cultured individually under 20% oxygen will also exhibit increased apoptosis (Brison and Schultz 1997), excessive reactive oxygen species (ROS) (Goto et al. 1993), increased percentage of glucose metabolised by glycolysis (Wale and Gardner 2012), and changes to gene expression (Kind et al. 2005, Rinaudo et al. 2006) and protein expression (Katz-Jaffe et al. 2005, Meuter et al. 2014), and increased rate of miscarriage (Karagenc et al. 2004) compared to those cultured under 5% oxygen in groups. It is yet to be determined if mouse blastocysts cultured under 20% oxygen also exhibit changes in epigenetic marks (Gaspar et al. 2015, Li et al. 2016) or DNA damage (Takahashi et al. 2000) as observed in bovine embryos.

140

Chapter 7: General Discussion

Changes in preimplantation metabolism are indicative of lower viability post transfer (Lane and

Gardner 1996), therefore, an investigation into the effects of individual culture on postimplantation development was undertaken. After culture under 5% oxygen, there was no difference in the size of the outgrowth formed by group-cultured and individually cultured embryos, nor any difference in implantation rate, miscarriage rate, fetal and placental weight, or fetal development score. However, individual culture under 5% oxygen was associated with a smaller placental labyrinth. Furthermore, this does not rule out more subtle effects of individual culture on fetal and placental epigenetics and postnatal development, and given the changes observed in preimplantation metabolism, this is plausible. However, after culture under

20% oxygen, individually cultured embryos formed smaller outgrowths, smaller and thinner fetuses, and smaller placentas compared to embryos cultured in groups. Like the preimplantation results, this demonstrates that the effects of individual culture are more exaggerated when combined with 20% oxygen. The development of these foetuses could be investigated further by determining whether the culture conditions have influenced the epigenetic regulation of gene expression, which is then likely to determine postnatal development.

Embryos cultured individually or under atmospheric oxygen exhibited slower development, lower cell numbers, and changes in metabolism, all of which are indicative of stress (Feuer and

Rinaudo 2012, Puscheck et al. 2015). However, this study did not determine if individual culture or atmospheric oxygen activate specific stress response proteins that are known to be activated under other stressful culture conditions. Under low levels of stress, activation of stress response pathways can promote survival and suppress apoptosis, but under prolonged or high levels of stress they prevent proliferation and promote cell death, and these mechanisms may be involved in the embryo’s response to individual culture or atmospheric oxygen. Culture stress, for example hyperosmotic stress (Xie et al. 2007b), shear stress (Xie et al. 2007a), or simple culture media (Wang et al. 2005, Xie et al. 2006), can activate several different stress-response

141

Chapter 7: General Discussion pathways in embryos, such as SAPK/JNK (MAPK8/9), which upregulates transcription factors that slow proliferation, increase apoptosis and induce differentiation. Culture stress can also upregulate AMPK (Xie et al. 2013), possibly because maintaining homeostasis in a stressful environment requires energy, which lowers the AMP: ATP ratio, activating AMPK. This could then shift the embryo from anabolic to catabolic pathways and influence mitochondrial function, resulting in slower proliferation and altered cell allocation, providing a link between stress and metabolism (Puscheck et al. 2015). Culture stress can also activate the transcription factor p53 which stops the cell cycle and initiates apoptosis (Chandrakanthan et al. 2006,

Ganeshan et al. 2017). Embryo-secreted PAF, via the PI3/AKT pathway, reduces the activation of p53 in mouse embryos (Jin et al. 2009). Inactivating p53 improves the blastocyst rate of individually cultured embryos (Chandrakanthan et al. 2006), indicating that p53 plays an important role in the stress response to individual culture. Embryos can cope with some level of stress, and stress response pathways can be important in normal development, but beyond a certain threshold of stress, the embryo is unable to maintain normal physiology and developmental program. This may be the result of a single high-level stressor or may be the culmination of multiple low-level stressors. To further determine the molecular mechanisms by which individual culture and atmospheric oxygen cause stress, these stress response pathways should be investigated.

The work presented in this thesis demonstrates that embryo density can determine the embryo’s response to other elements of the culture system. There are many unavoidable sources of potential stress to gametes and embryos during IVF treatment; from ovarian stimulation and gamete collection, to manipulations like ICSI, cryopreservation and biopsy, to simple pipetting and fluctuations in temperature, pH and oxygen during handling (Puscheck et al. 2015, Wale and Gardner 2016, Gardner and Kelley 2017). Furthermore, patient factors such as smoking, obesity, and aetiology of infertility also determine embryo quality, and multiple unfavourable patient factors are more detrimental than one (Finger et al. 2015, McPherson et

142

Chapter 7: General Discussion al. 2018). The stress from each of these elements could accumulate to reduce embryo quality, however minimisation of culture stress where possible would result in more embryos available for transfer or cryopreservation, higher implantation and pregnancy rates, and healthier babies and children. Since there are so many uncontrollable sources of stress to the embryo, it is critical to avoid any additional stresses where possible, including optimising embryo density and oxygen concentration.

The significance of this work goes beyond the initial findings; the fact that the stress an embryo is under determines its responses to other conditions is important for the interpretation of disputes in the scientific literature. For example, embryos cultured in suboptimal conditions have a stronger positive response to growth factors (Karagenc et al. 2005, Ziebe et al. 2013) or antioxidants (Truong et al. 2016), and a stronger negative response to stress such as peroxides

(Hughes et al. 2010). Unfortunately, there is inconsistency in reporting experimental conditions, with researchers omitting key information such as the oxygen concentration used during culture, making interpretation of all the variables difficult.

As demonstrated in Chapter 3, the beneficial effect of culturing embryos in groups is due, at least in part, to the microenvironment they create in vitro. Therefore, in Chapter 6 the concentration of embryo-secreted growth factors and cytokines in the in vitro microenvironment of the preimplantation embryo was investigated. After identifying IL-6 as a highly-secreted cytokine in post-compaction embryo-conditioned media, consistent with previous studies in humans and mice (Zolti et al. 1991, Austgulen et al. 1995, Dominguez et al.

2010, Yu et al. 2012, Dominguez et al. 2015, Lindgren et al. 2018), it was added to individually cultured embryos to determine if it would promote their growth in a similar manner to group culture. rmIL-6 increased hatching rates at 0.01 ng/ml and 10 ng/ml, and total cell number at

0.1 ng/ml, but without an increase in ICM. The highest dose of 100 ng/ml rmIL-6 was detrimental to cell number and ICM size, suggesting perhaps that an excess of IL-6 causes dysregulation of

143

Chapter 7: General Discussion cell signalling pathways. Previous reports on the effect of IL-6 on mouse embryos have used stressful culture environments with media lacking amino acids (Shen et al. 2009) or atmospheric oxygen (Desai et al. 1999, Yu et al. 2018), but this study shows that the development of high- quality individual embryos under optimised conditions can still be improved by supplementation with IL-6.

IL-6 has a wide range of target genes but one way in which it may result in higher cell numbers is by inhibiting apoptosis through STAT3 (Shen et al. 2009). This raises concerns since apoptosis is an important part of normal development and is required to eliminate cells that are developing abnormally (Brison and Schultz 1997). If, like GM-CSF (Chin et al. 2009), its growth- promoting effect is due to the inhibition of apoptosis, the potential long-term consequences should be thoroughly investigated before its addition to human culture media (Thouas et al.

2015). The higher rate of hatching blastocysts may be related to the higher cell number, or IL-6 may upregulate proteases (Mohamed et al. 2010, Seshagiri et al. 2016). These data show that

IL-6 is an embryotrophic cytokine, but it is just one of hundreds of embryo-secreted and tract- secreted molecules that can influence embryo development, and supplementation to culture would be more physiological in combination with other growth factors. Furthermore, expression of IL-6 in the tract is dynamic (Sanford et al. 1992), and therefore timing of exposure to IL-6 may be important for appropriate development.

Conclusions The data presented in this thesis demonstrate that group culture of embryos is preferable to individual culture, especially under suboptimal culture conditions, and that single culture increases susceptibility to other stressors. However, group culture is now becoming less common and is likely to be superseded in the near future, with ESHRE guidelines now advocating single embryo culture (Labs et al. 2016). The need to culture embryos individually for tracking and monitoring generates a moral obligation to optimise the culture environment for individual

144

Chapter 7: General Discussion embryos in order to reduce their vulnerability to stress. This may be through the development of new media, including the addition of embryo-secreted and tract-secreted factors, or it may be through the development of culture dishes such as microwell dishes or microfluidic dishes.

These advances should improve the likelihood of patients achieving a pregnancy, and the health of children born from IVF.

145

Appendices

146

Appendices

Appendix A

Table 1. Stocks for media preparation

Component Molecular Weight (g) for Final weight stock concentration in media (mM) Salt stock 100 mL X10 Use for 3 months NaCl 58.44 5.2643 90.08 KCl 74.56 0.4108 5.5 NaH2PO4 119.98 0.02995 0.25 MgSO4.7H20 246.47 0.24647 1

Bicarbonate stock 84.01 20 mL X10 NaHCO3 0.42006 25 Use for 1 month

Calcium stock 5 mL X100 CaCl2 147.02 0.13232 1.8 Use for 1 month

Carbohydrate stock for G1 5 mL X100 Use for 1 month Glucose 180.16 0.04504 0.5 Sodium L-Lactate acid 112.06 0.5883 10.5 Sodium pyruvate 110.04 0.01761 0.32

Carbohydrate stock for G2 5 mL X100 Use for 1 month Glucose 180.16 0.28375 3.15 Sodium L-Lactate acid 112.06 0.3289 5.87 Sodium pyruvate 110.04 0.0055 0.1

EDTA stock 5 mL X100 292.24 Use for 1 month EDTA 0.00185 0.01

Aln-Gln stock Alanyl-glutamine 217.2 0.2172 0.5

147

Appendices

5 mL X100 Use for 3 months

NEAA stock* 20 mL X100 Use for 3 months L-Alanine 89.09 0.0178 0.1 L-Aspartatic acid 133.1 0.0266 0.1 L-Asparagine-H2O 150.14 0.03003 0.1 L-Glutamatic acid 169.1 0.03382 0.1 Glycine 75.07 0.01501 0.1 L-Proline 115.13 0.02301 0.1 L-Serine 105.09 0.02102 0.1

Taurine stock 10 mL X100 125.14 0.0125 0.1 Use for 3 months Taurine

Vitamin stock 20 mL X100 D-Ca 238.3 Use for 3 months Pantothenate 0.002 0.0042 Pyridoxal-HCl 203.62 0.002 0.0049 Thiamine-HCl 337.27 0.00202 0.0030 Riboflavin 367.37 0.0002 0.0003

Hyaluronan stock 50 mL X25 0.16076 0.125 mg/ml Use for 3 months Hyaluronan

Gentomycin stock 1 mL X4000 Gentomycin 0.04 0.01 g/ml

* Vortex 10-15 minutes until completely dissolved.

148

Appendices

Table 2. Preparation of culture media from stock solutions Stock G1 G2 (mL) (mL) H2O 6.50 6.4 Salts 1 1 Bicarb 1 1 Calcium 0.1 0.1 Carbs (G1) 0.1 0 Carbs (G2) 0 0.1 EDTA 0.1 0 Aln-Gln 0.1 0.2 NEAAs 0.1 0.1 Taurine 0.1 0 EAAs* 0 0.1 Vitamins 0 0.1 Hyaluronan 0.4 0.4 Recombinant albumin# 0.5 0.5 Gentomycin 0.00375 0.00375

*MEM Cellgro (Corning Life Sciences)

#G-MM (Vitrolife)

149

References

150

References

Adamson, G.D., de Mouzon, J., Chambers, G.M., Zegers-Hochschild, F., Mansour, R., Ishihara, O., Banker, M. and Dyer, S., 2018. International Committee for Monitoring Assisted Reproductive Technology: world report on assisted reproductive technology, 2011. Fertil Steril. 110, 1067-1080

Aiken, C.E., Swoboda, P.P., Skepper, J.N. and Johnson, M.H., 2004. The direct measurement of embryogenic volume and nucleo-cytoplasmic ratio during mouse pre-implantation development. Reproduction. 128, 527-535

Ainsworth, A.J., Fredrickson, J.R. and Morbeck, D.E., 2017. Improved detection of mineral oil toxicity using an extended mouse embryo assay. J Assist Reprod Genet. 34, 391-397

Aitken, R.J., 1977. Changes in the protein content of mouse uterine flushings during normal pregnancy and delayed implantation, and after ovariectomy and oestradiol administration. J Reprod Fertil. 50, 29-36

Almagor, M., Bejar, C., Kafka, I. and Yaffe, H., 1996. Pregnancy rates after communal growth of preimplantation human embryos in vitro. Fertil Steril. 66, 394-397

Alminana, C., Heath, P.R., Wilkinson, S., Sanchez-Osorio, J., Cuello, C., Parrilla, I., Gil, M.A., Vazquez, J.L., Vazquez, J.M., Roca, J., Martinez, E.A. and Fazeli, A., 2012. Early developing pig embryos mediate their own environment in the maternal tract. PLoS One. 7, e33625

Anas, M.K., Hammer, M.A., Lever, M., Stanton, J.A. and Baltz, J.M., 2007. The organic osmolytes betaine and proline are transported by a shared system in early preimplantation mouse embryos. J Cell Physiol. 210, 266-277

Andrae, U., Singh, J. and Ziegler-Skylakakis, K., 1985. Pyruvate and related alpha-ketoacids protect mammalian cells in culture against hydrogen peroxide-induced cytotoxicity. Toxicol Lett. 28, 93-98

Aplin, J.D. and Ruane, P.T., 2017. Embryo-epithelium interactions during implantation at a glance. J Cell Sci. 130, 15-22

Austgulen, R., Arntzen, K.J., Vatten, L.J., Kahn, J. and Sunde, A., 1995. Detection of cytokines (interleukin-1, interleukin-6, transforming growth factor-beta) and soluble tumour necrosis factor receptors in embryo culture fluids during in-vitro fertilization. Hum Reprod. 10, 171-176

Awonuga, A.O., Yang, Y. and Rappolee, D.A., 2013. When stresses collide. Biol Reprod. 89, 74

Banrezes, B., Sainte-Beuve, T., Canon, E., Schultz, R.M., Cancela, J. and Ozil, J.P., 2011. Adult body weight is programmed by a redox-regulated and energy-dependent process during the pronuclear stage in mouse. PLoS One. 6, e29388

Bar-Or, D., Bar-Or, R., Rael, L.T., Gardner, D.K., Slone, D.S. and Craun, M.L., 2005. Heterogeneity and oxidation status of commercial human albumin preparations in clinical use. Crit Care Med. 33, 1638-1641

Baranao, R.I., Piazza, A., Rumi, L.S. and Polak de Fried, E., 1997. Determination of IL-1 and IL-6 levels in human embryo culture-conditioned media. Am J Reprod Immunol. 37, 191-194

Barbehenn, E.K., Wales, R.G. and Lowry, O.H., 1974. The explanation for the blockade of glycolysis in early mouse embryos. Proc Natl Acad Sci U S A. 71, 1056-1060

151

References

Barcroft, L.C., Offenberg, H., Thomsen, P. and Watson, A.J., 2003. Aquaporin proteins in murine trophectoderm mediate transepithelial water movements during cavitation. Dev Biol. 256, 342-354

Barker, D.J., Winter, P.D., Osmond, C., Margetts, B. and Simmonds, S.J., 1989. Weight in infancy and death from ischaemic heart disease. Lancet. 2, 577-580

Barnea, E.R., Simon, J., Levine, S.P., Coulam, C.B., Taliadouros, G.S. and Leavis, P.C., 1999. Progress in characterization of pre-implantation factor in embryo cultures and in vivo. Am J Reprod Immunol. 42, 95-99

Barnea, E.R., Lubman, D.M., Liu, Y.H., Absalon-Medina, V., Hayrabedyan, S., Todorova, K., Gilbert, R.O., Guingab, J. and Barder, T.J., 2014. Insight into PreImplantation Factor (PIF*) mechanism for embryo protection and development: target oxidative stress and protein misfolding (PDI and HSP) through essential RIKP [corrected] binding site. PLoS One. 9, e100263

Bean, C.J., Hassold, T.J., Judis, L. and Hunt, P.A., 2002. Fertilization in vitro increases non- disjunction during early cleavage divisions in a mouse model system. Hum Reprod. 17, 2362- 2367

