Dissertations / Theses on the topic 'Embryo implantation'

Human pre-implantation embryos display a high prevalence of aneuploidy and chromosomal mosaicism, unique from any other species. The decreasing incidence of aneuploidy observed between the cleavage and blastocyst stages of preimplantation embryo development infers a degree of 'self-correction' following activation of the embryonic genome. However, contrary to the previous assumption that only euploid embryos should be considered 'normal', new evidence has confirmed that mosaic embryos can result in the birth of healthy babies. Thus, aneuploidy should be viewed as an intrinsic feature of human pre-implantation embryo development, which presents a novel challenge at implantation. The endometrium must implement both positive and negative selection, in order to limit maternal investment to only viable embryos. The ability of the endometrium to act as a 'biosensor' of embryo quality has been well documented yet there is little direct evidence for the key regulators of this process. For the first time, we demonstrate a biological context for embryo biosensoring. Firstly, we discover novel embryo-secreted proteases that are enhanced at the blastocyst stage and relate to implantation outcome upon embryo transfer. Secondly, we identify corresponding protease-sensitive receptors in the endometrium, heightened during the window of implantation. By demonstrating the cleavage and de-activation of endometrial toll-like receptor 4 (TLR4) by embryo conditioned medium, we link successful implantation to a diminished inflammatory response. Furthermore, we demonstrate that TLR4 levels in the endometrium constitute a selectivity checkpoint, which is supressed in women suffering from recurrent miscarriage (RM).

Embryo implantation represents a complex process vital in ensuring the normal development of pregnancy. Whether embryo implantation is the goal of natural conception or assisted reproductive treatment, the environment within the uterine cavity must be optimised in order to increase the chance of pregnancy. This thesis uses a mixture of research methods to investigate potential approaches to improving embryo implantation. Below are the key findings from this thesis: 1. The vitamin D status in women undergoing assisted reproductive treatment is important. An interventional trial would prove or disprove the merits of vitamin D deficiency treatment in these women. 2. There is not enough evidence to suggest a clear association between vitamin D and recurrent miscarriage, however there is a strong argument for biological plausibility. 3. The use of endometrial fluid collected at the time of embryo transfer in women undergoing assisted reproductive treatments for metabolomics analysis is possible. 4. Women with hydrosalpinx associated tubal infertility should be offered salpingostomy as a treatment option as the natural conception rates are similar to that achieved in in vitro fertilisation treatment.

Identification of embryo implantation-related proteins Mitra Arianmanesh Embryo implantation is a complex process involving an active dialogue between the endometrium and embryo. Tightly controlled communication between the hypothalamus, pituitary, corpus luteum (CL), endometrium and embryo is essential for implantation. Unravelling molecules involved in embryo implantation is essential since implantation failure is one of the main causes of female infertility. Therefore, identification of molecular events during embryo implantation may result in enhancing implantation rates in both natural and assisted reproductive cycles, improving contraceptive design and reducing the rate of multiple pregnancies following embryo transfer in IVF cycles. Thus, in this study, sheep was used as an animal model in order to study endometrial, corpus luteal and plasma proteome changes during embryo implantation and early pregnancy. Endometrium, CLs and plasma were harvested from cyclic ewes on days 12 and 16 of the oestrous cycle (n=4 ewes/group) and from pregnant ewes on days 12, 16 and 20 of pregnancy (n=4 ewes/group). Furthermore, ovine endometrium were collected from pregnant and non-pregnant horns on days 16 (n=4) and 20 (n=4) of pregnancy to compare endometrial protein profiles of the gravid horn (in the presence of the conceptus) with the non-gravid horn (in the absence of the conceptus) in response to the conceptus to elucidate local embryo-endometrial signalling. 2DE gel, LC-MS/MS, Western blot, IHC and qRT-PCR were employed to quantify implantation processes. This study has identified proteins in the CL and endometrium with involvement in biological pathways that are fundamental for embryo implantation and gestation. In addition, it was found that the implanting embryo is capable of regulating the expression of endometrial proteins to establish an ideal environment for its implantation and establishment of pregnancy. These findings provide an addition to the field and a solid base for targeted studies to improve our understanding of implantation and its regulation.

Failure of the embryo to implant into the uterine lining results in infertility and is also the rate limiting step in FVF. The tethered, glycoprotein MUC1 is a protective cell surface receptor that has been associated with infertility. Evidence suggests MUC1 and other endometrial mucins regulate embryo implantation. Endometrial epithelia must be shielded from infection whilst permitting the recognition and implantation of the embryo. The protein backbone of MUC1 is thought to act as a scaffold for L-selectin ligands and may be integral to the initial tethering of the embryo during early implantation. Objectives The remit of this thesis was to model the expression of MUC1 and other mucins in the human endometrium using endometrial cell lines and to use atomic force microscopy to understand the role played by these proteins in shaping the non-specific and embryo specific adhesive characteristics of endometrial monolayers. If possible the proposed L-selectin implantation mechanism was to be identified and functionally characterised. Methodology This project successfully married the traditional molecular tools of quantitative PCR and immunocytochemistry with novel application of INCELL analyzer high content screening protein analysis, peak force quantitative nano-mechanical mapping atomic force microscopy and single molecule force spectroscopy atomic force microscopy to characterise the specific and non-specific surface adhesion on live endometrial monolayers. Results Firstly, immunocytochemistry and qRTPCR revealed that basal MUC1 expression was significantly higher in Hec-1-B relative to Hec-IA, Ishikawa and Hec50. Secondly, INCELL analysis qualified a distinct heterogeneous expression of MUC1 across the endometrial monolayer and delineated altered patterning following treatment with estradiol and progestins. Thirdly, we have shown a direct and proportional correlation between MUC1 expression and adhesion in live Hec-IA and Hec-IB cell monolayers. Fourthly, this work has confirmed and characterised binding of recombinant L-selectin to the endometrial epithelial cell surface. Fifthly, it is shown that L-selectin surface binding decreases following a reduction in MUC1 surface presentation. Conclusions The results implicate MUC1 as a key component of endometrial adhesion and an initial mediator of implantation with a functional patterned expression suggesting areas of altered receptivity exist across endometrial monolayers. Abnormal MUC1 expression has been shown in endometrial pathologies and unexplained infertility. The current investigation suggests MUC1 protein may assist embryo attachment by retarding it sufficiently through mechanical impedance to allow specific L-selectin binding further securing the embryo. A non-receptive endometrium may contribute towards the infertile phenotype despite repeated IVF treatments, thus novel examination of potential embryo adhesion molecules such as MUC1 may aid understanding of endometrial characteristics which prevent embryo implantation and contribute towards IVF failure.