Beardsley, A.J., Li, Y. and O'Neill, C., 2010. Characterization of a diverse secretome generated by the mouse preimplantation embryo in vitro. Reprod Biol Endocrinol. 8, 71

Bellver, J., De Los Santos, M.J., Alama, P., Castello, D., Privitera, L., Galliano, D., Labarta, E., Vidal, C., Pellicer, A. and Dominguez, F., 2015. Day-3 embryo metabolomics in the spent culture media is altered in obese women undergoing in vitro fertilization. Fertil Steril. 103, 1407-1415 e1401

Beltman, M.E., Forde, N., Furney, P., Carter, F., Roche, J.F., Lonergan, P. and Crowe, M.A., 2010. Characterisation of endometrial gene expression and metabolic parameters in beef heifers yielding viable or non-viable embryos on Day 7 after insemination. Reprod Fertil Dev. 22, 987-999

Benos, D.J., Biggers, J.D., Balaban, R.S., Mills, J.W. and Overstrom, E.W., 1985. Developmental aspects of sodium-dependent transport processes of preimplantation rabbit embryos. Soc Gen Physiol Ser. 39, 211-235

Bhusane, K., Bhutada, S., Chaudhari, U., Savardekar, L., Katkam, R. and Sachdeva, G., 2016. Secrets of Endometrial Receptivity: Some Are Hidden in Uterine Secretome. Am J Reprod Immunol. 75, 226-236

Biggers, J.D., Whittingham, D.G. and Donahue, R.P., 1967. The pattern of energy metabolism in the mouse oocyte and zygote. Proc Natl Acad Sci U S A. 58, 560-567

Binder, N.K., Evans, J., Gardner, D.K., Salamonsen, L.A. and Hannan, N.J., 2014. Endometrial signals improve embryo outcome: functional role of vascular endothelial growth factor isoforms on embryo development and implantation in mice. Hum Reprod. 29, 2278-2286

Bloor, D.J., Wilson, Y., Kibschull, M., Traub, O., Leese, H.J., Winterhager, E. and Kimber, S.J., 2004. Expression of connexins in human preimplantation embryos in vitro. Reprod Biol Endocrinol. 2, 25

152

References

Bolnick, A., Awonuga, A.O., Yang, Y., Abdulhasan, M., Xie, Y., Zhou, S., Puscheck, E.E. and Rappolee, D.A., 2017. Using stem cell oxygen physiology to optimize blastocyst culture while minimizing hypoxic stress. J Assist Reprod Genet. 34, 1251-1259

Bongso, A. and Fong, C.Y., 1993. The effect of coculture on human zygote development. Curr Opin Obstet Gynecol. 5, 585-593

Bontekoe, S., Mantikou, E., van Wely, M., Seshadri, S., Repping, S. and Mastenbroek, S., 2012. Low oxygen concentrations for embryo culture in assisted reproductive technologies. Cochrane Database Syst Rev. 7, CD008950

Boomsma, C.M., Kavelaars, A., Eijkemans, M.J., Amarouchi, K., Teklenburg, G., Gutknecht, D., Fauser, B.J., Heijnen, C.J. and Macklon, N.S., 2009. Cytokine profiling in endometrial secretions: a non-invasive window on endometrial receptivity. Reprod BioMed Online. 18, 85-94

Booth, P.J., Humpherson, P.G., Watson, T.J. and Leese, H.J., 2005. Amino acid depletion and appearance during porcine preimplantation embryo development in vitro. Reproduction. 130, 655-668

Booth, P.J., Watson, T.J. and Leese, H.J., 2007. Prediction of porcine blastocyst formation using morphological, kinetic, and amino acid depletion and appearance criteria determined during the early cleavage of in vitro-produced embryos. Biol Reprod. 77, 765-779

Borgatti, M., Rizzo, R., Canto, M.B., Fumagalli, D., Renzini, M.M., Fadini, R., Stignani, M., Baricordi, O.R. and Gambari, R., 2008. Release of sICAM-1 in oocytes and in vitro fertilized human embryos. PLoS One. 3, e3970

Borges, E., Jr., Braga, D.P., Setti, A.S., Montanni, D.A., Cabral, E.C., Eberlin, M.N., Turco, E.G. and Iaconelli, A., Jr., 2016. Non-invasive prediction of blastocyst implantation, ongoing pregnancy and live birth, by mass spectrometry lipid fingerprinting. JBRA Assist Reprod. 20, 227-231

Braga, D.P., Setti, A.S., Cabral, E.C., Eberlin, M., Loturco, E.G. and Borges, E., Jr., 2015. Non- Invasive Prediction of Blastocyst Formation by Day Three Embryo Culture Medium Mass Spectrometry Lipid Fingerprinting. JBRA Assist Reprod. 19, 119-124

Brinster, R.L., 1967. Carbon dioxide production from glucose by the preimplantation mouse embryo. Exp Cell Res. 47, 271-277

Brison, D.R. and Schultz, R.M., 1997. Apoptosis during mouse blastocyst formation: evidence for a role for survival factors including transforming growth factor alpha. Biol Reprod. 56, 1088-1096

Brison, D.R., Houghton, F.D., Falconer, D., Roberts, S.A., Hawkhead, J., Humpherson, P.G., Lieberman, B.A. and Leese, H.J., 2004. Identification of viable embryos in IVF by non-invasive measurement of amino acid turnover. Hum Reprod. 19, 2319-2324

Brosens, J.J., Salker, M.S., Teklenburg, G., Nautiyal, J., Salter, S., Lucas, E.S., Steel, J.H., Christian, M., Chan, Y.W., Boomsma, C.M., Moore, J.D., Hartshorne, G.M., Sucurovic, S., Mulac- Jericevic, B., Heijnen, C.J., Quenby, S., Koerkamp, M.J., Holstege, F.C., Shmygol, A. and Macklon, N.S., 2014. Uterine selection of human embryos at implantation. Sci Rep. 4, 3894

153

References

Burkus, J., Kacmarova, M., Kubandova, J., Kokosova, N., Fabianova, K., Fabian, D., Koppel, J. and Cikos, S., 2015. Stress exposure during the preimplantation period affects blastocyst lineages and offspring development. J Reprod Dev. 61, 325-331

Calarco, P.G. and Epstein, C.J., 1973. Cell surface changes during preimplantation development in the mouse. Dev Biol. 32, 208-213

Campbell, J.M., Nottle, M.B., Vassiliev, I., Mitchell, M. and Lane, M., 2012. Insulin increases epiblast cell number of in vitro cultured mouse embryos via the PI3K/GSK3/p53 pathway. Stem Cells Dev. 21, 2430-2441

Campbell, J.M., Lane, M., Vassiliev, I. and Nottle, M.B., 2013. Epiblast cell number and primary embryonic stem cell colony generation are increased by culture of cleavage stage embryos in insulin. J Reprod Dev. 59, 131-138

Capalbo, A., Romanelli, V., Patassini, C., Poli, M., Girardi, L., Giancani, A., Stoppa, M., Cimadomo, D., Ubaldi, F.M. and Rienzi, L., 2018. Diagnostic efficacy of blastocoel fluid and spent media as sources of DNA for preimplantation genetic testing in standard clinical conditions. Fertil Steril. 110, 870-879 e875

Carson, D.D., Dutt, A. and Tang, J.P., 1987. Glycoconjugate synthesis during early pregnancy: hyaluronate synthesis and function. Dev Biol. 120, 228-235

Casado-Vela, J., Rodriguez-Suarez, E., Iloro, I., Ametzazurra, A., Alkorta, N., Garcia-Velasco, J.A., Matorras, R., Prieto, B., Gonzalez, S., Nagore, D., Simon, L. and Elortza, F., 2009. Comprehensive proteomic analysis of human endometrial fluid aspirate. J Proteome Res. 8, 4622-4632

Casan, E.M., Raga, F., Bonilla-Musoles, F. and Polan, M.L., 2000. Human oviductal gonadotropin-releasing hormone: possible implications in fertilization, early embryonic development, and implantation. J Clin Endocrinol Metab. 85, 1377-1381

Casslen, B. and Ohlsson, K., 1981. Cyclic variation of proteinase inhibitors in human uterine fluid and influence of an IUD. Contraception. 23, 425-434

Catt, J.W. and Henman, M., 2000. Toxic effects of oxygen on human embryo development. Hum Reprod. 15 Suppl 2, 199-206

Ceelen, M., van Weissenbruch, M.M., Roos, J.C., Vermeiden, J.P., van Leeuwen, F.E. and Delemarre-van de Waal, H.A., 2007. Body composition in children and adolescents born after in vitro fertilization or spontaneous conception. J Clin Endocrinol Metab. 92, 3417-3423

Ceelen, M., van Weissenbruch, M.M., Vermeiden, J.P., van Leeuwen, F.E. and Delemarre-van de Waal, H.A., 2008. Cardiometabolic differences in children born after in vitro fertilization: follow-up study. J Clin Endocrinol Metab. 93, 1682-1688

Chambers, G.M., Paul, R.C., Harris, K., Fitzgerald, O., Boothroyd, C.V., Rombauts, L., Chapman, M.G. and Jorm, L., 2017. Assisted reproductive technology in Australia and New Zealand: cumulative live birth rates as measures of success. Med J Aust. 207, 114-118

Chandrakanthan, V., Li, A., Chami, O. and O'Neill, C., 2006. Effects of in vitro fertilization and embryo culture on TRP53 and Bax expression in B6 mouse embryos. Reprod Biol Endocrinol. 4, 61

154

References

Chang, H.S., Cheng, W.T., Wu, H.K. and Choo, K.B., 2000. Identification of genes expressed in the epithelium of porcine oviduct containing early embryos at various stages of development. Mol Reprod Dev. 56, 331-335

Chin, P.Y., Macpherson, A.M., Thompson, J.G., Lane, M. and Robertson, S.A., 2009. Stress response genes are suppressed in mouse preimplantation embryos by granulocyte- macrophage colony-stimulating factor (GM-CSF). Hum Reprod. 24, 2997-3009

Choi, B.I., Harvey, A.J. and Green, M.P., 2016. Bisphenol A affects early bovine embryo development and metabolism that is negated by an oestrogen receptor inhibitor. Sci Rep. 6, 29318

Choudhary, C., Weinert, B.T., Nishida, Y., Verdin, E. and Mann, M., 2014. The growing landscape of lysine acetylation links metabolism and cell signalling. Nat Rev Mol Cell Biol. 15, 536-550

Christianson, M.S., Zhao, Y., Shoham, G., Granot, I., Safran, A., Khafagy, A., Leong, M. and Shoham, Z., 2014. Embryo catheter loading and embryo culture techniques: results of a worldwide Web-based survey. J Assist Reprod Genet. 31, 1029-1036

Chronopoulou, E. and Harper, J.C., 2015. IVF culture media: past, present and future. Hum Reprod Update. 21, 39-55

Ciray, H.N., Aksoy, T., Goktas, C., Ozturk, B. and Bahceci, M., 2012. Time-lapse evaluation of human embryo development in single versus sequential culture media--a sibling oocyte study. J Assist Reprod Genet. 29, 891-900

Coan, P.M., Angiolini, E., Sandovici, I., Burton, G.J., Constancia, M. and Fowden, A.L., 2008. Adaptations in placental nutrient transfer capacity to meet fetal growth demands depend on placental size in mice. J Physiol. 586, 4567-4576

Cole, R.J., Edwards, R.G. and Paul, J., 1965. Cytodifferentiation in Cell Colonies and Cell Strains Derived from Cleaving Ova and Blastocysts of the Rabbit. Exp Cell Res. 37, 501-504

Collier, M., O'Neill, C., Ammit, A.J. and Saunders, D.M., 1988. Biochemical and pharmacological characterization of human embryo-derived platelet activating factor. Hum Reprod. 3, 993-998

Comes, S., Gagliardi, M., Laprano, N., Fico, A., Cimmino, A., Palamidessi, A., De Cesare, D., De Falco, S., Angelini, C., Scita, G., Patriarca, E.J., Matarazzo, M.R. and Minchiotti, G., 2013. L- Proline induces a mesenchymal-like invasive program in embryonic stem cells by remodeling H3K9 and H3K36 methylation. Stem Cell Reports. 1, 307-321

Cortezzi, S.S., Garcia, J.S., Ferreira, C.R., Braga, D.P., Figueira, R.C., Iaconelli, A., Jr., Souza, G.H., Borges, E., Jr. and Eberlin, M.N., 2011. Secretome of the preimplantation human embryo by bottom-up label-free proteomics. Anal Bioanal Chem. 401, 1331-1339

Costa-Borges, N., Belles, M., Meseguer, M., Galliano, D., Ballesteros, A. and Calderon, G., 2016. Blastocyst development in single medium with or without renewal on day 3: a prospective cohort study on sibling donor oocytes in a time-lapse incubator. Fertil Steril. 105, 707-713

Criscuoli, L., Rizzo, R., Fuzzi, B., Melchiorri, L., Menicucci, A., Cozzi, C., Dabizzi, S., Branconi, F., Evangelisti, P., Baricordi, O.R. and Noci, I., 2005. Lack of Histocompatibility Leukocyte Antigen-

155

References

G expression in early embryos is not related to germinal defects or impairment of interleukin- 10 production by embryos. Gynecol Endocrinol. 20, 264-269

Cuffe, J.S., Dickinson, H., Simmons, D.G. and Moritz, K.M., 2011. Sex specific changes in placental growth and MAPK following short term maternal dexamethasone exposure in the mouse. Placenta. 32, 981-989

Cuman, C., Menkhorst, E.M., Rombauts, L.J., Holden, S., Webster, D., Bilandzic, M., Osianlis, T. and Dimitriadis, E., 2013. Preimplantation human blastocysts release factors that differentially alter human endometrial epithelial cell adhesion and gene expression relative to IVF success. Hum Reprod. 28, 1161-1171

Cuman, C., Van Sinderen, M., Gantier, M.P., Rainczuk, K., Sorby, K., Rombauts, L., Osianlis, T. and Dimitriadis, E., 2015. Human Blastocyst Secreted microRNA Regulate Endometrial Epithelial Cell Adhesion. EBioMedicine. 2, 1528-1535

Cuthbertson, K.S. and Cobbold, P.H., 1985. Phorbol ester and sperm activate mouse oocytes by inducing sustained oscillations in cell Ca2+. Nature. 316, 541-542

D'Souza, F., Pudakalakatti, S.M., Uppangala, S., Honguntikar, S., Salian, S.R., Kalthur, G., Pasricha, R., Appajigowda, D., Atreya, H.S. and Adiga, S.K., 2016. Unraveling the association between genetic integrity and metabolic activity in pre-implantation stage embryos. Sci Rep. 6, 37291

Dai, S.J., Xu, C.L., Wang, J., Sun, Y.P. and Chian, R.C., 2012. Effect of culture medium volume and embryo density on early mouse embryonic development: tracking the development of the individual embryo. J Assist Reprod Genet. 29, 617-623

Das, S.K., Wang, X.N., Paria, B.C., Damm, D., Abraham, J.A., Klagsbrun, M., Andrews, G.K. and Dey, S.K., 1994. Heparin-Binding Egf-Like Growth-Factor Gene Is Induced in the Mouse Uterus Temporally by the Blastocyst Solely at the Site of Its Apposition - a Possible Ligand for Interaction with Blastocyst Egf-Receptor in Implantation. Development. 120, 1071-1083

Davies, M.J., Moore, V.M., Willson, K.J., Van Essen, P., Priest, K., Scott, H., Haan, E.A. and Chan, A., 2012. Reproductive technologies and the risk of birth defects. N Engl J Med. 366, 1803-1813

Dawson, K.M. and Baltz, J.M., 1997. Organic osmolytes and embryos: substrates of the Gly and beta transport systems protect mouse zygotes against the effects of raised osmolarity. Biol Reprod. 56, 1550-1558

Daya, S. and Clark, D.A., 1986. Immunosuppressive factor (or factors) produced by human embryos in vitro. N Engl J Med. 315, 1551-1552