The morphogenetic events that give rise to the early post-implantation mouse embryo (egg cylinder) have not been thoroughly studied and our knowledge is restricted to "snap-shot" descriptions of embryos recovered at different stages of implantation from the mother. A central feature of the egg cylinder is the pro-amniotic cavity, which spans the embryo and participates in formation of the extraembryonic membranes. The major aims of my PhD studies have been to reveal how this cavity is formed (Aim 1) and then how the egg cylinder grows (Aim 2). In order to address how the pro-amniotic cavity forms (Aim 1), I first characterised in detail development of the architecture of the extra-embryonic ectoderm (ExE), which has to be remodelled to permit cavity formation. My findings indicate that the ExE comprises cells in direct contact with a basement membrane and cells that lie deeper in the tissue. The ExE originates in the polar trophectoderm, a monolayer covering the epiblast of the blastocyst, which expands and undergoes invagination to form a slit-like cavity. By carrying out analyses of fixed specimens and live imaging of cultured embryos, I have found that the epiblast and ExE cavity extend towards each other through the formation and resolution of multiple rosette structures. This leads to the fusion of the ExE and epiblast cavities to form the unified pro-amniotic cavity. I show that this process is dependent on signalling cues stemming from the underlying basement membrane that activate the b1-integrin signalling pathway to regulate cell polarity, ExE tissue architecture and rosette formation. In addition to the basement membrane's role in b1-integrin signalling, it also has physical functions that I characterise in the second part of my study (Aim 2). High resolution imaging revealed that the basement membrane underlying the epiblast is highly perforated during the implantation stages. These perforations are initially evenly distributed and then accumulate asymmetrically at the future posterior part of the embryo, just prior to gastrulation. Finally, I demonstrate that remodelling of the basement membrane requires the expression of matrix metalloproteinases (MMPs) in the epiblast under the control of Nodal. The anterior visceral endoderm inhibits Nodal signalling and hence MMP inhibition in the anterior. I demonstrate that activity of the MMPs and perforations in the basement membrane are essential for embryo growth. The domain of posterior basement membrane perforations persists beyond gastrulation suggesting a potential role for these perforations in primitive streak formation and extension. Together, my studies bring new important insights into the understanding of early mouse embryo morphogenesis.

Implantation is a critical step in reproduction. It is complicated and well-coordinated consisting of apposition, attachment and invasion of embryo into the endometrium. The mechanism of implantation is unclear. Our previous proteomic study showed an increase of annexin A2 in the endometrium during the implantation window of mice, consistent with the increased annexin A2 expression in the receptive human endometrium. The hypothesis of this project was that annexin A2 mediatedthe embryo-endometrium attachment. The first objective was to study the spatio-temporal expression of endometrial annexin A2 immunoreactivities in humans and mice. The cyclical change in annexin A2 expression in the mouse and human reproductive cycle suggested the involvement of a steroid regulatory mechanism. Interestingly, annexinA2 was transiently expressed on the membrane between the mouse uterine luminal epithelium and the implanting embryos from Day 4 (pre-implantation) to Day 5 (post-implantation) of pregnancy. No such signal change was observed at the inter-implantation sites, showing that the implanting embryos partially regulated annexin A2 expression. These observations and the high expression of the molecule in the luminal epithelium of human endometrium in the mid-and late luteal phase were consistent with a role of annexin A2 in implantation. The second objective was to verify the action of steroids on annexin A2 expression. It was found that a combination of 6675 pmol/L of estrogen and 429.8nmol/L of progesterone increased the total and apical surface expression of annexin A2. In mice, estrogen but not progesterone, increased annexin A2 expression in the uterine luminal epithelium of ovariectomized mice. The third objective was to study the function of annexin A2 in embryo-endometrium attachment using an Ishikawa (endometrial epithelial cells)-JEG-3 trophoblast spheroids (embryo surrogate) coculture model. Knockdown of the expression of annexin A2 in either or both cell lines significantly decreased the attachment rate of the spheroids onto the endometrial cells. The suppressive action on the two cell lines was additive. The attachment was also suppressed in the presence of anti-annexin A2 antibody during coculture. Annexin A2 was also involved in mouse implantation as demonstrated by a significant decrease in implantation sites after injection of anti-annexin A2 antibody into the mouse uterine horn. The final objective was to study the action of annexin A2 as an adhesive molecule in embryo attachment. It was found that loss of P11, the binding partner of annexin A2, reduced the attachment rate of the JEG-3 spheroids probably by decreasing the translocation of annexin A2 to the surface of the endometrial cells. Recombinant P11 and annexin A2 protein failed to bind significantly to the Ishikawa cells and the JEG-3 cells. In summary, this study demonstrates the involvement of annexin A2 as an adherent molecule in the embryo-endometrium interaction.
published_or_final_version
Obstetrics and Gynaecology
Doctoral
Doctor of Philosophy

Background: In the last two decades the discovery of the Toll-Like Receptor (TLR) family in humans revealed the importance of innate immunity in providing host defence against invading pathogens. Moreover, TLRs have a vital role in mediating the interactions between the reproductive and immune systems. Similar to TLRs, (NOD)-like receptors (NLRs); retinoic acid-inducible gene-1, RIG-1-like receptors (RLRs) are also important pattern recognition receptors in the female reproductive system. Successful implantation requires effective reciprocal interactions between receptive endometrium and competent embryo. The endometrial innate immunity during implantation has been intensively investigated. However, little is known about the expression of innate immunity during the early stages of human embryo development. Objective: To investigate the expression of innate immunity molecules during the early stages of human embryo development and to determine the functions of TLRs in blastocysts. Material and methods: The expression of TLRs, a panel of downstream signalling molecules, NLRs, RLRs, inflammatory cytokines, chemokines and the hyaluronic acid system were investigated in the following developmental stages: oocyte, 4- cell, blastomeres, 8- cell, blastocyst, inner cell mass and trophectoderm using Affymetrix Human Genome U133 Plus 2.0 arrays. Microarray data were validated by Q-PCR. TLR function in human blastocysts was investigated by treating day five blastocysts with TLR3 or TLR5 specific ligands; Poly (I:C) and flagellin respectively, for 24 hours. The culture media was analysed for elevated cytokine and chemokine levels using cytometric bead array. Results: TLRs, NLRs, RLRs, TLR downstream signalling molecules, cytokines and chemokines involved during implantation event and the hyaluronic system were all found to be positively expressed in the early stages of human embryo development. The expression levels of the above molecules were generally moderate to low (CT 24-34) and varied across the embryonic developmental stages. Stimulation of TLR3 and TLR5 in day 5 blastocysts produced cytokines and chemokines. In addition, there were alterations in gene expression patterns in the Poly (I:C) and flagellin treated blastocysts in comparison to the untreated blastocysts. Conclusion: The varied expression levels of the investigated molecules involved in early embryonic developmental suggests a potential role for these molecules in early pregnancy loss and implantation failure. Specifically, the relationship between the level of TLR expression, function and embryo quality is worth investigating in the future as a potential marker for embryo competence.