De Munck, N., Santos-Ribeiro, S., Mateizel, I. and Verheyen, G., 2015. Reduced blastocyst formation in reduced culture volume. J Assist Reprod Genet. 32, 1365-1370 de Waal, E., Mak, W., Calhoun, S., Stein, P., Ord, T., Krapp, C., Coutifaris, C., Schultz, R.M. and Bartolomei, M.S., 2014. In vitro culture increases the frequency of stochastic epigenetic errors at imprinted genes in placental tissues from mouse concepti produced through assisted reproductive technologies. Biol Reprod. 90, 22 de Waal, E., Vrooman, L.A., Fischer, E., Ord, T., Mainigi, M.A., Coutifaris, C., Schultz, R.M. and Bartolomei, M.S., 2015. The cumulative effect of assisted reproduction procedures on

156

References placental development and epigenetic perturbations in a mouse model. Hum Mol Genet. 24, 6975-6985

Delle Piane, L., Lin, W., Liu, X., Donjacour, A., Minasi, P., Revelli, A., Maltepe, E. and Rinaudo, P.F., 2010. Effect of the method of conception and embryo transfer procedure on mid- gestation placenta and fetal development in an IVF mouse model. Hum Reprod. 25, 2039-2046

Desai, N., Scarrow, M., Lawson, J., Kinzer, D. and Goldfarb, J., 1999. Evaluation of the effect of interleukin-6 and human extracellullar matrix on embryonic development. Hum Reprod. 14, 1588-1592

Devreker, F., Hardy, K., Van den Bergh, M., Vannin, A.S., Emiliani, S. and Englert, Y., 2001. Amino acids promote human blastocyst development in vitro. Hum Reprod. 16, 749-756

Doherty, A.S., Mann, M.R., Tremblay, K.D., Bartolomei, M.S. and Schultz, R.M., 2000. Differential effects of culture on imprinted H19 expression in the preimplantation mouse embryo. Biol Reprod. 62, 1526-1535

Dolinko, A.V., Farland, L.V., Kaser, D.J., Missmer, S.A. and Racowsky, C., 2017. National survey on use of time-lapse imaging systems in IVF laboratories. J Assist Reprod Genet. 34, 1167-1172

Dominguez, F., Gadea, B., Esteban, F.J., Horcajadas, J.A., Pellicer, A. and Simon, C., 2008. Comparative protein-profile analysis of implanted versus non-implanted human blastocysts. Hum Reprod. 23, 1993-2000

Dominguez, F., Gadea, B., Mercader, A., Esteban, F.J., Pellicer, A. and Simon, C., 2010. Embryologic outcome and secretome profile of implanted blastocysts obtained after coculture in human endometrial epithelial cells versus the sequential system. Fertil Steril. 93, 774-782 e771

Dominguez, F., Meseguer, M., Aparicio-Ruiz, B., Piqueras, P., Quinonero, A. and Simon, C., 2015. New strategy for diagnosing embryo implantation potential by combining proteomics and time-lapse technologies. Fertil Steril. 104, 908-914

Donjacour, A., Liu, X., Lin, W., Simbulan, R. and Rinaudo, P.F., 2014. In vitro fertilization affects growth and glucose metabolism in a sex-specific manner in an outbred mouse model. Biol Reprod. 90, 80

Donohoe, D.R. and Bultman, S.J., 2012. Metaboloepigenetics: interrelationships between energy metabolism and epigenetic control of gene expression. J Cell Physiol. 227, 3169-3177

Downing, S.J., Maguiness, S.D., Tay, J.I., Watson, A. and Leese, H.J., 2002. Effect of platelet- activating factor on the electrophysiology of the human Fallopian tube: early mediation of embryo-maternal dialogue? Reproduction. 124, 523-529

Dumoulin, J.C., van Wissen, L.C., Menheere, P.P., Michiels, A.H., Geraedts, J.P. and Evers, J.L., 1997. Taurine acts as an osmolyte in human and mouse oocytes and embryos. Biol Reprod. 56, 739-744

Dumoulin, J.C., Meijers, C.J., Bras, M., Coonen, E., Geraedts, J.P. and Evers, J.L., 1999. Effect of oxygen concentration on human in-vitro fertilization and embryo culture. Hum Reprod. 14, 465-469

157

References

Dunglison, G.F., Barlow, D.H. and Sargent, I.L., 1996. Leukaemia inhibitory factor significantly enhances the blastocyst formation rates of human embryos cultured in serum-free medium. Hum Reprod. 11, 191-196

Dyer, S., Chambers, G.M., de Mouzon, J., Nygren, K.G., Zegers-Hochschild, F., Mansour, R., Ishihara, O., Banker, M. and Adamson, G.D., 2016. International Committee for Monitoring Assisted Reproductive Technologies world report: Assisted Reproductive Technology 2008, 2009 and 2010. Hum Reprod. 31, 1588-1609

Dyrlund, T.F., Kirkegaard, K., Poulsen, E.T., Sanggaard, K.W., Hindkjaer, J.J., Kjems, J., Enghild, J.J. and Ingerslev, H.J., 2014. Unconditioned commercial embryo culture media contain a large variety of non-declared proteins: a comprehensive proteomics analysis. Hum Reprod. 29, 2421-2430

Ebner, T., Shebl, O., Moser, M., Mayer, R.B., Arzt, W. and Tews, G., 2010. Group culture of human zygotes is superior to individual culture in terms of blastulation, implantation and life birth. Reprod BioMed Online. 21, 762-768

Edwards, L.J., Williams, D.A. and Gardner, D.K., 1998. Intracellular pH of the mouse preimplantation embryo: amino acids act as buffers of intracellular pH. Hum Reprod. 13, 3441- 3448

El Mouatassim, S., Guerin, P. and Menezo, Y., 2000. Mammalian oviduct and protection against free oxygen radicals: expression of genes encoding antioxidant enzymes in human and mouse. Eur J Obstet Gynecol Reprod Biol. 89, 1-6

Elhassan, Y.M., Wu, G., Leanez, A.C., Tasca, R.J., Watson, A.J. and Westhusin, M.E., 2001. Amino acid concentrations in fluids from the bovine oviduct and uterus and in KSOM-based culture media. Theriogenology. 55, 1907-1918

Fawzy, M., Sabry, M., Nour, M., Abdelrahman, M.Y., Roshdy, E., Magdi, Y. and Abdelghafar, H., 2016. Integrating insulin into single-step culture medium regulates human embryo development in vitro. Fertil Steril.

Feichtinger, M., Vaccari, E., Carli, L., Wallner, E., Madel, U., Figl, K., Palini, S. and Feichtinger, W., 2017. Non-invasive preimplantation genetic screening using array comparative genomic hybridization on spent culture media: a proof-of-concept pilot study. Reprod BioMed Online. 34, 583-589

Feltrin, C., Forell, F., dos Santos, L. and Rodrigues, J.L., 2006. In vitro bovine embryo development asfter nuclear transfer by handmade cloning using a modified WOW culture system. Reprod Fertil Dev. 18, 126-126

Feltrin, C., 2015. Effectiveness of Microwell-Based In Vitro Culture Systems for Zona-Free Cloned Bovine Embryos. Acta Scientiae Veterinariae. 43, 1

Fernandez-Gonzalez, R., Moreira, P., Bilbao, A., Jimenez, A., Perez-Crespo, M., Ramirez, M.A., Rodriguez De Fonseca, F., Pintado, B. and Gutierrez-Adan, A., 2004. Long-term effect of in vitro culture of mouse embryos with serum on mRNA expression of imprinting genes, development, and behavior. Proc Natl Acad Sci U S A. 101, 5880-5885

158

References

Fernandez-Gonzalez, R., Ramirez, M.A., Pericuesta, E., Calle, A. and Gutierrez-Adan, A., 2010. Histone modifications at the blastocyst Axin1(Fu) locus mark the heritability of in vitro culture- induced epigenetic alterations in mice. Biol Reprod. 83, 720-727

Ferry, L., Mermillod, P., Massip, A. and Dessy, F., 1994. Bovine embryos cultured in serum-poor oviduct-conditioned medium need cooperation to reach the blastocyst stage. Theriogenology. 42, 445-453

Feuer, S. and Rinaudo, P., 2012. Preimplantation stress and development. Birth Defects Res C Embryo Today. 96, 299-314

Feuer, S., Liu, X., Donjacour, A., Simbulan, R., Maltepe, E. and Rinaudo, P., 2016. Common and specific transcriptional signatures in mouse embryos and adult tissues induced by in vitro procedures. Reproduction. 153, 107-122

Feuer, S. and Rinaudo, P., 2016. From Embryos to Adults: A DOHaD Perspective on In Vitro Fertilization and Other Assisted Reproductive Technologies. Healthcare (Basel). 4, E51

Feuer, S.K., Donjacour, A., Simbulan, R.K., Lin, W., Liu, X., Maltepe, E. and Rinaudo, P.F., 2014. Sexually dimorphic effect of in vitro fertilization (IVF) on adult mouse fat and liver metabolomes. Endocrinology. 155, 4554-4567

Finger, B.J., Harvey, A.J., Green, M.P. and Gardner, D.K., 2015. Combined parental obesity negatively impacts preimplantation mouse embryo development, kinetics, morphology and metabolism. Hum Reprod. 30, 2084-2096

Fischer, B. and Bavister, B.D., 1993. Oxygen tension in the oviduct and uterus of rhesus monkeys, hamsters and rabbits. J Reprod Fertil. 99, 673-679

Fleming, T.P., McConnell, J., Johnson, M.H. and Stevenson, B.R., 1989. Development of tight junctions de novo in the mouse early embryo: control of assembly of the tight junction-specific protein, ZO-1. J Cell Biol. 108, 1407-1418

Fleming, T.P., Velazquez, M.A. and Eckert, J.J., 2015. Embryos, DOHaD and David Barker. J Dev Orig Health Dis. 6, 377-383

Foresta, C., Ubaldi, F.M., Rienzi, L., Franchin, C., Pivato, M., Romano, S., Guidolin, D., De Caro, R., Ferlin, A. and De Toni, L., 2016. Early protein profile of human embryonic secretome. Front Biosci (Landmark Ed). 21, 620-634

Franasiak, J.M. and Scott, R.T., Jr., 2015. Reproductive tract microbiome in assisted reproductive technologies. Fertil Steril. 104, 1364-1371

Fry, R.C., 1992. The effect of leukaemia inhibitory factor (LIF) on embryogenesis. Reprod Fertil Dev. 4, 449-458

Fujita, T., Umeki, H., Shimura, H., Kugumiya, K. and Shiga, K., 2006. Effect of group culture and embryo-culture conditioned medium on development of bovine embryos. J Reprod Dev. 52, 137-142

Furnus, C.C., de Matos, D.G. and Martinez, A.G., 1998. Effect of hyaluronic acid on development of in vitro produced bovine embryos. Theriogenology. 49, 1489-1499

159

References

Ganeshan, L., Jin, X.L. and O'Neill, C., 2017. The induction of tumour suppressor protein P53 limits the entry of cells into the pluripotent inner cell mass lineage in the mouse embryo. Exp Cell Res. 358, 227-233

Garcia-Martinez, S., Sanchez Hurtado, M.A., Gutierrez, H., Sanchez Margallo, F.M., Romar, R., Latorre, R., Coy, P. and Lopez Albors, O., 2018. Mimicking physiological O2 tension in the female reproductive tract improves assisted reproduction outcomes in pig. Mol Hum Reprod. 24, 260-270

Gardner, D.K. and Leese, H.J., 1986. Non-invasive measurement of nutrient uptake by single cultured pre-implantation mouse embryos. Hum Reprod. 1, 25-27

Gardner, D.K. and Leese, H.J., 1987. Assessment of embryo viability prior to transfer by the noninvasive measurement of glucose uptake. J Exp Zool. 242, 103-105

Gardner, D.K. and Leese, H.J., 1988. The role of glucose and pyruvate transport in regulating nutrient utilization by preimplantation mouse embryos. Development. 104, 423-429

Gardner, D.K. and Leese, H.J., 1990. Concentrations of nutrients in mouse oviduct fluid and their effects on embryo development and metabolism in vitro. J Reprod Fertil. 88, 361-368

Gardner, D.K. and Lane, M., 1993. Amino acids and ammonium regulate mouse embryo development in culture. Biol Reprod. 48, 377-385

Gardner, D.K., Lane, M., Spitzer, A. and Batt, P.A., 1994. Enhanced rates of cleavage and development for sheep zygotes cultured to the blastocyst stage in vitro in the absence of serum and somatic cells: amino acids, vitamins, and culturing embryos in groups stimulate development. Biol Reprod. 50, 390-400

Gardner, D.K., Lane, M., Calderon, I. and Leeton, J., 1996. Environment of the preimplantation human embryo in vivo: metabolite analysis of oviduct and uterine fluids and metabolism of cumulus cells. Fertil Steril. 65, 349-353

Gardner, D.K. and Lane, M., 1998. Culture of viable human blastocysts in defined sequential serum-free media. Hum Reprod. 13 Suppl 3, 148-159; discussion 160

Gardner, D.K., Rodriegez-Martinez, H. and Lane, M., 1999. Fetal development after transfer is increased by replacing protein with the glycosaminoglycan hyaluronan for mouse embryo culture and transfer. Human Reproduction. 14, 2575-2580

Gardner, D.K., Surrey, E., Minjarez, D., Leitz, A., Stevens, J. and Schoolcraft, W.B., 2004b. Single blastocyst transfer: a prospective randomized trial. Fertil Steril. 81, 551-555

Gardner, D.K. and Lane, M., 2005. Ex vivo early embryo development and effects on gene expression and imprinting. Reprod Fertil Dev. 17, 361-370

Gardner, D.K., Reed, L., Linck, D., Sheehan, C. and Lane, M., 2005. Quality control in human in vitro fertilization. Semin Reprod Med. 23, 319-324

Gardner, D.K. and Lane, M., 2007. Embryo culture systems, in: Gardner, D.K., In Vitro Fertilization: A Practical Approach. Informa Healthcare, New York, 221-222

160

References

Gardner, D.K., 2008. Dissection of culture media for embryos: the most important and less important components and characteristics. Reproduction Fertility and Development. 20, 9-18

Gardner, D.K., Wale, P.L., Collins, R. and Lane, M., 2011. Glucose consumption of single post- compaction human embryos is predictive of embryo sex and live birth outcome. Hum Reprod. 26, 1981-1986

Gardner, D.K. and Lane, M., 2014. Mammalian preimplantation embryo culture. Methods Mol Biol. 1092, 167-182

Gardner, D.K., 2015. Lactate production by the mammalian blastocyst: manipulating the microenvironment for uterine implantation and invasion? Bioessays. 37, 364-371

Gardner, D.K. and Harvey, A.J., 2015. Blastocyst metabolism. Reprod Fertil Dev. 27, 638-654

Gardner, D.K., 2016. The impact of physiological oxygen during culture, and vitrification for cryopreservation, on the outcome of extended culture in human IVF. Reprod BioMed Online. 32, 137-141

Gardner, D.K. and Kelley, R.L., 2017. Impact of the IVF laboratory environment on human preimplantation embryo phenotype. J Dev Orig Health Dis. 8, 418-435

Gaspar, R.C., Arnold, D.R., Correa, C.A., da Rocha, C.V., Jr., Penteado, J.C., Del Collado, M., Vantini, R., Garcia, J.M. and Lopes, F.L., 2015. Oxygen tension affects histone remodeling of in vitro-produced embryos in a bovine model. Theriogenology. 83, 1408-1415

Ghosh, J., Coutifaris, C., Sapienza, C. and Mainigi, M., 2017. Global DNA methylation levels are altered by modifiable clinical manipulations in assisted reproductive technologies. Clin Epigenetics. 9, 14

Giacomini, E., Vago, R., Sanchez, A.M., Podini, P., Zarovni, N., Murdica, V., Rizzo, R., Bortolotti, D., Candiani, M. and Vigano, P., 2017. Secretome of in vitro cultured human embryos contains extracellular vesicles that are uptaken by the maternal side. Sci Rep. 7, 5210

Gomes Sobrinho, D.B., Oliveira, J.B., Petersen, C.G., Mauri, A.L., Silva, L.F., Massaro, F.C., Baruffi, R.L., Cavagna, M. and Franco, J.G., Jr., 2011. IVF/ICSI outcomes after culture of human embryos at low oxygen tension: a meta-analysis. Reprod Biol Endocrinol. 9, 143