Hypothesis: Pelvic perfusion is the pivotal factor for the outcome of in vitro fertilisation (IVF) treatment once clinical and embryological variables are controlled for their effect.;Demonstration of Hypothesis: In a series of three studies, the clinical aspects of embryo implantation were examined from the perspective of tissue perfusion.;Epidemiological Study: Clinical and embryological data were evaluated to predict multiplicity of implantation and ongoing pregnancy in IVF treatment. Oocyte and embryo quality were appraised and the impact of the number of embryos transferred was assessed.;Study on In-vivo Vascular Physiology: The prognostic role of utero-ovarian perfusion and its pharmacological manipulation with low dose aspirin was evaluated in the outcome of IVF treatment. Serum, follicular fluid vascular endothelial growth factor (VEGF) and vascular endothelial growth factor receptor (VEGFR) concentrations were correlated with Doppler indices.;Study on In-vivo Endometrial Physiology: Endometrial receptivity (in terms of endometrial thickness and echo-pattern) and VEGF-VEGFR concentrations were evaluated with regards to the outcome of frozen-thawed embryo replacement (FTER) during natural and hormone replacement cycles.;Results: The epidemiological study showed that the outcome of IVF treatment was closely associated with the severity of subfertility. Ovarian reserve and response to stimulation were the key factors. The probability of pregnancy was affected by the number and quality of oocytes and by their fertilisation rate and the cleavage rate of the resulting embryos. The potential to provide mature oocytes and high quality embryos was an inherent characteristic of the ovaries and independent of stimulation protocols. When embryo quality was taken into consideration, the number of embryos transferred no longer affected the chance of pregnancy. The clinical study showed that the chance of pregnancy was directly dependant upon tissue perfusion. Pregnancy rates were very low with uterine artery pulsatility index >3 (PI) and peri-follicular PI >1. Better ovarian reserve, response to stimulation, endometrial development, implantation and pregnancy rates were associated with low follicular fluid VEGF-VEGFR levels and this was also associated with good uterine and endometrial perfusion. Aspirin (150 mg/day) had no beneficial effect on Doppler indices, ovarian response to stimulation, implantation or pregnancy rates. Pregnancy rates were similar with naturally and hormonally prepared endometrium in frozen-thawed embryo replacement cycles. Higher serum VEGF and lower VEGFR levels were observed in pregnant cycles, but the differences were not significant. Endometrial echo-pattern and thickness did not affect conception.;Conclusions: Tissue perfusion plays a key role in the physiological steps leading to conception and implantation. Aspirin (150 mg/day) improved neither tissue perfusion nor the outcome of fresh embryo transfer. The type of endometrial preparation did not affect the outcome of frozen-thawed embryo replacement cycles.

The use of assisted reproductive technologies (ART) such as in vitro fertilisation (IVF) is increasing and today close to 4 percent of babies born in Australia, are as a result of ART. IVF procedures involve the culture of resultant embryos in medium that routinely lacks the growth factors that are present in the reproductive tract. Cultured embryos develop at a slower rate and have higher levels of developmental arrest, with fewer than 50% of embryos reaching the blastocyst stage. Furthermore, once an embryo is transferred back into the uterus, it faces the hurdle of implantation, with implantation failure being a major cause of IVF failure. This thesis examines whether the addition of growth factors to the embryo culture medium can increase blastocyst development and adhesion competency, using mouse embryos as a model system. In particular, the effect of insulin-like growth factor 1(IGF1) and insulin-like binding protein 3 (IGFBP3) on preimplantation mouse embryo development in vitro and the role of IGF1 on implantation in vitro was examined. In the present study, the culture of preimplantation embryos in the presence of a physiological concentration of IGF1 improved development from compaction onwards, resulting in improved blastocyst development. Conversely, high levels of IGF1 negatively impacted on development by decreasing hatching probably due to these high levels of IGF1 causing IGF1 receptor (IGF1R) down-regulation and apoptosis in the mouse embryo, as shown previously. Blocking the IGF1R with a neutralising antibody was shown to decrease blastocyst development, hatching and cell numbers and to increase apoptosis. Furthermore, treatment of blastocysts with IGF1 caused phosphorylation of Akt, which regulates cell survival by activating anti-apoptotic pathways. Therefore, IGF1 may act as a survival factor in the preimplantation embryo. During early implantation integrins accumulate on the surface of the blastocyst and endometrium and these interact with each other via extracellular matrix proteins such as fibronectin. These interactions are important for the attachment of blastocysts to the endometrium. In the present study treatment of blastocysts with IGF1 increased fibronectin on the surface of the blastocyst via activation of the Phosphoinositide 3 Kinase (PI3K) pathway. As a result, blastocysts had increased attachment to cultured uterine epithelial cells and increased outgrowth. In addition to IGF1, the reproductive tract produces IGFBP3, which is also thought to improve development of the embryo. However, to the best of our knowledge this is the first study to examine the effect of exogenous IGFBP3 on embryo development in vitro. IGFBP3 caused an increase rate of progression of embryos through the early stages of division (5-8cells) and activation of Akt and ribosomal protein S6 (rpS6) proteins as well as induction of calcium signalling. In the present study, it appears that IGFBP3 signalling in the embryo requires the IGF1R, as the use of an IGF1R neutralising antibody blocked IGFBP3 from enhancing early stages of division and the induction of calcium signalling. In other cell types, IGFBP3 signals through the IGF1R following a transactivation event involving the Sphingosine 1-Phosphate (S1P) pathway. The complex interactions and signalling of IGFBP3 are beginning to emerge in a number of different cell types and further investigation of IGFBP3 function is required in the embryo. As growth factors are generally absent from embryo culture media there is a potential avenue for the improvement of embryo culture and ART outcomes by addition of IGF1 and or IGFBP3 to the culture medium. The requirements of the embryo are complex and understanding the role of growth factors in embryo development is essential in order to optimise embryo culture and develop culture media for use in ART.

Les lymphocytes T régulateurs naturels (Treg) établissent la tolérance à l’émergence de la tumeur. Les tumeurs entraînent la réponse rapide des Treg déjà activés/en mémoire (amTreg), qui domine la réponse des cellules T effecteurs naïves (Teff). Nous faisons l’hypothèse que des mécanismes similaires mènent à la tolérance des fœtus. Cette thèse se concentre sur l’analyse des parallèles entre les réponses immunitaires aux tumeurs et à l’implantation de l’embryon. Nous avons identifié une sous-population de Treg qui apparait comme un régulateur dans l’implantation de la tumeur/embryon, les similarités étant frappantes. Comme les tumeurs, les amTreg spécifiques aux autoantigènes sont rapidement recrutés et activés au niveau des ganglions drainant l’embryon, ceci peu après l’implantation. Mais à l’inverse, la seule élimination des Treg n’est pas toujours suffisante pour entraîner l’avortement ; en parallèle, la pré immunisation maternelle contre les antigènes du fœtus n’est pas assez puissante pour induire la perte des fœtus, sauf si la délétion des Treg est imposée au même moment – contrairement aux tumeurs, puisque la pré-immunisation contre les antigènes des tumeurs conduit au rejet complet de la tumeur même quand les Treg sont présents. Ceci révèle un réseau protecteur robuste qui assure le succès de la grossesse. Les fonctionnalités partagées par les réponses immunitaires dans les grossesses et cancers peuvent avoir des implications réciproques dans des applications thérapeutiques. Nous avons aussi testé sur des souris les effets d’injections d’IL2 à faible dose sur de courtes périodes sur la régression des avortements spontanés, qui a montré des résultats prometteurs.

“The role of L-proline in pre-implantation mouse embryo development in vitro” Amino acids are known to play important roles in pre-implantation embryo development, including regulation of cell volume and metabolism. Inclusion of L-glutamine, glycine and betaine in embryo culture medium has been shown to improve development in vitro by acting as organic osmolytes, thereby regulating cell volume. The purpose of the present study was to determine the role of L-proline in pre-implantation mouse embryo development in vitro. In order to ensure the osmotic effect of L-proline, the osmolality of culture medium was prepared in different ranges (258, 270, 296 and 336 mOsm/kg). One-cell embryos were cultured in amino acid free modified human tubal fluid medium and potassium simplex optimization medium, at low density (1embryo/100μl) and high density (1embryo/μl). Amino acids (L-glutamine, L-proline, D-proline, glycine, L-alanine, betaine) were added to the culture medium and development of the embryos was scored every 24h until day 6 of pregnancy (blastocyst stage). Blastocysts were fixed, stained with DAPI and imaged on a confocal microscope. Cell numbers in each blastocyst were then counted. At low density, with 270−336mOsm/kg L-proline increased development at day 6 (p<0.001) to the blastocyst stage, and also increased the proportion of blastocysts that were hatching at day 6 (p<0.01) compared to embryos cultured in the absence of L-proline. However, there was no change in the number of cells in the embryos that reached the blastocyst stage. In contrast, at high density, L-proline had no effect on development compared to the absence of L-proline. L-proline improved development and blastocyst expansion despite when embryos were cultured in upon and sub-optimal (hyper osmolality and low density) conditions.

One of the critical periods of time for the establishment of pregnancy is after the onset of implantation but before the establishment of the placenta. In fact, evidence strongly suggests that when abnormalities occur during this period of pregnancy in humans there may be dire consequences such as termination of pregnancy, pre-eclampsia later in pregnancy and small for gestational weight offspring that are less healthy in later life. Therefore, what occurs in the endometrium during the progression of implantation can be crucial for proper fetal development later in pregnancy as well as the health of the offspring. One of the evolving ideas coming from research on the biology of what occurs during early pregnancy is the existence of key bi-directional communications between the conceptus and uterus that may be required for successful pregnancy. Amazingly, some common aspects of this signaling might exist to some extent between mammalian species that have very different modes of implantation and where quite different placentas are formed. Although some conceptus-uterine communication during the progression of decidualization is starting to be worked out, we still have much to learn about the precise details of the entire repertoire of the molecular signals responsible. In the rodent we still have to clearly find and define the biology of important paracrine factors produced by the conceptus that target cells of the endometrium during this period of time when the endometrium is undergoing decidualization. This review focuses on some of the approaches that have been used in the past and what has been learned about the effect of the conceptus on the decidualization of the rodent uterus. Where possible, this is compared and contrasted to what is currently known in other species that exhibit quite different modes of implantation.

In mammals, development of a new organism requires fertilisation of the female egg by sperm. The resulting zygote develops into the blastocyst stage as it travels towards the uterus. Within the uterus, the blastocyst invades the maternal tissues and establishes access to the maternal blood supply. This process is called implantation and is absolutely essential for the further development of the conceptus and establishment of pregnancy. Successful implantation requires a proper preparation of the uterus and the embryo as well as a molecular dialogue between the embryo and the uterine tissues. Female mice that have a disruption in the Plcβ1 gene are infertile. In the course of this Thesis it became apparent that the main cause of their infertility is their inability to implant their embryos. PLCβ1 protein is a mediator of G-protein coupled receptor (GPCR) signalling and it is involved in the production of second messengers essential for downstream transmission of signals. A host of reproductive functions are under the control of GPCR signalling. In this PhD Thesis the infertile phenotype of Plcβ1 knockout (KO) female mice was investigated to identify the reproductive processes affected by the lack of a functional PLCβ1 protein. A combination of histological, molecular biology and in vivo techniques were utilised to show that at the time of implantation, embryos fail to attach to the uterine epithelium of KO uteri. In addition, it was demonstrated that estrogen signalling and components of the endocannabinoid metabolism, both key processes for successful implantation are severely altered in KO uteri. These observations show that KO uteri fail to prepare for implantation. In addition, the KO reproductive tract exerts a detrimental effect on pre- and peri- implantation embryo development. Currently, failure of implantation is thought to be one of the major causes of infertility in women and up to this date there are no successful treatments. The results of this project expand our current knowledge on the physiology of implantation and provide cues for the development of diagnostic markers and treatments for the women who are unable to conceive.

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Title page, contents and abstract only. The complete thesis in print form is available from the University Library.
Thesis (Ph.D.)--University of Adelaide, Dept. of Obstetrics and Gynaecology, 1993