Gomez, E., Caamano, J.N., Corrales, F.J., Diez, C., Correia-Alvarez, E., Martin, D., Trigal, B., Carrocera, S., Mora, M.I., Pello-Palma, J., Moreno, J.F. and Munoz, M., 2013. Embryonic sex induces differential expression of proteins in bovine uterine fluid. J Proteome Res. 12, 1199- 1210

Gomez, E., Munoz, M., Simo, C., Ibanez, C., Carrocera, S., Martin-Gonzalez, D. and Cifuentes, A., 2016. Non-invasive metabolomics for improved determination of embryonic sex markers in chemically defined culture medium. J Chromatogr A. 1474, 138-144

Gonzales, D.S., Bavister, B.D. and Mese, S.A., 2001. In utero and in vitro proteinase activity during the Mesocricetus auratus embryo zona escape time window. Biol Reprod. 64, 222-230

Goodale, L.F., Hayrabedran, S., Todorova, K., Roussev, R., Ramu, S., Stamatkin, C., Coulam, C.B., Barnea, E.R. and Gilbert, R.O., 2017. PreImplantation factor (PIF) protects cultured embryos

161

References against oxidative stress: relevance for recurrent pregnancy loss (RPL) therapy. Oncotarget. 8, 32419-32432

Gopichandran, N. and Leese, H.J., 2003. Metabolic characterization of the bovine blastocyst, inner cell mass, trophectoderm and blastocoel fluid. Reproduction. 126, 299-308

Gopichandran, N. and Leese, H.J., 2006. The effect of paracrine/autocrine interactions on the in vitro culture of bovine preimplantation embryos. Reproduction. 131, 269-277

Goto, Y., Noda, Y., Mori, T. and Nakano, M., 1993. Increased generation of reactive oxygen species in embryos cultured in vitro. Free Radic Biol Med. 15, 69-75

Gould, J.M., Smith, P.J., Airey, C.J., Mort, E.J., Airey, L.E., Warricker, F.D.M., Pearson-Farr, J.E., Weston, E.C., Gould, P.J.W., Semmence, O.G., Restall, K.L., Watts, J.A., McHugh, P.C., Smith, S.J., Dewing, J.M., Fleming, T.P. and Willaime-Morawek, S., 2018. Mouse maternal protein restriction during preimplantation alone permanently alters brain neuron proportion and adult short-term memory. Proc Natl Acad Sci U S A. 115, E7398-E7407

Green, M.P., Mouat, F., Miles, H.L., Hopkins, S.A., Derraik, J.G., Hofman, P.L., Peek, J.C. and Cutfield, W.S., 2013. Phenotypic differences in children conceived from fresh and thawed embryos in in vitro fertilization compared with naturally conceived children. Fertil Steril. 99, 1898-1904

Greening, D.W., Nguyen, H.P., Elgass, K., Simpson, R.J. and Salamonsen, L.A., 2016. Human Endometrial Exosomes Contain Hormone-Specific Cargo Modulating Trophoblast Adhesive Capacity: Insights into Endometrial-Embryo Interactions. Biol Reprod. 94, 38

Griffin, J., Emery, B.R., Huang, I., Peterson, C.M. and Carrell, D.T., 2006. Comparative analysis of follicle morphology and oocyte diameter in four mammalian species (mouse, hamster, pig, and human). J Exp Clin Assist Reprod. 3, 2

Gross, N., Kropp, J. and Khatib, H., 2017. Sexual Dimorphism of miRNAs Secreted by Bovine In vitro-produced Embryos. Front Genet. 8, 39

Guerin, P., El Mouatassim, S. and Menezo, Y., 2001. Oxidative stress and protection against reactive oxygen species in the pre-implantation embryo and its surroundings. Hum Reprod Update. 7, 175-189

Guo, H., Zhu, P., Yan, L., Li, R., Hu, B., Lian, Y., Yan, J., Ren, X., Lin, S., Li, J., Jin, X., Shi, X., Liu, P., Wang, X., Wang, W., Wei, Y., Li, X., Guo, F., Wu, X., Fan, X., Yong, J., Wen, L., Xie, S.X., Tang, F. and Qiao, J., 2014. The DNA methylation landscape of human early embryos. Nature. 511, 606- 610

Ha, H.C., Sirisoma, N.S., Kuppusamy, P., Zweier, J.L., Woster, P.M. and Casero, R.A., Jr., 1998. The natural polyamine spermine functions directly as a free radical scavenger. Proc Natl Acad Sci U S A. 95, 11140-11145

Haimovici, F., Anderson, J.L., Bates, G.W., Racowsky, C., Ginsburg, E.S., Simovici, D. and Fichorova, R.N., 2018. Stress, anxiety, and depression of both partners in infertile couples are associated with cytokine levels and adverse IVF outcome. Am J Reprod Immunol. 79, e12832

Hamatani, T., Carter, M.G., Sharov, A.A. and Ko, M.S., 2004. Dynamics of global gene expression changes during mouse preimplantation development. Dev Cell. 6, 117-131

162

References

Hammond, E.R., McGillivray, B.C., Wicker, S.M., Peek, J.C., Shelling, A.N., Stone, P., Chamley, L.W. and Cree, L.M., 2017. Characterizing nuclear and mitochondrial DNA in spent embryo culture media: genetic contamination identified. Fertil Steril. 107, 220-228 e225

Hannan, N.J., Paiva, P., Meehan, K.L., Rombauts, L.J., Gardner, D.K. and Salamonsen, L.A., 2011. Analysis of fertility-related soluble mediators in human uterine fluid identifies VEGF as a key regulator of embryo implantation. Endocrinology. 152, 4948-4956

Hardarson, T., Bungum, M., Conaghan, J., Meintjes, M., Chantilis, S.J., Molnar, L., Gunnarsson, K. and Wikland, M., 2015. Noninferiority, randomized, controlled trial comparing embryo development using media developed for sequential or undisturbed culture in a time-lapse setup. Fertil Steril. 104, 1452-1459 e1451-1454

Hardy, K., Handyside, A.H. and Winston, R.M., 1989a. The human blastocyst: cell number, death and allocation during late preimplantation development in vitro. Development. 107, 597-604

Hardy, K., Hooper, M.A., Handyside, A.H., Rutherford, A.J., Winston, R.M. and Leese, H.J., 1989b. Non-invasive measurement of glucose and pyruvate uptake by individual human oocytes and preimplantation embryos. Hum Reprod. 4, 188-191

Harris, S.E., Gopichandran, N., Picton, H.M., Leese, H.J. and Orsi, N.M., 2005. Nutrient concentrations in murine follicular fluid and the female reproductive tract. Theriogenology. 64, 992-1006

Harvey, A.J., Kind, K.L. and Thompson, J.G., 2002. REDOX regulation of early embryo development. Reproduction. 123, 479-486

Harvey, A.J., Kind, K.L., Pantaleon, M., Armstrong, D.T. and Thompson, J.G., 2004. Oxygen- regulated gene expression in bovine blastocysts. Biol Reprod. 71, 1108-1119

Harvey, A.J., Rathjen, J. and Gardner, D.K., 2016. Metaboloepigenetic Regulation of Pluripotent Stem Cells. Stem Cells Int. 2016, 1816525

Harvey, M.B. and Kaye, P.L., 1990. Insulin increases the cell number of the inner cell mass and stimulates morphological development of mouse blastocysts in vitro. Development. 110, 963- 967

Harvey, M.B. and Kaye, P.L., 1991. Mouse blastocysts respond metabolically to short-term stimulation by insulin and IGF-1 through the insulin receptor. Mol Reprod Dev. 29, 253-258

Harvey, M.B. and Kaye, P.L., 1992. IGF-2 stimulates growth and metabolism of early mouse embryos. Mech Dev. 38, 169-173

Hashimoto, S., Kato, N., Saeki, K. and Morimoto, Y., 2012. Selection of high-potential embryos by culture in poly(dimethylsiloxane) microwells and time-lapse imaging. Fertil Steril. 97, 332- 337

Hemmings, R., Langlais, J., Falcone, T., Granger, L., Miron, P. and Guyda, H., 1992. Human embryos produce transforming growth factors alpha activity and insulin-like growth factors II. Fertil Steril. 58, 101-104

163

References

Hermoso, M., Barrera, N., Morales, B., Perez, S. and Villalon, M., 2001. Platelet activating factor increases ciliary activity in the hamster oviduct through epithelial production of prostaglandin E2. Pflugers Arch. 442, 336-345

Hewitson, L.C. and Leese, H.J., 1993. Energy metabolism of the trophectoderm and inner cell mass of the mouse blastocyst. J Exp Zool. 267, 337-343

Heyner, S., Rao, L.V., Jarett, L. and Smith, R.M., 1989. Preimplantation mouse embryos internalize maternal insulin via receptor-mediated endocytosis: pattern of uptake and functional correlations. Dev Biol. 134, 48-58

Hillman, N. and Flynn, T.J., 1980. The metabolism of exogenous fatty acids by preimplantation mouse embryos developing in vitro. J Embryol Exp Morphol. 56, 157-168

Hoelker, M., Rings, F., Lund, Q., Phatsara, C., Schellander, K. and Tesfaye, D., 2010. Effect of embryo density on in vitro developmental characteristics of bovine preimplantative embryos with respect to micro and macroenvironments. Reprod Domest Anim. 45, e138-145

Holmquist-Mengelbier, L., Fredlund, E., Lofstedt, T., Noguera, R., Navarro, S., Nilsson, H., Pietras, A., Vallon-Christersson, J., Borg, A., Gradin, K., Poellinger, L. and Pahlman, S., 2006. Recruitment of HIF-1alpha and HIF-2alpha to common target genes is differentially regulated in neuroblastoma: HIF-2alpha promotes an aggressive phenotype. Cancer Cell. 10, 413-423

Hong, K.H., Lee, H., Forman, E.J., Upham, K.M. and Scott, R.T., Jr., 2014. Examining the temperature of embryo culture in in vitro fertilization: a randomized controlled trial comparing traditional core temperature (37 degrees C) to a more physiologic, cooler temperature (36 degrees C). Fertil Steril. 102, 767-773

Houghton, F.D., Thompson, J.G., Kennedy, C.J. and Leese, H.J., 1996. Oxygen consumption and energy metabolism of the early mouse embryo. Mol Reprod Dev. 44, 476-485

Houghton, F.D., Hawkhead, J.A., Humpherson, P.G., Hogg, J.E., Balen, A.H., Rutherford, A.J. and Leese, H.J., 2002. Non-invasive amino acid turnover predicts human embryo developmental capacity. Hum Reprod. 17, 999-1005

Houghton, F.D., Humpherson, P.G., Hawkhead, J.A., Hall, C.J. and Leese, H.J., 2003. Na+, K+, ATPase activity in the human and bovine preimplantation embryo. Dev Biol. 263, 360-366

Houghton, F.D., 2006. Energy metabolism of the inner cell mass and trophectoderm of the mouse blastocyst. Differentiation. 74, 11-18

Hughes, P.M., Morbeck, D.E., Hudson, S.B., Fredrickson, J.R., Walker, D.L. and Coddington, C.C., 2010. Peroxides in mineral oil used for in vitro fertilization: defining limits of standard quality control assays. J Assist Reprod Genet. 27, 87-92

Ieda, S., Akai, T., Sakaguchi, Y., Shimamura, S., Sugawara, A., Kaneda, M., Matoba, S., Kagota, M., Sugimura, S. and Kaijima, H., 2018. A microwell culture system that allows group culture and is compatible with human single media. J Assist Reprod Genet. 35, 1869-1880

Ikeda, S., Sugimoto, M. and Kume, S., 2012. Importance of methionine metabolism in morula- to-blastocyst transition in bovine preimplantation embryos. J Reprod Dev. 58, 91-97

164

References

Ingman, W.V. and Jones, R.L., 2008. Cytokine knockouts in reproduction: the use of gene ablation to dissect roles of cytokines in reproductive biology. Hum Reprod Update. 14, 179-192

Inzunza, J., Danielsson, O., Lalitkumar, P.G., Larsson, O., Axelson, M., Tohonen, V., Danielsson, K.G. and Stavreus-Evers, A., 2010. Selective insulin-like growth factor-I antagonist inhibits mouse embryo development in a dose-dependent manner. Fertil Steril. 93, 2621-2626

Isobe, T., 2014. In vitro development of OPU-derived bovine embryos cultured either individually or in groups with the silk protein sericin and the viability of frozen-thawed embryos after transfer. Anim Sci J.

Jang, G., Lee, B.C., Kang, S.K. and Hwang, W.S., 2003. Effect of glycosaminoglycans on the preimplantation development of embryos derived from in vitro fertilization and somatic cell nuclear transfer. Reprod Fertil Dev. 15, 179-185

Jin, X.L., Chandrakanthan, V., Morgan, H.D. and O'Neill, C., 2009. Preimplantation embryo development in the mouse requires the latency of TRP53 expression, which is induced by a ligand-activated PI3 kinase/AKT/MDM2-mediated signaling pathway. Biol Reprod. 81, 234-242

Kallen, B., Finnstrom, O., Lindam, A., Nilsson, E., Nygren, K.G. and Olausson, P.O., 2010. Cancer risk in children and young adults conceived by in vitro fertilization. Pediatrics. 126, 270-276

Kang, S.S., Ofuji, S., Imai, K., Huang, W., Koyama, K., Yanagawa, Y., Takahashi, Y. and Nagano, M., 2015. The efficacy of the well of the well (WOW) culture system on development of bovine embryos in a small group and the effect of number of adjacent embryos on their development. Zygote. 23, 412-415

Karagenc, L., Sertkaya, Z., Ciray, N., Ulug, U. and Bahceci, M., 2004. Impact of oxygen concentration on embryonic development of mouse zygotes. Reprod BioMed Online. 9, 409- 417

Karagenc, L., Lane, M. and Gardner, D.K., 2005. Granulocyte-macrophage colony-stimulating factor stimulates mouse blastocyst inner cell mass development only when media lack human serum albumin. Reprod BioMed Online. 10, 511-518

Kato, Y. and Tsunoda, Y., 1994. Effects of the culture density of mouse zygotes on the development in vitro and in vivo. Theriogenology. 41, 1315-1322

Katz-Jaffe, M.G., Linck, D.W., Schoolcraft, W.B. and Gardner, D.K., 2005. A proteomic analysis of mammalian preimplantation embryonic development. Reproduction. 130, 899-905

Katz-Jaffe, M.G., Schoolcraft, W.B. and Gardner, D.K., 2006. Analysis of protein expression (secretome) by human and mouse preimplantation embryos. Fertil Steril. 86, 678-685

Katz-Jaffe, M.G., McCallie, B.R., Janesch, A., Filipovits, J.A., Schoolcraft, W.B. and Gardner, D.K., 2010. Blastocysts from patients with polycystic ovaries exhibit altered transcriptome and secretome. Reprod BioMed Online. 21, 520-526

Kaye, P.L., Bell, K.L., Beebe, L.F., Dunglison, G.F., Gardner, H.G. and Harvey, M.B., 1992. Insulin and the insulin-like growth factors (IGFs) in preimplantation development. Reprod Fertil Dev. 4, 373-386

165

References

Keefer, C.L., Stice, S.L., Paprocki, A.M. and Golueke, P., 1994. In-Vitro Culture of Bovine IVM- IVF Embryos - Cooperative Interaction among Embryos and the Role of Growth-Factors. Theriogenology. 41, 1323-1331

Kelley, R.L. and Gardner, D.K., 2016. Combined effects of individual culture and atmospheric oxygen on preimplantation mouse embryos in vitro. Reprod BioMed Online. 33, 537-549

Kelley, R.L. and Gardner, D.K., 2017. In vitro culture of individual mouse preimplantation embryos: the role of embryo density, microwells, oxygen, timing and conditioned media. Reprod BioMed Online. 34, 441-454

Khosla, S., Dean, W., Brown, D., Reik, W. and Feil, R., 2001. Culture of preimplantation mouse embryos affects fetal development and the expression of imprinted genes. Biol Reprod. 64, 918-926

Khurana, N.K. and Wales, R.G., 1989. Effects of oxygen concentration on the metabolism of [U- 14C]glucose by mouse morulae and early blastocysts in vitro. Reprod Fertil Dev. 1, 99-106

Kind, K.L., Collett, R.A., Harvey, A.J. and Thompson, J.G., 2005. Oxygen-regulated expression of GLUT-1, GLUT-3, and VEGF in the mouse blastocyst. Mol Reprod Dev. 70, 37-44

Kirkegaard, K., Hindkjaer, J.J. and Ingerslev, H.J., 2013. Effect of oxygen concentration on human embryo development evaluated by time-lapse monitoring. Fertil Steril. 99, 738-744 e734

Kitagawa, Y., Suzuki, K., Yoneda, A. and Watanabe, T., 2004. Effects of oxygen concentration and antioxidants on the in vitro developmental ability, production of reactive oxygen species (ROS), and DNA fragmentation in porcine embryos. Theriogenology. 62, 1186-1197

Kolle, S., Dubielzig, S., Reese, S., Wehrend, A., Konig, P. and Kummer, W., 2009. Ciliary transport, gamete interaction, and effects of the early embryo in the oviduct: ex vivo analyses using a new digital videomicroscopic system in the cow. Biol Reprod. 81, 267-274

Konecna, B., Laukova, L. and Vlkova, B., 2018. Immune activation by nucleic acids: A role in pregnancy complications. Scand J Immunol. 87, e12651

Kovacic, B., Sajko, M.C. and Vlaisavljevic, V., 2010. A prospective, randomized trial on the effect of atmospheric versus reduced oxygen concentration on the outcome of intracytoplasmic sperm injection cycles. Fertil Steril. 94, 511-519

Kropp, J., Salih, S.M. and Khatib, H., 2014. Expression of microRNAs in bovine and human preimplantation embryo culture media. Front Genet. 5, 91

Kropp, J. and Khatib, H., 2015a. mRNA fragments in in vitro culture media are associated with bovine preimplantation embryonic development. Front Genet. 6, 273

Kropp, J. and Khatib, H., 2015b. Characterization of microRNA in bovine in vitro culture media associated with embryo quality and development. J Dairy Sci. 98, 6552-6563

Kubisch, H.M. and Johnson, K.M., 2007. The effects of blastomere biopsy and oxygen tension on bovine embryo development, rate of apoptosis and interferon-tau secretion. Reprod Domest Anim. 42, 509-515

166

References

Kudo, M., Ikeda, S., Sugimoto, M. and Kume, S., 2015. Methionine-dependent histone methylation at developmentally important gene loci in mouse preimplantation embryos. J Nutr Biochem. 26, 1664-1669

Kurzawa, R., Glabowski, W. and Wenda-Rozewicka, L., 2001. Evaluation of mouse preimplantation embryos cultured in media enriched with insulin-like growth factors I and II, epidermal growth factor and tumor necrosis factor alpha. Folia Histochem Cytobiol. 39, 245- 251

Kuznyetsov, V., Madjunkova, S., Antes, R., Abramov, R., Motamedi, G., Ibarrientos, Z. and Librach, C., 2018. Evaluation of a novel non-invasive preimplantation genetic screening approach. PLoS One. 13, e0197262

Kwong, W.Y., Wild, A.E., Roberts, P., Willis, A.C. and Fleming, T.P., 2000. Maternal undernutrition during the preimplantation period of rat development causes blastocyst abnormalities and programming of postnatal hypertension. Development. 127, 4195-4202

Kwong, W.Y., Adamiak, S.J., Gwynn, A., Singh, R. and Sinclair, K.D., 2010. Endogenous folates and single-carbon metabolism in the ovarian follicle, oocyte and pre-implantation embryo. Reproduction. 139, 705-715

Labs, E.G.G.o.G.P.i.I., De los Santos, M.J., Apter, S., Coticchio, G., Debrock, S., Lundin, K., Plancha, C.E., Prados, F., Rienzi, L., Verheyen, G., Woodward, B. and Vermeulen, N., 2016. Revised guidelines for good practice in IVF laboratories (2015). Hum Reprod. 31, 685-686

Lachance, C., Bailey, J.L. and Leclerc, P., 2007. Expression of Hsp60 and Grp78 in the human endometrium and oviduct, and their effect on sperm functions. Hum Reprod. 22, 2606-2614

Lamar Jones, M., Bancroft, J.D. and Gamble, M., 2008. Connective Tissues and Stains, in: Bancroft, J.D. and Gamble, M., Theory and Practice of Histological Techniques. Churchill Livingstone, 135-160

Lamb, V.K. and Leese, H.J., 1994. Uptake of a mixture of amino acids by mouse blastocysts. J Reprod Fertil. 102, 169-175

Lamy, J., Gatien, J., Dubuisson, F., Nadal-Desbarats, L., Salvetti, P., Mermillod, P. and Saint- Dizier, M., 2018. Metabolomic profiling of bovine oviductal fluid across the oestrous cycle using proton nuclear magnetic resonance spectroscopy. Reprod Fertil Dev. 10.1071/RD17389

Lande, Y., Seidman, D.S., Maman, E., Baum, M. and Hourvitz, A., 2015. Why do couples discontinue unlimited free IVF treatments? Gynecol Endocrinol. 31, 233-236

Lane, M. and Gardner, D.K., 1992. Effect of incubation volume and embryo density on the development and viability of mouse embryos in vitro. Hum Reprod. 7, 558-562

Lane, M. and Gardner, D.K., 1994. Increase in postimplantation development of cultured mouse embryos by amino acids and induction of fetal retardation and exencephaly by ammonium ions. J Reprod Fertil. 102, 305-312

Lane, M. and Gardner, D.K., 1996. Selection of viable mouse blastocysts prior to transfer using a metabolic criterion. Hum Reprod. 11, 1975-1978

167

References

Lane, M. and Gardner, D.K., 1997. Differential regulation of mouse embryo development and viability by amino acids. J Reprod Fertil. 109, 153-164

Lane, M. and Gardner, D.K., 1998. Amino acids and vitamins prevent culture-induced metabolic perturbations and associated loss of viability of mouse blastocysts. Hum Reprod. 13, 991-997

Lane, M. and Gardner, D.K., 2000. Lactate regulates pyruvate uptake and metabolism in the preimplantation mouse embryo. Biol Reprod. 62, 16-22

Lane, M. and Gardner, D.K., 2005a. Mitochondrial malate-aspartate shuttle regulates mouse embryo nutrient consumption. J Biol Chem. 280, 18361-18367

Lane, M. and Gardner, D.K., 2005b. Understanding cellular disruptions during early embryo development that perturb viability and fetal development. Reproduction Fertility and Development. 17, 371-378

Lane, M. and Gardner, D.K., 2007. Embryo culture medium: which is the best? Best Pract Res Clin Obstet Gynaecol. 21, 83-100

Larson, M.A. and Kubisch, H.M., 1999. The effects of group size on development and interferon-tau secretion by in-vitro fertilized and cultured bovine blastocysts. Hum Reprod. 14, 2075-2079

Larson, R.C., Ignotz, G.G. and Currie, W.B., 1992. Platelet derived growth factor (PDGF) stimulates development of bovine embryos during the fourth cell cycle. Development. 115, 821-826

Lawitts, J.A. and Biggers, J.D., 1992. Joint effects of sodium chloride, glutamine, and glucose in mouse preimplantation embryo culture media. Mol Reprod Dev. 31, 189-194

Lee, K.F., Yao, Y.Q., Kwok, K.L., Xu, J.S. and Yeung, W.S., 2002. Early developing embryos affect the gene expression patterns in the mouse oviduct. Biochem Biophys Res Commun. 292, 564- 570

Lee, Y.S., Thouas, G.A. and Gardner, D.K., 2015. Developmental kinetics of cleavage stage mouse embryos are related to their subsequent carbohydrate and amino acid utilization at the blastocyst stage. Hum Reprod. 30, 543-552

Leese, H.J. and Barton, A.M., 1984. Pyruvate and glucose uptake by mouse ova and preimplantation embryos. J Reprod Fertil. 72, 9-13

Leese, H.J., Biggers, J.D., Mroz, E.A. and Lechene, C., 1984. Nucleotides in a single mammalian ovum or preimplantation embryo. Anal Biochem. 140, 443-448

Leese, H.J., 1998. Human embryo culture: back to nature. J Assist Reprod Genet. 15, 466-468

Lehner, A., Kaszas, Z., Murber, A., Rigo, J., Jr., Urbancsek, J. and Fancsovits, P., 2017. Embryo density may affect embryo quality during in vitro culture in a microwell group culture dish. Arch Gynecol Obstet. 296, 345-353

Leonavicius, K., Royer, C., Preece, C., Davies, B., Biggins, J.S. and Srinivas, S., 2018. Mechanics of mouse blastocyst hatching revealed by a hydrogel-based microdeformation assay. Proc Natl Acad Sci U S A. 115, 10375-10380

168

References

Li, L., Lu, X. and Dean, J., 2013. The maternal to zygotic transition in mammals. Mol Aspects Med. 34, 919-938

Li, S.J., Wang, T.S., Qin, F.N., Huang, Z., Liang, X.H., Gao, F., Song, Z. and Yang, Z.M., 2015. Differential regulation of receptivity in two uterine horns of a recipient mouse following asynchronous embryo transfer. Sci Rep. 5, 15897

Li, W., Goossens, K., Van Poucke, M., Forier, K., Braeckmans, K., Van Soom, A. and Peelman, L.J., 2016. High oxygen tension increases global methylation in bovine 4-cell embryos and blastocysts but does not affect general retrotransposon expression. Reprod Fertil Dev. 28, 948- 959

Lighten, A.D., Moore, G.E., Winston, R.M.L. and Hardy, K., 1998. Routine addition of human insulin-like growth factor-I ligand could benefit clinical in-vitro fertilization culture. Human Reproduction. 13, 3144-3150

Lima, A., Burgstaller, J., Sanchez-Nieto, J.M. and Rodriguez, T.A., 2018. The Mitochondria and the Regulation of Cell Fitness During Early Mammalian Development. Curr Top Dev Biol. 128, 339-363

Lindenbaum, A., 1973. A survey of naturally occurring chelating ligands. Adv Exp Med Biol. 40, 67-77

Lindgren, K.E., Gulen Yaldir, F., Hreinsson, J., Holte, J., Karehed, K., Sundstrom-Poromaa, I., Kaihola, H. and Akerud, H., 2018. Differences in secretome in culture media when comparing blastocysts and arrested embryos using multiplex proximity assay. Ups J Med Sci. 123, 143-152

Litzky, J.F., Deyssenroth, M.A., Everson, T.M., Armstrong, D.A., Lambertini, L., Chen, J. and Marsit, C.J., 2017. Placental imprinting variation associated with assisted reproductive technologies and subfertility. Epigenetics. 12, 653-661

Liu, W., Liu, J., Du, H., Ling, J., Sun, X. and Chen, D., 2017. Non-invasive pre-implantation aneuploidy screening and diagnosis of beta thalassemia IVSII654 mutation using spent embryo culture medium. Ann Med. 49, 319-328

Lopera-Vasquez, R., Hamdi, M., Fernandez-Fuertes, B., Maillo, V., Beltran-Brena, P., Calle, A., Redruello, A., Lopez-Martin, S., Gutierrez-Adan, A., Yanez-Mo, M., Ramirez, M.A. and Rizos, D., 2016. Extracellular Vesicles from BOEC in In Vitro Embryo Development and Quality. PLoS One. 11, e0148083

Lv, C., Yu, W.X., Wang, Y., Yi, D.J., Zeng, M.H. and Xiao, H.M., 2018. MiR-21 in extracellular vesicles contributes to the growth of fertilized eggs and embryo development in mice. Biosci Rep. 38,

Ma, Y.Y., Chen, H.W. and Tzeng, C.R., 2017. Low oxygen tension increases mitochondrial membrane potential and enhances expression of antioxidant genes and implantation protein of mouse blastocyst cultured in vitro. J Ovarian Res. 10, 47

Macklon, N.S., Pieters, M.H., Hassan, M.A., Jeucken, P.H., Eijkemans, M.J. and Fauser, B.C., 2002. A prospective randomized comparison of sequential versus monoculture systems for in- vitro human blastocyst development. Hum Reprod. 17, 2700-2705

169

References

Mahsoudi, B., Li, A. and O'Neill, C., 2007. Assessment of the long-term and transgenerational consequences of perturbing preimplantation embryo development in mice. Biol Reprod. 77, 889-896

Maillo, V., Gaora, P.O., Forde, N., Besenfelder, U., Havlicek, V., Burns, G.W., Spencer, T.E., Gutierrez-Adan, A., Lonergan, P. and Rizos, D., 2015. Oviduct-Embryo Interactions in Cattle: Two-Way Traffic or a One-Way Street? Biol Reprod. 92, 144

Maillo, V., Sanchez-Calabuig, M.J., Lopera-Vasquez, R., Hamdi, M., Gutierrez-Adan, A., Lonergan, P. and Rizos, D., 2016. Oviductal response to gametes and early embryos in mammals. Reproduction. 152, R127-141

Mains, L.M., Christenson, L., Yang, B., Sparks, A.E., Mathur, S. and Van Voorhis, B.J., 2011. Identification of apolipoprotein A1 in the human embryonic secretome. Fertil Steril. 96, 422- 427 e422

Malizia, B.A., Hacker, M.R. and Penzias, A.S., 2009. Cumulative live-birth rates after in vitro fertilization. N Engl J Med. 360, 236-243

Mann, M.R., Lee, S.S., Doherty, A.S., Verona, R.I., Nolen, L.D., Schultz, R.M. and Bartolomei, M.S., 2004. Selective loss of imprinting in the placenta following preimplantation development in culture. Development. 131, 3727-3735

Mantikou, E., Bontekoe, S., van Wely, M., Seshadri, S., Repping, S. and Mastenbroek, S., 2013. Low oxygen concentrations for embryo culture in assisted reproductive technologies. Hum Reprod Update. 19, 209

Marei, W.F., Salavati, M. and Fouladi-Nashta, A.A., 2013. Critical role of hyaluronidase-2 during preimplantation embryo development. Mol Hum Reprod. 19, 590-599

Martin, K.L., Barlow, D.H. and Sargent, I.L., 1998. Heparin-binding epidermal growth factor significantly improves human blastocyst development and hatching in serum-free medium. Hum Reprod. 13, 1645-1652

Mascarenhas, M.N., Flaxman, S.R., Boerma, T., Vanderpoel, S. and Stevens, G.A., 2012. National, regional, and global trends in infertility prevalence since 1990: a systematic analysis of 277 health surveys. PLoS Med. 9, e1001356

Masson, P., 1929. Some histological methods: trichrome stainings and their preliminary technique. J Tech Methods. 12, 75-90

Mastroianni, L., Jr. and Jones, R., 1965. Oxygen Tension within the Rabbit Fallopian Tube. J Reprod Fertil. 9, 99-102

Mather, E.C., 1975. "In vivo" uterine lumen pH values of the bovine. Theriogenology. 3, 113- 119

Matsuura, K., 2014. Numerical calculations for diffusion effects in the well-of-the-well culture system for mammalian embryos. Reprod Fertil Dev. 26, 742-751

McPherson, N.O., Bell, V.G., Zander-Fox, D.L., Fullston, T., Wu, L.L., Robker, R.L. and Lane, M., 2015. When two obese parents are worse than one! Impacts on embryo and fetal development. Am J Physiol Endocrinol Metab. 309, E568-581

170

References

McPherson, N.O., Zander-Fox, D., Vincent, A.D. and Lane, M., 2018. Combined advanced parental age has an additive negative effect on live birth rates-data from 4057 first IVF/ICSI cycles. J Assist Reprod Genet. 35, 279-287

McReynolds, S., Vanderlinden, L., Stevens, J., Hansen, K., Schoolcraft, W.B. and Katz-Jaffe, M.G., 2011. Lipocalin-1: a potential marker for noninvasive aneuploidy screening. Fertil Steril. 95, 2631-2633

Meintjes, M., Chantilis, S.J., Douglas, J.D., Rodriguez, A.J., Guerami, A.R., Bookout, D.M., Barnett, B.D. and Madden, J.D., 2009a. A controlled randomized trial evaluating the effect of lowered incubator oxygen tension on live births in a predominantly blastocyst transfer program. Hum Reprod. 24, 300-307

Meintjes, M., Chantilis, S.J., Ward, D.C., Douglas, J.D., Rodriguez, A.J., Guerami, A.R., Bookout, D.M., Barnett, B.D. and Madden, J.D., 2009b. A randomized controlled study of human serum albumin and serum substitute supplement as protein supplements for IVF culture and the effect on live birth rates. Hum Reprod. 24, 782-789