Thesis (PhD)--Stellenbosch University, 2012.
ENGLISH ABSTRACT: CHAPTER 1 In this chapter the aim is to outline the different chapters under section A. Against this background, we will conduct a literature review of relevant studies performed, and evaluate their comments regarding identifying embryo markers which can be utilized to improve overall ART outcome. We will evaluate the embryo marker sHLA-G in detail, using a prospective randomized study as well as a retrospective multi-centre study. The role of the morphology and genetic profile of an oocyte, zygote and embryo and subsequent blastocyst formation will be evaluated with the help of WGA/CGH. The work will then be summarized and conclusions will be made as well as possible suggestions for future directions will be indicated. In section B the methodology of the studies explaining the role of the candidate is illustrated. CHAPTER 2 In this chapter the impact of the oocyte/zygote and the embryo on implantation/pregnancy rate was discussed. The morphologic characteristics of the oocyte, the cumulus–oocyte-complex (COC), the zona pellucida, the perivitelline space, cytoplasm and meiotic spindle and the polar body and its appearance were discussed in detail. The morphologic characteristics of embryo fragmentation and its effect on embryo development, ploidy and blastocyst formation were also studied. Embryo markers to predict pregnancy outcome were researched based on the international literature. The pronuclear morphology and early cleavage were highlighted as non-invasive embryo markers to predict outcome. A non-invasive biochemical marker, soluble Human Leucocyte-Antigen-G (sHLA-G), that is expressed by developing embryos was researched. The value of blastocyst transfer and the improved ongoing pregnancy rate compared to cleavage stage embryos were highlighted based on a recent meta-analysis. A detailed discussion on sHLA-G as well as Array-CGH and the future of these tests followed. CHAPTER 3 In this chapter the aim was to compare pregnancy and implantation rates when embryos are selected based on a single Day 3 (D 3) morphology score vs. a GES score plus sHLA-G expression. This was a prospective randomized study (n=214) undergoing fresh ICSI cycles. Embryos were selected for transfer based on either Day 3 morphology score (Group A) or GES-scoring plus sHLA-G expression (Group B). The following results were reported: Clinical [35/107 (33%) vs. 52/107 (49%)] and ongoing pregnancy [20/107 (19%) vs. 52/107 (49%)] rates were significantly different between Group A and Group B (p<0.05). Implantation rates were not significantly different between Group A [52/353 (15%)] and Group B [73/417 (18%)] (p<0.05). The number of pregnancies lost during the first trimester was nearly 12 times higher in Group A [25/52 (48%)]. It was concluded that the miscarriage rate was significantly lower in Group B than Group A and the pregnancy results were superior when embryos were selected based on GES plus sHLA-G expression. CHAPTER 4 Several studies have reported an association between the presence of soluble human leukocyte antigen-G (sHLA-G) in human embryo culture supernatants (ES) with implantation and pregnancy outcome in vitro. However, the actual presence role during implantation and effect on implantation and pregnancy outcome are still controversial. A retrospective multi-centre study was performed on 2040 ICSI patients in six different centers. All embryos were individually cultured and a chemiluminescence enzyme-linked immunosorbent assay (ELISA) was used to detect the presence of sHLA-G in culture medium surrounding embryos. In all centers, a positive sHLA-G result was associated with an increase in odds of multiple clinical implantations (OR: 1.48, 95% CI: 1.07 to 2.05, p-value: 0.0170), and an increased odds of multiple on-going pregnancies (OR: 1.66, 95% CI: 1.10, 2.51, p-value: 0.0170). Data from this multi-centre study emphasize that sHLA-G expression is a valuable non-invasive embryo marker to assist in improving pregnancy outcome with the theoretical potential to reduce multiple pregnancies. A combination of sHLA-G expression and extended embryo culture to the blastocyst stage might provide future tools by which to select single embryos for transfer and reduce the risk of multiple gestational, without compromising their pregnancy rates. CHAPTER 5 In this chapter the ploidy status of first and second polar bodies and Day 3 blastomere, embryo morphology and biochemical (sHLA-G) characteristics were correlated with blastocyst development and subsequent pregnancy outcome. All oocytes/zygotes and embryos were individually cultured to the blastocyst stage. PB-I, PB-II and blastomeres underwent whole genome amplification (WGA) and comparative genome hybridization (CGH) and complete karyotyping. Each embryo‟s culture medium supernatant was collected and analyzed for sHLA-G expression on Day 2. The following results were reported: Fifty seven mature (MII) donor oocytes were obtained, 33/57 (57.9%) were aneuploid, 21/57 (36.8%) were euploid and 3/57 (5%) were “inconclusive”. No correlation was found between CGH status of PB-I, PB-II and the GES-score. Furthermore, no correlation was established between PB-I CGH results and blastocyst morphology grade. There was a significant correlation between PB-I CGH and blastomere CGH results. Euploid and aneuploid PB-I developed into 58% and 67% blastocysts, respectively. Kappa statistics (>0.7) revealed a positive correlation between the ploidy of PB-I, PB-II and the blastomeres. It was concluded that following ICSI and sequential genetic karyotyping of the oocyte/zygote and subsequent blastomeres, the majority of oocytes fertilized and subsequent zygotes developed into blastocysts, despite their ploidy status. We therefore conclude that blastocyst development is not associated with ploidy. CHAPTER 6 Identifying a developmentally competent embryo to transfer that has the highest probability to develop into a live baby has been an issue of debate and continues research. The aim of this chapter is to discuss the morphological, biochemical and genetic features of an embryo that has been shown to be predictive of implantation and pregnancy outcome in ART using most current evidence. A literature search was performed looking at the correlation between pronuclear morphology, early cleavage, cleavage stage embryos, blastocyst development, the presence of sHLA-G, CGH, embryo development and implantation/pregnancy rates in ART. Based on the available literature, a combination of observations could assist the scientist with embryo selection. The pronuclear stage morphology, the early embryo division, cleavage embryo stage and quality of the day 3 embryos provides limited guidance. However, choosing a blastocyst with a positive sHLA-G result on Day 5 is the optimal combination to make the final selection before embryo transfer or freezing. This non-invasive approach should improve pregnancy outcome and reduce multiple pregnancy rates. As far as the use of the more invasive technology such as aCGH is concerned, more research on pregnancy outcome is needed. CHAPTER 7 A combination of observations for embryo selection, starting with oocyte grading, pronuclear stage morphology, early zygote cleaving and cleavage-stage embryo morphology/quality on Day-3, however, ultimately using extended embryo culture and choosing a blastocyst on Day 5 with positive sHLA-G values available, will assist the scientist in making the final decision before selecting an embryo for transfer or cryopreservation. The use of aCGH (for chromosomal analysis) is invasive and is still considered experimental. Finally we conclude that despite all the above mentioned parameters to select an embryo for transfer that will develop into a live baby, more extensive research and international corroboration is needed in order to improve and standardize embryo selection criteria.
AFRIKAANSE OPSOMMING: HOOFSTUK 1 Die doel in hierdie hoofstuk is om die verskillende hoofstukke onder Afdeling A uiteen te sit. Daar word beplan om „n literatuur oorsig te doen van toepaslike studies rakend embriomerkers wat swangerskap-uitkoms in in vitro bevrugting kan verbeter. Verder sal die embriomerker sHLA-G deeglike bestudeer word met behulp van „n prospektiewe gerandomiseerde studie, asook „n retrospektiewe multisentrum studie. Die rol van embrio morfologie en die genetiese profiel van die ovum, sigoot asook die embrio en die daaropvolgende blastosist vorming sal geëvalueer word met behulp van WGA/CGH. Alle bevindings sal daarna opgesom word, gevolg deur „n sinvolle gevolgtrekking en laastens sal voorstelle gemaak word vir toekomstige navorsing op die gebied. In Afdeling B sal die metodiek van die studies verduidelik word, asook „n beskrywing gegee word van die kandidaat se rol gedurende die navorsings projekte in hierdie tesis. HOOFSTUK 2 In hierdie hoofstuk word die impak van die oösiet en die embrio op die inplanting/swangerskap-koers bespreek. Die morfologiese eienskappe van die oösiet, die kumulus-oösiet kompleks, die sona pellucida, die perivitelline spasie, sitoplasma en meiotiese spoel, die poolliggaam en die se voorkoms word breedvoerig bespreek. Die morfologiese eienskappe van die embrio, fragmentasie en die invloed daarvan op die embrio, ploïdie, en blastosistvorming word bespreek. Embriomerkers om swangerskapsuitkoms te voorspel, gebaseer op internasionale literatuur, is ook nagevors. Die pronukleêre morfologie en vroeë deling word as nie-indringende embriomerkers uitgelig om swangerskapsuitkoms te voorspel. „n Biochemiese, nie-indringende merker wat deur ontwikkelende embrios uitgedruk word, oplosbare menslike leukosiet antigeen-G (sHLA-G), word bespreek. Die waarde van blastosist oordrag en die verbeterde koers van voortgaande swangerskappe in vergelyking met verdelende embrios, is ook uitgelig, gebaseer op „n onlangse metanalise. „n Breedvoerige bespreking van sHLA-G asook “Array-CGH” en die toekoms van hierdie toetse word behandel. HOOFSTUK 3 Die doel van hierdie hoofstuk is om swangerskap en inplantingskoerse te vergelyk wanneer embrios geselekteer word op „n enkel Dag 3 (D 3) morfologie beoordeling, teenoor „n kumulatiewe GES-telling plus sHLA-G uitdrukking. Hierdie was „n prospektiewe ewekansige studie (n=214) waar pasiënte ICSI-siklusse ondergaan het. Embrios is geselekteer vir terugplasing gebaseer op óf Dag 3 morfologie telling (Groep A), óf „n kumulatiewe GES-telling plus sHLA-G uitdrukking (Groep B). Die volgende resultate is gerapporteer: kliniese swangerskappe [35/107 (33%) vs 52/107 (49%)] en voortgaande swangerskappe [20/107 (19%) vs. 52/107 (49%)] se sukses koerse is beduidend verskillend tussen Groep A en Groep B (p<0.05). Inplantingskoerse is nie beduidend verskillend tussen Groep A [52/353 (15%)] en Groep B [73/417 (18%)] (p<0.05) nie. Die aantal swangerskappe wat tot niet gegaan het tydens die eerste trimester was bykans 12 keer hoër in Groep A [25/52 (48%)]. Die slotsom was dat die miskraamsyfer beduidend laer in Groep B as in Groep A is en die swangerskap syfer betekenisvol beter was wanneer die selektering van embrios op GES plus sHLA-G gebaseer is. HOOFSTUK 4 Verskeie studies het „n assosiasie getoon tussen die teenwoordigheid van oplosbare menslike leukosiet antigeen-G (sHLA-G) in menslike embrio kultuur en swangerskaps uitkoms in vitro. „n Retrospektiewe studie is op 2040 ICSI pasiënte by 6 verskillende sentra gedoen om die effek van s-HLAG verder te bestudeer. Alle embrios is individueel gekweek om die teenwoordigheid van sHLA-G in „n kultuurmedium rondom die embrios te identifiseer. In alle sentra is „n positiewe sHLA-G uitslag met „n toename in die waarskynlikheid van veelvuldige inplantings geassosieer (OR: 1.48, 95% CI: 1.07 tot 2.05, p-waarde: 0.0170), asook „n toename in waarskynlikheid van meervoudige swangerskappe wat voortduur (OR: 1.66, 95% CI: 1.10, 2.51, p-waarde: 0.0170). Data uit die multisentriese studie beklemtoon dat sHLA-G uitdrukking „n waardevolle nie-indringende embriomerker is om by te dra tot die verbetering van swangerskapsuitkoms, asook die teoretiese potensiaal om meervoudige swangerskappe te verminder. „n Kombinasie van sHLA-G uitdrukking en verlengde embrio kultuur tot die blastosist stadium mag moontlik „n toekomstige hulpmiddel wees waardeur enkele embrios vir terugplasing geselekteer kan word. Daardeur kan die risiko van meervoudige swangerskappe beperk word sonder om die swangerskapkoerse in gevaar te stel. HOOFSTUK 5 In dié hoofstuk word die ploïdie status van die eerste en tweede poolliggaampies en Dag 3 blastomere, embrio morfologie en biochemiese (sHLA-G) eienskappe gekorrelleer met blastosist ontwikkelling en uiteindelike swangerskapsuitkoms. Alle oösiete/sigote en embrios is individueel tot die blastosist stadium gevolg. PB-I, PB-II en blastomere het “volledige kariotipering ondergaan deur gebruik te maak van die toets “comparative genome hybridization (CGH)”. Elke embrio se kultuurmedium supernatant is versamel en ontleed vir sHLA-G uitdrukking op Dag 2. Die volgende uitslae is gerapporteer: Sewe-en-vyftig mature (MII) donor oösiete is verkry; 33/57 (57.9%) is aneuploïd, 21/57 (36.8%) is euploïd en 3/57 (5%) is onbeslis. Geen verwantskap is gevind tussen CGH status van PB-I, PB-II en die GES-telling. Geen verwantskap is gevind tussen CGH status van sHLA-G. Verder was daar geen verwantskap gevind tussen PB-I CGH uitslae en blastosist morfologie graad nie. Daar was „n beduidende korrelasie tussen PB-I CGH en blastomeer CGH uitslae. Euploïde en aneuploïde PB-I het onderskeidelik in 58% en 67% blastosiste ontwikkel. Daar is „n positiewe verwantskap tussen die ploïdie van PB-I, PB-II en die blastomere aangetoon [Kappa (>0.7)]. Dit is afgelei dat na ICSI en sekwensiële genetiese kariotipering van die oösiet/sigoot en daaropvolgende blastomere, die meerderheid oösiete bevrug is en die daaropvolgende sigote ontwikkel het tot blastosiste, ongeag hul ploïdie status. Ons afleiding is dus dat blastosist ontwikkelling nie aan ploïdie verwant is nie. HOOFSTUK 6 In hierdie hoofstuk bespreek ons waarnemings wat betref seleksie kriteria om die beste embrios te kies vir terugplasing wat uiteindelik tot „n suksesvolle swangeskap sal lei. Morfologiese, biochemiese en genetiese faktore is ondersoek. „n Onderskeiding is gemaak tussen nie-indringende (mikroskopiese en biochemiese) en indringende (embrio biopsie, aCGH) tegnieke. 'n Kombinasie van nie-indringende observasies, wat insluit pronukliere mofologie, vroee sigoot verdeling en vroeë embrio morfologie/kwalitieit op Dag-3 het beperkte inligting verskaf wat betref swangerskapkans. Verlengde embrio kweking tot die blastosist stadium (Dag-5) plus „n positiewe sHLA-G resultaat gee egter veel meer voordelige inligting aan die embrioloog met die embrio seleksie proses, voor embrio terugplasing of bevriesing. Laasgenoemde inligting sal die swangerskap syfer bevoordeel en die meervoudige swangerskap kans verlaag. Wat die indringende tegniek (aCGH) betref, word veel meer data benodig rakend die potensiele voor- en nadele wat betref swangerskap uitkoms, voordat „n sinvolle gevolgtrekking gemaak kan word. HOOFSTUK 7 „n Volledige literatuur oorsig dui daarop dat alle beskikbare riglyne om embrios te kies vir terugplasing, ingespan moet word. In die studie is daar gekyk na „n kombinasie van hierdie voorstelle. Daar is begin met die morfologie van die pronukliere stadium, gevolg deur vroeë sigoot-verdeling, asook beoordeling van embrios se morfologie/kwaliteit op Dag-3 van ontwikkeling. Daar word voorgestel dat die keuse van „n blastosist op Dag 5, gekombineerd met „n positiewe oplosbare menslike leukosiet antigeen G (shla-G) die embrioloog van hulp kan wees om die beste embrio te kies vir terugplasing of bevriesing. Hierdie nie-indringende riglyn behoort swangerskap-uitkoms te verbeter asook meervoudige swangerskappe te verminder. Indringende tegnieke soos ACGH benodig verdere in diepte navorsing en data verkryging om die waarde van hierdie toets te kan beoordeel.