Menezo, Y., Khatchadourian, C., Gharib, A., Hamidi, J., Greenland, T. and Sarda, N., 1989. Regulation of S-adenosyl methionine synthesis in the mouse embryo. Life Sci. 44, 1601-1609

Menezo, Y.J., Servy, E., Veiga, A., Hazout, A. and Elder, K., 2012. Culture systems: embryo coculture. Methods Mol Biol. 912, 231-247

Menezo, Y.J., Silvestris, E., Dale, B. and Elder, K., 2016. Oxidative stress and alterations in DNA methylation: two sides of the same coin in reproduction. Reprod BioMed Online. 33, 668-683

Meseguer, M., Herrero, J., Tejera, A., Hilligsoe, K.M., Ramsing, N.B. and Remohi, J., 2011. The use of morphokinetics as a predictor of embryo implantation. Hum Reprod. 26, 2658-2671

Meuter, A., Rogmann, L.M., Winterhoff, B.J., Tchkonia, T., Kirkland, J.L. and Morbeck, D.E., 2014. Markers of cellular senescence are elevated in murine blastocysts cultured in vitro: molecular consequences of culture in atmospheric oxygen. J Assist Reprod Genet. 31, 1259- 1267

Milazzotto, M.P., Goissis, M.D., Chitwood, J.L., Annes, K., Soares, C.A., Ispada, J., Assumpcao, M.E. and Ross, P.J., 2016. Early cleavages influence the molecular and the metabolic pattern of individually cultured bovine blastocysts. Mol Reprod Dev. 83, 324-336

Miller, J.G. and Schultz, G.A., 1987. Amino acid content of preimplantation rabbit embryos and fluids of the reproductive tract. Biol Reprod. 36, 125-129

Mills, R.M. and Brinster, R.L., 1967. Oxygen consumption of preimplantation mouse embryos. Exp Cell Res. 47, 337-344

Minasi, M.G., Fabozzi, G., Casciani, V., Lobascio, A.M., Colasante, A., Scarselli, F. and Greco, E., 2015. Improved blastocyst formation with reduced culture volume: comparison of three different culture conditions on 1128 sibling human zygotes. J Assist Reprod Genet. 32, 215-220

Mitchell, M., Cashman, K.S., Gardner, D.K., Thompson, J.G. and Lane, M., 2009. Disruption of mitochondrial malate-aspartate shuttle activity in mouse blastocysts impairs viability and fetal growth. Biol Reprod. 80, 295-301

171

References

Moessner, J. and Dodson, W.C., 1995. The Quality of Human Embryo Growth Is Improved When Embryos Are Cultured in Groups Rather Than Separately. Fertil Steril. 64, 1034-1035

Mohamed, M.M., Cavallo-Medved, D., Rudy, D., Anbalagan, A., Moin, K. and Sloane, B.F., 2010. Interleukin-6 increases expression and secretion of cathepsin B by breast tumor-associated monocytes. Cell Physiol Biochem. 25, 315-324

Morbeck, D.E., Krisher, R.L., Herrick, J.R., Baumann, N.A., Matern, D. and Moyer, T., 2014a. Composition of commercial media used for human embryo culture. Fertil Steril. 102, 759-766 e759

Morbeck, D.E., Paczkowski, M., Fredrickson, J.R., Krisher, R.L., Hoff, H.S., Baumann, N.A., Moyer, T. and Matern, D., 2014b. Composition of protein supplements used for human embryo culture. J Assist Reprod Genet. 31, 1703-1711

Moreno, I., Codoner, F.M., Vilella, F., Valbuena, D., Martinez-Blanch, J.F., Jimenez-Almazan, J., Alonso, R., Alama, P., Remohi, J., Pellicer, A., Ramon, D. and Simon, C., 2016. Evidence that the endometrial microbiota has an effect on implantation success or failure. Am J Obstet Gynecol. 215, 684-703

Moussaieff, A., Rouleau, M., Kitsberg, D., Cohen, M., Levy, G., Barasch, D., Nemirovski, A., Shen-Orr, S., Laevsky, I., Amit, M., Bomze, D., Elena-Herrmann, B., Scherf, T., Nissim-Rafinia, M., Kempa, S., Itskovitz-Eldor, J., Meshorer, E., Aberdam, D. and Nahmias, Y., 2015. Glycolysis- mediated changes in acetyl-CoA and histone acetylation control the early differentiation of embryonic stem cells. Cell Metab. 21, 392-402

Munoz, M., Corrales, F.J., Caamano, J.N., Diez, C., Trigal, B., Mora, M.I., Martin, D., Carrocera, S. and Gomez, E., 2012. Proteome of the early embryo-maternal dialogue in the cattle uterus. J Proteome Res. 11, 751-766

Nagao, Y., Saeki, K. and Nagai, M., 1998. Effects of embryo density, oxygen concentration and medium composition on in vitro development of bovine early embryos. Theriogenology. 49, 210-210

Nastri, C.O., Nobrega, B.N., Teixeira, D.M., Amorim, J., Diniz, L.M.M., Barbosa, M.W.P., Giorgi, V.S.I., Pileggi, V.N. and Martins, W.P., 2016. Low versus atmospheric oxygen tension for embryo culture in assisted reproduction: a systematic review and meta-analysis. Fertil Steril. 106, 95-104 e117

Navarrete Santos, A., Tonack, S., Kirstein, M., Pantaleon, M., Kaye, P. and Fischer, B., 2004. Insulin acts via mitogen-activated protein kinase phosphorylation in rabbit blastocysts. Reproduction. 128, 517-526

Navarrete Santos, A., Ramin, N., Tonack, S. and Fischer, B., 2008. Cell lineage-specific signaling of insulin and insulin-like growth factor I in rabbit blastocysts. Endocrinology. 149, 515-524

Ng, K.Y.B., Mingels, R., Morgan, H., Macklon, N. and Cheong, Y., 2018. In vivo oxygen, temperature and pH dynamics in the female reproductive tract and their importance in human conception: a systematic review. Hum Reprod Update. 24, 15-34

Nichol, R., Hunter, R.H. and Cooke, G.M., 1997. Oviduct fluid pH in intact and unilaterally ovariectomized pigs. Can J Physiol Pharmacol. 75, 1069-1074

172

References

Nieder, J. and Augustin, W., 1986. Prostaglandin E and F profiles in human fallopian tubes during different phases of the menstrual cycle. Gynecol Obstet Invest. 21, 202-207

Niederberger, C., Pellicer, A., Cohen, J., Gardner, D.K., Palermo, G.D., O'Neill, C.L., Chow, S., Rosenwaks, Z., Cobo, A., Swain, J.E., Schoolcraft, W.B., Frydman, R., Bishop, L.A., Aharon, D., Gordon, C., New, E., Decherney, A., Tan, S.L., Paulson, R.J., Goldfarb, J.M., Brannstrom, M., Donnez, J., Silber, S., Dolmans, M.M., Simpson, J.L., Handyside, A.H., Munne, S., Eguizabal, C., Montserrat, N., Izpisua Belmonte, J.C., Trounson, A., Simon, C., Tulandi, T., Giudice, L.C., Norman, R.J., Hsueh, A.J., Sun, Y., Laufer, N., Kochman, R., Eldar-Geva, T., Lunenfeld, B., Ezcurra, D., D'Hooghe, T., Fauser, B., Tarlatzis, B.C., Meldrum, D.R., Casper, R.F., Fatemi, H.M., Devroey, P., Galliano, D., Wikland, M., Sigman, M., Schoor, R.A., Goldstein, M., Lipshultz, L.I., Schlegel, P.N., Hussein, A., Oates, R.D., Brannigan, R.E., Ross, H.E., Pennings, G., Klock, S.C., Brown, S., Van Steirteghem, A., Rebar, R.W. and LaBarbera, A.R., 2018. Forty years of IVF. Fertil Steril. 110, 185-324 e185

Nomm, M., Porosk, R., Parn, P., Kilk, K., Soomets, U., Koks, S. and Jaakma, U., 2018. Invitro culture and non-invasive metabolic profiling of single bovine embryos. Reprod Fertil Dev. 10.1071/RD17446

O'Doherty, E.M., Wade, M.G., Hill, J.L. and Boland, M.P., 1997. Effects of culturing bovine oocytes either singly or in groups on development to blastocysts. Theriogenology. 48, 161-169

O'Neill, C., 1985. Partial characterization of the embryo-derived platelet-activating factor in mice. J Reprod Fertil. 75, 375-380

O'Neill, C., 1998. Autocrine mediators are required to act on the embryo by the 2-cell stage to promote normal development and survival of mouse preimplantation embryos in vitro. Biol Reprod. 58, 1303-1309

O'Neill, C., 2005. The role of paf in embryo physiology. Hum Reprod Update. 11, 215-228

O'Neill, C., Li, Y. and Jin, X.L., 2015. Survival signalling in the preimplantation embryo. Adv Exp Med Biol. 843, 129-149

O'Sullivan, C.M., Rancourt, S.L., Liu, S.Y. and Rancourt, D.E., 2001. A novel murine tryptase involved in blastocyst hatching and outgrowth. Reproduction. 122, 61-71

Orsi, N.M. and Leese, H.J., 2004. Ammonium exposure and pyruvate affect the amino acid metabolism of bovine blastocysts in vitro. Reproduction. 127, 131-140

Orsi, N.M. and Reischl, J.B., 2007. Mammalian embryo co-culture: trials and tribulations of a misunderstood method. Theriogenology. 67, 441-458

Ortiz, M.E., Llados, C. and Croxatto, H.B., 1989. Embryos of different ages transferred to the rat oviduct enter the uterus at different times. Biol Reprod. 41, 381-384

Orvieto, R., Bar-Hava, I., Schwartz, A., Shelef, M. and Ben-Rafael, Z., 1997. Interleukin-2 production by human pre-implantation embryos. Gynecol Endocrinol. 11, 331-334

Ottosen, L.D., Hindkaer, J., Husth, M., Petersen, D.E., Kirk, J. and Ingerslev, H.J., 2006. Observations on intrauterine oxygen tension measured by fibre-optic microsensors. Reprod BioMed Online. 13, 380-385

173

References

Ozornek, M.H., Bielfeld, P., Krussel, J.S., Moustafa, M., Mikat-Drozdzynski, B., Koldovsky, U. and Kuhn, U., 1995. Interferon gamma and interleukin 10 levels in preimplantation embryo culture media. J Assist Reprod Genet. 12, 590-593

Ozornek, M.H., Bielfeld, P., Krussel, J.S., Cupisti, S., Jeyendran, R.S. and Koldovsky, U., 1997. Interferon-gamma production by the human preimplantation embryo. Am J Reprod Immunol. 37, 435-437

Paiva, P., Hannan, N.J., Hincks, C., Meehan, K.L., Pruysers, E., Dimitriadis, E. and Salamonsen, L.A., 2011. Human chorionic gonadotrophin regulates FGF2 and other cytokines produced by human endometrial epithelial cells, providing a mechanism for enhancing endometrial receptivity. Hum Reprod. 26, 1153-1162

Pampfer, S., Wuu, Y.D., Vanderheyden, I. and De Hertogh, R., 1994. Expression of tumor necrosis factor-alpha (TNF alpha) receptors and selective effect of TNF alpha on the inner cell mass in mouse blastocysts. Endocrinology. 134, 206-212

Paria, B.C. and Dey, S.K., 1990. Preimplantation embryo development in vitro: cooperative interactions among embryos and role of growth factors. Proc Natl Acad Sci U S A. 87, 4756- 4760

Paria, B.C., Huet-Hudson, Y.M. and Dey, S.K., 1993. Blastocyst's state of activity determines the "window" of implantation in the receptive mouse uterus. Proc Natl Acad Sci U S A. 90, 10159- 10162

Parks, J.C., McCallie, B.R., Patton, A.L., Al-Safi, Z.A., Polotsky, A.J., Griffin, D.K., Schoolcraft, W.B. and Katz-Jaffe, M.G., 2018. The impact of infertility diagnosis on embryo-endometrial dialogue. Reproduction. 155, 543-552

Partridge, R.J. and Leese, H.J., 1996. Consumption of amino acids by bovine preimplantation embryos. Reprod Fertil Dev. 8, 945-950

Pena, F., Davalos, R., Rechkemmer, A., Ascenzo, A. and Gonzales, M., 2018. Embryo development until blastocyst stage with and without renewal of single medium on day 3. JBRA Assist Reprod. 22, 49-51

Pendeville, H., Carpino, N., Marine, J.C., Takahashi, Y., Muller, M., Martial, J.A. and Cleveland, J.L., 2001. The ornithine decarboxylase gene is essential for cell survival during early murine development. Molecular and Cellular Biology. 21, 6549-6558

Pereira, D.C., Dode, M.A. and Rumpf, R., 2005. Evaluation of different culture systems on the in vitro production of bovine embryos. Theriogenology. 63, 1131-1141

Perona, R.M. and Wassarman, P.M., 1986. Mouse blastocysts hatch in vitro by using a trypsin- like proteinase associated with cells of mural trophectoderm. Dev Biol. 114, 42-52

Picton, H.M., Elder, K., Houghton, F.D., Hawkhead, J.A., Rutherford, A.J., Hogg, J.E., Leese, H.J. and Harris, S.E., 2010. Association between amino acid turnover and chromosome aneuploidy during human preimplantation embryo development in vitro. Mol Hum Reprod. 16, 557-569

Piliszek, A. and Madeja, Z.E., 2018. Pre-implantation Development of Domestic Animals. Curr Top Dev Biol. 128, 267-294

174

References

Pontesilli, M., Painter, R.C., Grooten, I.J., van der Post, J.A., Mol, B.W., Vrijkotte, T.G., Repping, S. and Roseboom, T.J., 2015. Subfertility and assisted reproduction techniques are associated with poorer cardiometabolic profiles in childhood. Reprod BioMed Online. 30, 258-267

Puscheck, E.E., Awonuga, A.O., Yang, Y., Jiang, Z. and Rappolee, D.A., 2015. Molecular biology of the stress response in the early embryo and its stem cells. Adv Exp Med Biol. 843, 77-128

Qin, J., Sheng, X., Wu, D., Gao, S., You, Y., Yang, T. and Wang, H., 2017. Adverse Obstetric Outcomes Associated With In Vitro Fertilization in Singleton Pregnancies. Reprod Sci. 24, 595- 608

Qu, P., Qing, S., Liu, R., Qin, H., Wang, W., Qiao, F., Ge, H., Liu, J., Zhang, Y., Cui, W. and Wang, Y., 2017. Effects of embryo-derived exosomes on the development of bovine cloned embryos. PLoS One. 12, e0174535

Quinn, P. and Harlow, G.M., 1978. The effect of oxygen on the development of preimplantation mouse embryos in vitro. J Exp Zool. 206, 73-80

Raatikainen, K., Kuivasaari-Pirinen, P., Hippelainen, M. and Heinonen, S., 2012. Comparison of the pregnancy outcomes of subfertile women after infertility treatment and in naturally conceived pregnancies. Hum Reprod. 27, 1162-1169

Raff, M.C., 1992. Social controls on cell survival and cell death. Nature. 356, 397-400

Rebollar-Lazaro, I. and Matson, P., 2010. The culture of human cleavage stage embryos alone or in groups: effect upon blastocyst utilization rates and implantation. Reproductive Biology. 10, 227-234

Redel, B.K., Brown, A.N., Spate, L.D., Whitworth, K.M., Green, J.A. and Prather, R.S., 2012. Glycolysis in preimplantation development is partially controlled by the Warburg Effect. Mol Reprod Dev. 79, 262-271

Reed, M.L., 2012. Culture systems: embryo density, in: Smith, G.D., Swain, J.E. and Pool, T.B., Embryo Culture: Methods and Protocols. Humana Press, New York, 273-312

Reeve, W.J. and Ziomek, C.A., 1981. Distribution of microvilli on dissociated blastomeres from mouse embryos: evidence for surface polarization at compaction. J Embryol Exp Morphol. 62, 339-350

Renard, J.P., Philippon, A. and Menezo, Y., 1980. In-vitro uptake of glucose by bovine blastocysts. J Reprod Fertil. 58, 161-164

Richardson, L.L. and Oliphant, G., 1981. Steroid concentrations in rabbit oviducal fluid during oestrus and pseudopregnancy. J Reprod Fertil. 62, 427-431

Rijnders, P.M. and Jansen, C.A., 1999. Influence of group culture and culture volume on the formation of human blastocysts: a prospective randomized study. Hum Reprod. 14, 2333-2337

Rinaudo, P.F., Giritharan, G., Talbi, S., Dobson, A.T. and Schultz, R.M., 2006. Effects of oxygen tension on gene expression in preimplantation mouse embryos. Fertil Steril. 86, 1252-1265, 1265 e1251-1236

175

References

Roberts, C.T., White, C.A., Wiemer, N.G., Ramsay, A. and Robertson, S.A., 2003. Altered placental development in interleukin-10 null mutant mice. Placenta. 24 Suppl A, S94-99

Robertson, S.A., Chin, P.Y., Schjenken, J.E. and Thompson, J.G., 2015. Female tract cytokines and developmental programming in embryos. Adv Exp Med Biol. 843, 173-213

Romundstad, L.B., Romundstad, P.R., Sunde, A., von During, V., Skjaerven, R. and Vatten, L.J., 2006. Increased risk of placenta previa in pregnancies following IVF/ICSI; a comparison of ART and non-ART pregnancies in the same mother. Hum Reprod. 21, 2353-2358

Rosenbluth, E.M., Shelton, D.N., Wells, L.M., Sparks, A.E. and Van Voorhis, B.J., 2014. Human embryos secrete microRNAs into culture media--a potential biomarker for implantation. Fertil Steril. 101, 1493-1500

Roussev, R.G., Coulam, C.B., Kaider, B.D., Yarkoni, M., Leavis, P.C. and Barnea, E.R., 1996. Embryonic origin of preimplantation factor (PIF): biological activity and partial characterization. Mol Hum Reprod. 2, 883-887

Ryan, J.P., O'Neill, C. and Wales, R.G., 1990. Oxidative metabolism of energy substrates by preimplantation mouse embryos in the presence of platelet-activating factor. J Reprod Fertil. 89, 301-307

Saadeldin, I.M., Kim, S.J., Choi, Y.B. and Lee, B.C., 2014. Improvement of cloned embryos development by co-culturing with parthenotes: a possible role of exosomes/microvesicles for embryos paracrine communication. Cell Reprogram. 16, 223-234

Sakatani, M., Yamanaka, K., Balboula, A.Z. and Takahashi, M., 2017. Different thermotolerances in in vitro-produced embryos derived from different maternal and paternal genetic backgrounds. Anim Sci J. 88, 1934-1942

Salahuddin, S., Ookutsu, S., Goto, K., Nakanishi, Y. and Nagata, Y., 1995. Effects of embryo density and co-culture of unfertilized oocytes on embryonic development of in-vitro fertilized mouse embryos. Hum Reprod. 10, 2382-2385

Salamonsen, L.A., Evans, J., Nguyen, H.P. and Edgell, T.A., 2016. The Microenvironment of Human Implantation: Determinant of Reproductive Success. Am J Reprod Immunol. 75, 218- 225

Salvaing, J., Peynot, N., Bedhane, M.N., Veniel, S., Pellier, E., Boulesteix, C., Beaujean, N., Daniel, N. and Duranthon, V., 2016. Assessment of 'one-step' versus 'sequential' embryo culture conditions through embryonic genome methylation and hydroxymethylation changes. Hum Reprod. 31, 2471-2483

Sanchez-Ribas, I., Riqueros, M., Vime, P., Puchades-Carrasco, L., Jonsson, T., Pineda-Lucena, A., Ballesteros, A., Dominguez, F. and Simon, C., 2012. Differential metabolic profiling of non-pure trisomy 21 human preimplantation embryos. Fertil Steril. 98, 1157-1164 e1151-1152

Sanford, T.R., De, M. and Wood, G.W., 1992. Expression of colony-stimulating factors and inflammatory cytokines in the uterus of CD1 mice during days 1 to 3 of pregnancy. J Reprod Fertil. 94, 213-220

176

References

Saunders, C.M., Larman, M.G., Parrington, J., Cox, L.J., Royse, J., Blayney, L.M., Swann, K. and Lai, F.A., 2002. PLC zeta: a sperm-specific trigger of Ca(2+) oscillations in eggs and embryo development. Development. 129, 3533-3544

Scherrer, U., Rimoldi, S.F., Rexhaj, E., Stuber, T., Duplain, H., Garcin, S., de Marchi, S.F., Nicod, P., Germond, M., Allemann, Y. and Sartori, C., 2012. Systemic and pulmonary vascular dysfunction in children conceived by assisted reproductive technologies. Circulation. 125, 1890-1896

Schneider, E.G. and Hayslip, C.C., 1996. Globulin-enriched protein supplements shorten the pre-compaction mitotic interval and promote hatching of murine embryos. Am J Reprod Immunol. 36, 101-106

Schwarzer, C., Esteves, T.C., Arauzo-Bravo, M.J., Le Gac, S., Nordhoff, V., Schlatt, S. and Boiani, M., 2012. ART culture conditions change the probability of mouse embryo gestation through defined cellular and molecular responses. Hum Reprod. 27, 2627-2640

Scotchie, J.G., Fritz, M.A., Mocanu, M., Lessey, B.A. and Young, S.L., 2009. Proteomic analysis of the luteal endometrial secretome. Reprod Sci. 16, 883-893

Scott, K.A., Yamazaki, Y., Yamamoto, M., Lin, Y., Melhorn, S.J., Krause, E.G., Woods, S.C., Yanagimachi, R., Sakai, R.R. and Tamashiro, K.L., 2010. Glucose parameters are altered in mouse offspring produced by assisted reproductive technologies and somatic cell nuclear transfer. Biol Reprod. 83, 220-227

Seggers, J., Haadsma, M.L., La Bastide-Van Gemert, S., Heineman, M.J., Middelburg, K.J., Roseboom, T.J., Schendelaar, P., Van den Heuvel, E.R. and Hadders-Algra, M., 2014. Is ovarian hyperstimulation associated with higher blood pressure in 4-year-old IVF offspring? Part I: multivariable regression analysis. Hum Reprod. 29, 502-509

Seli, E., Botros, L., Sakkas, D. and Burns, D.H., 2008. Noninvasive metabolomic profiling of embryo culture media using proton nuclear magnetic resonance correlates with reproductive potential of embryos in women undergoing in vitro fertilization. Fertil Steril. 90, 2183-2189

Semenza, G.L., Roth, P.H., Fang, H.M. and Wang, G.L., 1994. Transcriptional regulation of genes encoding glycolytic enzymes by hypoxia-inducible factor 1. J Biol Chem. 269, 23757-23763

Seshagiri, P.B., Vani, V. and Madhulika, P., 2016. Cytokines and Blastocyst Hatching. Am J Reprod Immunol. 75, 208-217

Sharkey, A.M., Dellow, K., Blayney, M., Macnamee, M., Charnock-Jones, S. and Smith, S.K., 1995. Stage-specific expression of cytokine and receptor messenger ribonucleic acids in human preimplantation embryos. Biol Reprod. 53, 974-981

Sharma, N., Liu, S., Tang, L., Irwin, J., Meng, G. and Rancourt, D.E., 2006. Implantation Serine Proteinases heterodimerize and are critical in hatching and implantation. BMC Dev Biol. 6, 61

Shen, X.H., Han, Y.J., Zhang, D.X., Cui, X.S. and Kim, N.H., 2009. A link between the interleukin- 6/Stat3 anti-apoptotic pathway and microRNA-21 in preimplantation mouse embryos. Mol Reprod Dev. 76, 854-862

177

References

Sherwin, J.R., Sharkey, A.M., Cameo, P., Mavrogianis, P.M., Catalano, R.D., Edassery, S. and Fazleabas, A.T., 2007. Identification of novel genes regulated by chorionic gonadotropin in baboon endometrium during the window of implantation. Endocrinology. 148, 618-626

Shyh-Chang, N., Locasale, J.W., Lyssiotis, C.A., Zheng, Y., Teo, R.Y., Ratanasirintrawoot, S., Zhang, J., Onder, T., Unternaehrer, J.J., Zhu, H., Asara, J.M., Daley, G.Q. and Cantley, L.C., 2013. Influence of threonine metabolism on S-adenosylmethionine and histone methylation. Science. 339, 222-226

Sjoblom, C., Wikland, M. and Robertson, S.A., 1999. Granulocyte-macrophage colony- stimulating factor promotes human blastocyst development in vitro. Hum Reprod. 14, 3069- 3076

Smart, Y.C., Cripps, A.W., Clancy, R.L., Roberts, T.K., Lopata, A. and Shutt, D.A., 1981. Detection of an immunosuppressive factor in human preimplantation embryo cultures. Med J Aust. 1, 78- 79

Smith, Z.D., Chan, M.M., Mikkelsen, T.S., Gu, H., Gnirke, A., Regev, A. and Meissner, A., 2012. A unique regulatory phase of DNA methylation in the early mammalian embryo. Nature. 484, 339-344

Snyder, R.D., 1989. Polyamine depletion is associated with altered chromatin structure in HeLa cells. Biochem J. 260, 697-704

Souza, F., Asampille, G., Uppangala, S., Kalthur, G., Atreya, H.S. and Adiga, S.K., 2018. Sperm- mediated DNA lesions alter metabolite levels in spent embryo culture medium. Reprod Fertil Dev. 10.1071/RD18136

Spindler, R. and Wildt, D., 2001. Heterospecific (mouse) companion culture enhances cat embryo development in vitro, but the impact is embryo density dependent. Theriogenology. 55, 342-342

Spindler, R.E. and Wildt, D.E., 2002. Quality and age of companion felid embryos modulate enhanced development by group culture. Biol Reprod. 66, 167-173

Spindler, R.E., Crichton, E.G., Agca, Y., Loskutoff, N., Critser, J., Gardner, D.K. and Wildt, D.E., 2006. Improved felid embryo development by group culture is maintained with heterospecific companions. Theriogenology. 66, 82-92

Spyropoulou, I., Karamalegos, C. and Bolton, V.N., 1999. A prospective randomized study comparing the outcome of in-vitro fertilization and embryo transfer following culture of human embryos individually or in groups before embryo transfer on day 2. Human Reproduction. 14, 76-79

Stamatkin, C.W., Roussev, R.G., Stout, M., Absalon-Medina, V., Ramu, S., Goodman, C., Coulam, C.B., Gilbert, R.O., Godke, R.A. and Barnea, E.R., 2011a. PreImplantation Factor (PIF) correlates with early mammalian embryo development-bovine and murine models. Reprod Biol Endocrinol. 9, 63

Stamatkin, C.W., Roussev, R.G., Stout, M., Coulam, C.B., Triche, E., Godke, R.A. and Barnea, E.R., 2011b. Preimplantation factor negates embryo toxicity and promotes embryo development in culture. Reprod BioMed Online. 23, 517-524

178

References

Steeves, C.L. and Baltz, J.M., 2005. Regulation of intracellular glycine as an organic osmolyte in early preimplantation mouse embryos. J Cell Physiol. 204, 273-279

Stern, S., Biggers, J.D. and Anderson, E., 1971. Mitochondria and early development of the mouse. J Exp Zool. 176, 179-191

Stigliani, S., Anserini, P., Venturini, P.L. and Scaruffi, P., 2013. Mitochondrial DNA content in embryo culture medium is significantly associated with human embryo fragmentation. Hum Reprod. 28, 2652-2660

Stoddart, N.R., Wild, A.E. and Fleming, T.P., 1996. Stimulation of development in vitro by platelet-activating factor receptor ligands released by mouse preimplantation embryos. J Reprod Fertil. 108, 47-53

Stokes, P.J., Abeydeera, L.R. and Leese, H.J., 2005. Development of porcine embryos in vivo and in vitro; evidence for embryo 'cross talk' in vitro. Dev Biol. 284, 62-71

Sturmey, R.G., Brison, D.R. and Leese, H.J., 2008. Symposium: innovative techniques in human embryo viability assessment. Assessing embryo viability by measurement of amino acid turnover. Reprod BioMed Online. 17, 486-496

Sturmey, R.G., Hawkhead, J.A., Barker, E.A. and Leese, H.J., 2009. DNA damage and metabolic activity in the preimplantation embryo. Hum Reprod. 24, 81-91

Sugimura, S., Akai, T., Hashiyada, Y., Aikawa, Y., Ohtake, M., Matsuda, H., Kobayashi, S., Kobayashi, E., Konishi, K. and Imai, K., 2013. Effect of embryo density on in vitro development and gene expression in bovine in vitro-fertilized embryos cultured in a microwell system. J Reprod Dev. 59, 115-122

Sun, H., Kang, J., Su, J., Zhang, J., Zhang, L., Liu, X., Zhang, J., Wang, F., Lu, Z., Xing, X., Chen, H. and Zhang, Y., 2018. Methionine adenosyltransferase 2A regulates mouse zygotic genome activation and morula to blastocyst transition. Biol Reprod. 10.1093/biolre/ioy194

Sunderam, S., Kissin, D.M., Crawford, S.B., Folger, S.G., Boulet, S.L., Warner, L. and Barfield, W.D., 2018. Assisted Reproductive Technology Surveillance - United States, 2015. MMWR Surveill Summ. 67, 1-28

Suzuki, C., Yoshioka, K., Sakatani, M. and Takahashi, M., 2007. Glutamine and hypotaurine improves intracellular oxidative status and in vitro development of porcine preimplantation embryos. Zygote. 15, 317-324

Svalander, P.C., Holmes, P.V., Olovsson, M., Wikland, M., Gemzell-Danielsson, K. and Bygdeman, M., 1991. Platelet-derived growth factor is detected in human blastocyst culture medium but not in human follicular fluid - a preliminary report. Fertility and Sterility. 56, 367- 369

Swain, J.E., 2012. Media composition: pH and buffers. Methods Mol Biol. 912, 161-175

Swain, J.E., Lai, D., Takayama, S. and Smith, G.D., 2013. Thinking big by thinking small: application of microfluidic technology to improve ART. Lab Chip. 13, 1213-1224

179

References

Taka, M., Iwayama, H. and Fukui, Y., 2005. Effect of the well of the well (WOW) system on in vitro culture for porcine embryos after intracytoplasmic sperm injection. J Reprod Dev. 51, 533-537

Takahashi, M., Keicho, K., Takahashi, H., Ogawa, H., Schultz, R.M. and Okano, A., 2000. Effect of oxidative stress on development and DNA damage in in-vitro cultured bovine embryos by comet assay. Theriogenology. 54, 137-145

Tan, B.S., Lonic, A., Morris, M.B., Rathjen, P.D. and Rathjen, J., 2011. The amino acid transporter SNAT2 mediates L-proline-induced differentiation of ES cells. Am J Physiol Cell Physiol. 300, C1270-1279

Tan, B.S., Rathjen, P.D., Harvey, A.J., Gardner, D.K. and Rathjen, J., 2016. Regulation of amino acid transporters in pluripotent cell populations in the embryo and in culture; novel roles for sodium-coupled neutral amino acid transporters. Mech Dev. 141, 32-39

Tanikawa, M., Harada, T., Ito, M., Yoshida, S., Iwabe, T. and Terakawa, N., 1999. Globulins in protein supplements promote the development of preimplantation embryos. J Assist Reprod Genet. 16, 555-557

Tao, T., Robichaud, A., Mercier, J. and Ouellette, R., 2013. Influence of group embryo culture strategies on the blastocyst development and pregnancy outcome. J Assist Reprod Genet. 30, 63-68

Tervit, H.R., Whittingham, D.G. and Rowson, L.E., 1972. Successful culture in vitro of sheep and cattle ova. J Reprod Fertil. 30, 493-497

Thouas, G.A., Dominguez, F., Green, M.P., Vilella, F., Simon, C. and Gardner, D.K., 2015. Soluble ligands and their receptors in human embryo development and implantation. Endocr Rev. 36, 92-130

Tiemann, U., Neels, P., Kuchenmeister, U., Walzel, H. and Spitschak, M., 1996. Effect of ATP and platelet-activating factor on intracellular calcium concentrations of cultured oviductal cells from cows. J Reprod Fertil. 108, 1-9

Tribulo, P., Balzano-Nogueira, L., Conesa, A., Siqueira, L.G. and Hansen, P.J., 2018. Changes in the uterine metabolome of the cow during the first 7 days after estrus. Mol Reprod Dev. 10.1002/mrd.23082