El fracaso de la implantación es una causa importante de infertilidad humana y, en la actualidad, el paso más limitante en las Tecnologías de Reproducción Asistida (del inglés, ART). Puede ser causado por factores maternos y embrionarios, así como por una comunicación defectuosa entre ellos. Debido a limitaciones técnicas y éticas, no es posible estudiar la implantación humana in vivo. El conocimiento sobre la implantación se ha obtenido principalmente a partir de modelos animales que no representan la fisiología del proceso humano. Además, varios estudios in vitro han investigado los mecanismos moleculares subyacentes a la implantación, centrándose principalmente en el embrión o el endometrio de manera unilateral. Muchos autores también han intentado utilizar enfoques indirectos y no invasivos para predecir el éxito de la implantación. A pesar de estos esfuerzos, los determinantes moleculares y ambientales que imposibilitan el éxito del embarazo siguen siendo en gran medida desconocidos. En esta tesis, utilizamos un modelo in vitro para estudiar las interacciones embrión-endometrio durante las primeras etapas de la implantación. En un primer enfoque, nos centramos en las diferentes respuestas transcripcionales del sistema tras la adhesión de acuerdo a la receptividad epitelial (co-cultivo de esferoides trofoblásticos con epitelio endometrial receptivo vs. no receptivo). Los resultados mostraron que el epitelio receptivo es capaz de desencadenar una respuesta transcripcional al contacto con el trofoblasto por el contrario silenciada cuando no es receptivo. Además, caracterizamos la dinámica transcripcional en tiempos más tempranos durante la unión del trofoblasto al epitelio receptivo, tratando de mimetizar el establecimiento exitoso del embarazo. Esto resultó en una serie de cambios dinámicos de expresión génica, caracterizados por una regulación transcripcional positiva temprana y transitoria en el epitelio receptivo, mientras que la respuesta del trofoblasto fue más dinámica. Usando una estrategia de integración in silico, predijimos los pares de proteínas del trofoblasto y del endometrio que interactúan durante estas fases de tiempo y que podrían mediar en la adhesión e invasión temprana durante la implantación. Finalmente, utilizamos un enfoque indirecto para investigar los determinantes ambientales que afectan la implantación mediante la evaluación de la composición de la microbiota vaginal en el día de la transferencia embrionaria y su relación con los resultados reproductivos. Nuestros datos sugirieron que el perfil de microbiota vaginal en la transferencia de embriones no afecta directamente la implantación en mujeres sometidas a fecundación in vitro (del inglés, IVF) con ovocitos donados
Implantation failure is a major cause of human infertility and currently the most limiting step in Assisted Reproductive Technologies (ART). It can be caused by both maternal and embryonic factors, as well as by defective crosstalk between them. Due to technical and ethical limitations, it is not possible to study human implantation in vivo. The knowledge about implantation has been mainly obtained from animal models which fail to represent the physiology of the human process. Additionally, a number of in vitro studies have investigated the molecular mechanisms underlying implantation, mostly focussing on either the embryo or the endometrium in a unilateral manner. Many authors have also tried to use indirect, non-invasive approaches to predict implantation success. Despite these efforts, the molecular and environmental determinants precluding pregnancy success remain largely unknown. In this thesis, we used an in vitro model to study the embryo-endometrium interactions during the early stages of implantation. In a first approach, we focused on the different transcriptional responses of the system upon attachment according to the epithelial receptivity (co-culture of trophoblast spheroids with receptive vs. non-receptive endometrial epithelium). The results showed that the receptive epithelium is able to trigger a transcriptional response to the trophoblast challenge otherwise muted when it is non-receptive. We further characterized the transcriptional dynamics at earlier time points during the attachment of the trophoblast to the receptive epithelium, aiming to mimic the successful establishment of pregnancy. It resulted in a series of dynamic changes in gene expression, characterized by an early and transient transcriptional up-regulation in the receptive epithelium, while the trophoblast response was more dynamic. Using an in silico integrative strategy, we predicted the trophoblast and endometrial protein pairs that interact during these different time points and could mediate attachment and early invasion during implantation. Finally, we used an indirect approach to investigate the environmental determinants influencing implantation by evaluating the vaginal microbiota composition at the day of embryo transfer and its relationship with the reproductive outcomes. Our data suggested that vaginal microbiota profile at the embryo transfer does not directly affect implantation in women undergoing IVF with donated oocytes