Trimarchi, J.R., Liu, L., Porterfield, D.M., Smith, P.J. and Keefe, D.L., 2000. Oxidative phosphorylation-dependent and -independent oxygen consumption by individual preimplantation mouse embryos. Biol Reprod. 62, 1866-1874

Truong, T.T., Soh, Y. and Gardner, D.K., 2015. Combined antioxidants signficantly increase mouse embryo and fetal development following culture in atmospheric oxygen. Fertil Steril. 104, e59-e60

Truong, T.T., Soh, Y.M. and Gardner, D.K., 2016. Antioxidants improve mouse preimplantation embryo development and viability. Hum Reprod. 31, 1445-1454

Tsukada, Y., Fang, J., Erdjument-Bromage, H., Warren, M.E., Borchers, C.H., Tempst, P. and Zhang, Y., 2006. Histone demethylation by a family of JmjC domain-containing proteins. Nature. 439, 811-816

180

References

Ueda, O., Yorozu, K., Kamada, N., Jishage, K., Kawase, Y., Toyoda, Y. and Suzuki, H., 2003. Possible expansion of "Window of Implantation" in pseudopregnant mice: time of implantation of embryos at different stages of development transferred into the same recipient. Biol Reprod. 69, 1085-1090

Urbanski, J.P., Johnson, M.T., Craig, D.D., Potter, D.L., Gardner, D.K. and Thorsen, T., 2008. Noninvasive metabolic profiling using microfluidics for analysis of single preimplantation embryos. Anal Chem. 80, 6500-6507

Vajta, G., Peura, T.T., Holm, P., Paldi, A., Greve, T., Trounson, A.O. and Callesen, H., 2000. New method for culture of zona-included or zona-free embryos: the Well of the Well (WOW) system. Mol Reprod Dev. 55, 256-264

Vajta, G., Korosi, T., Du, Y., Nakata, K., Ieda, S., Kuwayama, M. and Nagy, Z.P., 2008. The Well- of-the-Well system: an efficient approach to improve embryo development. Reprod BioMed Online. 17, 73-81

Van Soom, A., Yuan, Y.Q., Peelman, L.J., de Matos, D.G., Dewulf, J., Laevens, H. and de Kruif, A., 2002. Prevalence of apoptosis and inner cell allocation in bovine embryos cultured under different oxygen tensions with or without cysteine addition. Theriogenology. 57, 1453-1465

Van Winkle, L.J., Haghighat, N. and Campione, A.L., 1990. Glycine protects preimplantation mouse conceptuses from a detrimental effect on development of the inorganic ions in oviductal fluid. J Exp Zool. 253, 215-219

Van Winkle, L.J., Tesch, J.K., Shah, A. and Campione, A.L., 2006. System B0,+ amino acid transport regulates the penetration stage of blastocyst implantation with possible long-term developmental consequences through adulthood. Hum Reprod Update. 12, 145-157

Velasquez, L.A., Aguilera, J.G. and Croxatto, H.B., 1995. Possible role of platelet-activating factor in embryonic signaling during oviductal transport in the hamster. Biol Reprod. 52, 1302- 1306

Vera-Rodriguez, M., Diez-Juan, A., Jimenez-Almazan, J., Martinez, S., Navarro, R., Peinado, V., Mercader, A., Meseguer, M., Blesa, D., Moreno, I., Valbuena, D., Rubio, C. and Simon, C., 2018. Origin and composition of cell-free DNA in spent medium from human embryo culture during preimplantation development. Hum Reprod. 33, 745-756

Vilella, F., Moreno-Moya, J.M., Balaguer, N., Grasso, A., Herrero, M., Martinez, S., Marcilla, A. and Simon, C., 2015. Hsa-miR-30d, secreted by the human endometrium, is taken up by the pre-implantation embryo and might modify its transcriptome. Development. 142, 3210-3221

Vousden, K.H. and Prives, C., 2009. Blinded by the Light: The Growing Complexity of p53. Cell. 137, 413-431

Wahlsten, D. and Wainwright, P., 1977. Application of a Morphological Time Scale to Hereditary Differences in Prenatal Mouse Development. J Embryol Exp Morph. 42, 79-92

Wakai, T., Vanderheyden, V. and Fissore, R.A., 2011. Ca2+ signaling during mammalian fertilization: requirements, players, and adaptations. Cold Spring Harb Perspect Biol. 3,

181

References

Wakuda, K., Takakura, K., Nakanishi, K., Kita, N., Shi, H., Hirose, M. and Noda, Y., 1999. Embryo- dependent induction of embryo receptivity in the mouse endometrium. J Reprod Fertil. 115, 315-324

Waldenstrom, U., Engstrom, A.B., Hellberg, D. and Nilsson, S., 2009. Low-oxygen compared with high-oxygen atmosphere in blastocyst culture, a prospective randomized study. Fertil Steril. 91, 2461-2465

Wale, P.L. and Gardner, D.K., 2010. Time-lapse analysis of mouse embryo development in oxygen gradients. Reprod BioMed Online. 21, 402-410

Wale, P.L. and Gardner, D.K., 2012. Oxygen regulates amino acid turnover and carbohydrate uptake during the preimplantation period of mouse embryo development. Biol Reprod. 87, 24, 21-28

Wale, P.L. and Gardner, D.K., 2013. Oxygen affects the ability of mouse blastocysts to regulate ammonium. Biol Reprod. 89, 75

Wale, P.L. and Gardner, D.K., 2016. The effects of chemical and physical factors on mammalian embryo culture and their importance for the practice of assisted human reproduction. Hum Reprod Update. 22, 2-22

Wales, R.G., 1969. Accumulation of carboxylic acids from glucose by the pre-implantation mouse embryo. Aust J Biol Sci. 22, 701-707

Wamaitha, S.E. and Niakan, K.K., 2018. Human Pre-gastrulation Development. Curr Top Dev Biol. 128, 295-338

Wang, J., Alexander, P., Wu, L., Hammer, R., Cleaver, O. and McKnight, S.L., 2009. Dependence of mouse embryonic stem cells on threonine catabolism. Science. 325, 435-439

Wang, Y., Puscheck, E.E., Lewis, J.J., Trostinskaia, A.B., Wang, F. and Rappolee, D.A., 2005. Increases in phosphorylation of SAPK/JNK and p38MAPK correlate negatively with mouse embryo development after culture in different media. Fertil Steril. 83 Suppl 1, 1144-1154

Warburg, O., 1956. On respiratory impairment in cancer cells. Science. 124, 269-270

Washington, J.M., Rathjen, J., Felquer, F., Lonic, A., Bettess, M.D., Hamra, N., Semendric, L., Tan, B.S., Lake, J.A., Keough, R.A., Morris, M.B. and Rathjen, P.D., 2010. L-Proline induces differentiation of ES cells: a novel role for an amino acid in the regulation of pluripotent cells in culture. Am J Physiol Cell Physiol. 298, C982-992

Watkins, A.J., Platt, D., Papenbrock, T., Wilkins, A., Eckert, J.J., Kwong, W.Y., Osmond, C., Hanson, M. and Fleming, T.P., 2007. Mouse embryo culture induces changes in postnatal phenotype including raised systolic blood pressure. Proc Natl Acad Sci U S A. 104, 5449-5454

Watkins, A.J., Lucas, E.S., Wilkins, A., Cagampang, F.R. and Fleming, T.P., 2011. Maternal periconceptional and gestational low protein diet affects mouse offspring growth, cardiovascular and adipose phenotype at 1 year of age. PLoS One. 6, e28745

Watson, A.J. and Kidder, G.M., 1988. Immunofluorescence assessment of the timing of appearance and cellular distribution of Na/K-ATPase during mouse embryogenesis. Dev Biol. 126, 80-90

182

References

Watson, A.J., Damsky, C.H. and Kidder, G.M., 1990. Differentiation of an epithelium: factors affecting the polarized distribution of Na+,K(+)-ATPase in mouse trophectoderm. Dev Biol. 141, 104-114

Weathersbee, P.S., Pool, T.B. and Ord, T., 1995. Synthetic serum substitute (SSS): a globulin- enriched protein supplement for human embryo culture. J Assist Reprod Genet. 12, 354-360

Weitlauf, H.M., 1989. Embryonic signaling at implantation in the mouse. Progress in clinical and biological research. 294, 359-376

Wellen, K.E., Hatzivassiliou, G., Sachdeva, U.M., Bui, T.V., Cross, J.R. and Thompson, C.B., 2009. ATP-citrate lyase links cellular metabolism to histone acetylation. Science. 324, 1076-1080

Whitten, W.K., 1971. Nutrient requirements for the culture of preimplantation embryos in vitro. Advances in the Biosciences. 6, 129-139

Wiley, L.M., Yamami, S. and Van Muyden, D., 1986. Effect of potassium concentration, type of protein supplement, and embryo density on mouse preimplantation development in vitro. Fertil Steril. 45, 111-119

Williams, C.L., Teeling, J.L., Perry, V.H. and Fleming, T.P., 2011. Mouse maternal systemic inflammation at the zygote stage causes blunted cytokine responsiveness in lipopolysaccharide-challenged adult offspring. BMC Biol. 9, 49

Winger, Q.A., de los Rios, P., Han, V.K., Armstrong, D.T., Hill, D.J. and Watson, A.J., 1997. Bovine oviductal and embryonic insulin-like growth factor binding proteins: possible regulators of "embryotrophic" insulin-like growth factor circuits. Biol Reprod. 56, 1415-1423

Witkin, S.S., Liu, H.-C., Davis, O.K. and Rosenwaks, Z., 1991. Tumor necrosis factor is present in maternal sera and embryo culture fluids during in vitro fertilization. J Reprod Immunol. 19, 85- 93

Wright, R.W., Watson, J.G. and Chaykin, S., 1978. Factors influencing the in vitro hatching of mouse blastocysts. Anim Reprod Sci. 1, 181-188

Wullschleger, S., Loewith, R. and Hall, M.N., 2006. TOR signaling in growth and metabolism. Cell. 124, 471-484

Wydooghe, E., Vandaele, L., Heras, S., De Sutter, P., Deforce, D., Peelman, L., De Schauwer, C. and Van Soom, A., 2017. Autocrine embryotropins revisited: how do embryos communicate with each other in vitro when cultured in groups? Biol Rev Camb Philos Soc. 92, 505-520

Xiao, M., Yang, H., Xu, W., Ma, S., Lin, H., Zhu, H., Liu, L., Liu, Y., Yang, C., Xu, Y., Zhao, S., Ye, D., Xiong, Y. and Guan, K.L., 2012. Inhibition of alpha-KG-dependent histone and DNA demethylases by fumarate and succinate that are accumulated in mutations of FH and SDH tumor suppressors. Genes Dev. 26, 1326-1338

Xie, Y., Puscheck, E.E. and Rappolee, D.A., 2006. Effects of SAPK/JNK inhibitors on preimplantation mouse embryo development are influenced greatly by the amount of stress induced by the media. Mol Hum Reprod. 12, 217-224

183

References

Xie, Y., Wang, F., Puscheck, E.E. and Rappolee, D.A., 2007a. Pipetting causes shear stress and elevation of phosphorylated stress-activated protein kinase/jun kinase in preimplantation embryos. Mol Reprod Dev. 74, 1287-1294

Xie, Y., Zhong, W., Wang, Y., Trostinskaia, A., Wang, F., Puscheck, E.E. and Rappolee, D.A., 2007b. Using hyperosmolar stress to measure biologic and stress-activated protein kinase responses in preimplantation embryos. Mol Hum Reprod. 13, 473-481

Xie, Y., Awonuga, A., Liu, J., Rings, E., Puscheck, E.E. and Rappolee, D.A., 2013. Stress induces AMPK-dependent loss of potency factors Id2 and Cdx2 in early embryos and stem cells [corrected]. Stem Cells Dev. 22, 1564-1575

Xu, J. and Sinclair, K.D., 2015. One-carbon metabolism and epigenetic regulation of embryo development. Reprod Fertil Dev. 27, 667-676

Yamada, M., Takanashi, K., Hamatani, T., Hirayama, A., Akutsu, H., Fukunaga, T., Ogawa, S., Sugawara, K., Shinoda, K., Soga, T., Umezawa, A., Kuji, N., Yoshimura, Y. and Tomita, M., 2012. A medium-chain fatty acid as an alternative energy source in mouse preimplantation development. Sci Rep. 2, 930

Yang, L., Lv, Q., Chen, W., Sun, J., Wu, Y., Wang, Y., Chen, X., Chen, X. and Zhang, Z., 2017. Presence of embryonic DNA in culture medium. Oncotarget. 8, 67805-67809

Yoon, J., Juhn, K.M., Ko, J.K., Yoon, S.H., Ko, Y., Lee, C.Y. and Lim, J.H., 2013. Effects of oxygen tension and IGF-I on HIF-1alpha protein expression in mouse blastocysts. J Assist Reprod Genet. 30, 99-105

Yoshida, S., Hong, S., Suzuki, T., Nada, S., Mannan, A.M., Wang, J., Okada, M., Guan, K.L. and Inoki, K., 2011. Redox regulates mammalian target of rapamycin complex 1 (mTORC1) activity by modulating the TSC1/TSC2-Rheb GTPase pathway. J Biol Chem. 286, 32651-32660

Yu, C., Wang, L., Li, J., Guo, C., Guo, X., Zhang, X., Xu, Y. and Yao, Y., 2012. Ovarian stimulation reduces IL-6 release from mouse and human pre-implantation embryos. Am J Reprod Immunol. 68, 199-204

Yu, C., Zhang, X., Wang, L., Liu, Y., Li, N., Li, M., Chen, L., Liu, Y. and Yao, Y., 2018. Interleukin-6 regulates expression of Fos and Jun genes to affect the development of mouse preimplantation embryos. J Obstet Gynaecol Res. 44, 253-262

Yuan, Y.Q., Van Soom, A., Coopman, F.O., Mintiens, K., Boerjan, M.L., Van Zeveren, A., de Kruif, A. and Peelman, L.J., 2003. Influence of oxygen tension on apoptosis and hatching in bovine embryos cultured in vitro. Theriogenology. 59, 1585-1596

Zhang, J., Wang, X., Vikash, V., Ye, Q., Wu, D., Liu, Y. and Dong, W., 2016. ROS and ROS- Mediated Cellular Signaling. Oxid Med Cell Longev. 2016, 4350965

Zhang, L.F., Jiang, S. and Liu, M.F., 2017. MicroRNA regulation and analytical methods in cancer cell metabolism. Cell Mol Life Sci. 74, 2929-2941

Zhao, T., Goh, K.J., Ng, H.H. and Vardy, L.A., 2012. A role for polyamine regulators in ESC self- renewal. Cell Cycle. 11, 4517-4523

184

References

Ziebe, S., Loft, A., Povlsen, B.B., Erb, K., Agerholm, I., Aasted, M., Gabrielsen, A., Hnida, C., Zobel, D.P., Munding, B., Bendz, S.H. and Robertson, S.A., 2013. A randomized clinical trial to evaluate the effect of granulocyte-macrophage colony-stimulating factor (GM-CSF) in embryo culture medium for in vitro fertilization. Fertil Steril. 99, 1600-1609

Zolti, M., Ben-Rafael, Z., Meirom, R., Shemesh, M., Bider, D., Mashiach, S. and Apte, R.N., 1991. Cytokine involvement in oocytes and early embryos. Fertil Steril. 56, 265-272

Zwierzchowski, L., Czlonkowska, M. and Guszkiewicz, A., 1986. Effect of polyamine limitation on DNA synthesis and development of mouse preimplantation embryos in vitro. J Reprod Fertil. 76, 115-121

185

Minerva Access is the Institutional Repository of The University of Melbourne

Author/s: Kelley, Rebecca Lauren

Title: Group culture of preimplantation embryos creates a microenvironment that determines development, viability, and response to external stressors

Date: 2018

Persistent Link: http://hdl.handle.net/11343/224106

File Description: Group culture of preimplantation embryos creates a microenvironment that determines development, viability, and response to external stressors

Terms and Conditions: Terms and Conditions: Copyright in works deposited in Minerva Access is retained by the copyright owner. The work may not be altered without permission from the copyright owner. Readers may only download, print and save electronic copies of whole works for their own personal non-commercial use. Any use that exceeds these limits requires permission from the copyright owner. Attribution is essential when quoting or paraphrasing from these works.