Early post-implantation vertebrate embryos are shaped by complex cellular and molecular mechanisms. In mice, the visceral endoderm, an extraembryonic cell lineage that appears before gastrulation, provides several important functions such as nutrition and mechanical protection. My thesis research focused on the role of the visceral endoderm in embryo patterning, a newly discovered function for this tissue. My results showed that an interplay between two subpopulations of visceral endoderm the anterior and posterior visceral endoderm, located on the opposite sides of the developing conceptus, are critical for the establishment of the anteroposterior body axis of the embryo. I also found that senescence-associated β-galactosidase activity delineates the visceral endoderm marking apical vacuole, a lysosomal-like organelle. This however indicates the nutritional function of visceral endoderm cells rather than a senescent population. My studies highlight the fundamental role of extraembryonic tissues in patterning mammalian embryos as opposed to housekeeping roles. They also reveal important difference when conducting studies at the organismal level rather than in cells in culture.

Early post-implantation vertebrate embryos are shaped by complex cellular and molecular mechanisms. In mice, the visceral endoderm, an extraembryonic cell lineage that appears before gastrulation, provides several important functions such as nutrition and mechanical protection. My thesis research focused on the role of the visceral endoderm in embryo patterning, a newly discovered function for this tissue. My results showed that an interplay between two subpopulations of visceral endoderm the anterior and posterior visceral endoderm, located on the opposite sides of the developing conceptus, are critical for the establishment of the anteroposterior body axis of the embryo. I also found that senescence-associated β-galactosidase activity delineates the visceral endoderm marking apical vacuole, a lysosomal-like organelle. This however indicates the nutritional function of visceral endoderm cells rather than a senescent population. My studies highlight the fundamental role of extraembryonic tissues in patterning mammalian embryos as opposed to housekeeping roles. They also reveal important difference when conducting studies at the organismal level rather than in cells in culture.

Dissertação de Mestrado Integrado em Medicina Veterinária
A mortalidade embrionária precoce em humanos, que ocorre frequentemente devido a erros antes, durante ou imediatamente após a implantação, é uma preocupação mundial com grandes implicações sociais e económicas. A via das prostaglandinas (PGs) é uma das vias de sinalização mais estudada com influência nos processos de implantação e de espaçamento dos embriões. A via Wnt canónica desempenha um papel importante em vários eventos do desenvolvimento embrionário e na preparação do útero para a receção do embrião. Desta forma, o objetivo deste trabalho foi inferir sobre uma possível interação entre estas duas vias de sinalização, já reportada noutros sistemas biológicos, e caracterizar o padrão de transcrição dos elementos da via de produção das PGs no embrião murino ao longo do desenvolvimento embrionário pré-implantação. Em relação à transcrição dos genes associados à via das PGs nos embriões, verificou-se um padrão crescente do nível de transcrição da COX-1 ao longo do desenvolvimento embrionário e ausência de transcrição de COX-2 em todos os estadios analisados. Observou-se ainda um aumento significativo (p<0,05) na transcrição da cPGES e mPGES-2 nos blastocistos eclodidos, e da mPGES-1 e PGFS nos blastocistos expandidos. No que diz respeito ao bloqueio da via Wnt pelo Dkk1, um inibidor da via Wnt canónica, não foram registadas diferenças estatisticamente significativas (p>0,05) entre os grupos estudados para nenhum dos genes ou dos estadios de desenvolvimento analisados. O padrão de transcrição dos genes associados à via das PGs no embrião murino foi, tanto quanto sabemos, descrito pela primeira vez neste trabalho, tendo-se concluído que a PGE2 parece ser a PG com maior nível de transcrição nos estadios estudados, indicando a sua importância no desenvolvimento embrionário pré-implantação, no processo de eclosão e na aquisição de competência do embrião para a implantação. Além disso, parece existir nesta espécie uma preferência pela via COX-1/PGES para a síntese de PGE2, tanto no embrião como no endométrio. A análise da transcrição nos locais de implantação, também evidenciou a PGE2 como a PG com maior importância. Por fim, não foi encontrada nenhuma interação entre as vias de sinalização Wnt-PG no desenvolvimento e competência do embrião para a implantação.
ABSTRACT - Wnt-PG pathways communication in the murine embryo development and competence - Early pregnancy loss in humans, which is often due to defects that occur before, during or immediately after implantation, is a worldwide social and economic concern. The prostaglandin (PG) pathway is one of the most extensively studied signal pathways that influence embryo implantation and embryo spacing. The canonical Wnt pathway plays an important role in multiple embryo development events and in uterine decidualization. Therefore, the objective of this work was to evaluate the communication of Wnt and PG pathways, that was already established for other biological systems, and to characterize the transcriptional pattern of PG associated genes in the murine pre-implantation embryo. In this work, we observed a pattern of increasing transcription of COX-1 mRNA throughout pre-implantation embryo development and an absence of COX-2 transcription in all the analyzed embryos. Moreover, there was a significant increase (p<0,05) of cPGES and mPGES-2 transcription in hatched blastocysts, and a significant increase (p<0,05) of mPGES-1 and PGFS in expanded blastocysts. Regarding the blockade of the canonical Wnt pathway with Dkk1, there were no significant differences (p>0,05) for any of the genes in any of the observed embryo developmental stages. To the best of our knowledge, the transcriptional pattern of PG associated genes in the murine pre-implantation embryo was described here for the first time. PGE2 seems to be the major PG involved in mouse pre-implantation embryo development, hatching process and acquisition of competence for implantation. Furthermore, our results suggest a preference for the COX-1/PGES pathway in PGE2 synthesis in both the embryo and uterus. The transcriptional analyses of the implantation sites also revealed PGE2 as the major PG involved in pre-implantation embryo development. Finally, there was no evidence of an interaction between Wnt and PG signaling pathways in the murine embryo development and competence.
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Unsuccessful fertilization, aberrant embryo development, implantation failure and recurrent miscarriages can occur despite any obvious reasons during assisted reproduction. DNA fragmentation and aneuploidy in gametes have been implicated as a possible cause for infertility, but the use of DNA fragmentation tests is controversial. The SCD-FISH test claims to analyse DNA fragmentation and aneuploidy in the same cell. In this thesis SCD-FISH was compared to single FISH and SCD, which showed that SCD-FISH was an unreliable method to study these parameters. Sperm preparation techniques in assisted reproduction technologies (ART) potentially generate exogenous stresses that cause additional DNA damage. This study subjected mature sperm to environmental insults that normally occur during ART, and highlighted the significant increase in sperm DNA fragmentation due to heat, freezing and oxidative stress. Since it is not possible to investigate the level of DNA damage in the egg or sperm, and still maintain its viability for use in fertilization, DNA damage in cumulus and granulosa cells were studied. There was no relationship between DNA fragmentation in these cells and fertilization or pregnancy outcome. DNA fragmentation was significantly higher in cumulus than granulosa cells. Allegedly minimising DNA damage, GM-CSF is a component added to IVF culture media. Its effect on murine embryo DNA fragmentation was studied and it was found to have no significant effect. The exposure of human embryonic stem cells to pre-tested toxins and its impact on DNA fragmentation and aneuploidy, and the correlation between these two parameters were also investigated. These studies emphasise the belief that the introduction of DNA fragmentation assays to the clinical arena is premature as its role is unsubstantiated, the lack of a transparent relationship between DNA fragmentation and pregnancy outcome and the importance of minimising damage to sperm and embryos due to external stresses during laboratory research and ART. Overall, the aim of this thesis was to examine the hypotheses that DNA fragmentation, aneuploidy and phosphatidylserine translocation are potential biomarkers of reproductive potential that exist in variable degrees of at different stages of gametogenesis and preimplantation embryo development, and are susceptible to environmental stresses.

MicroRNAs are small regulatory RNAs that bind to the seeding regions within the 3’-untranslated region (3’-UTR) of their target transcripts to modulate transcript stability and/or inhibit protein translation. MicroRNA Let-7a belonging to the Lethal-7 (Let-7) family is down-regulated at the blastocyst stage, suggesting its suppression is crucial for embryo implantation. Yet, the underlying mechanism on how Let-7a modulates blastocyst implantation remains largely unknown. In silico analysis identified attachment related integrin beta-3, outgrowth related vav3, and the microRNA processing dicer, as Let-7a targets. Therefore, it is hypothesized that down-regulation of Let-7a promotes embryo implantation by stimulating these target proteins. Let-7a is down-regulated during blastulation and at 3-hour post-estradiol activation of the dormant blastocysts. Force-expression of Let-7a in mouse blastocysts suppressed blastocyst attachment, outgrowth on fibronectin-coated plates and compromised pregnancy in vivo. Dual luciferase assay using the 3’-UTR reporter constructs of the integrin beta-3, vav3, and dicer confirmed the interaction between Let-7a and the three targets. Force-expressing or inhibiting Let-7a expression in mouse blastocysts by electroporating the Let-7a precursor or inhibitor respectively illustrated post-transcriptional regulation of Let-7a on integrin beta-3 and vav3, and transcriptional regulation on dicer. Dormant blastocysts retrieved from the delayed implanting mice expressed high Let-7a levels, which was suppressed in the first 12-hours of estradiol activation. Concomitantly, dormant blastocysts expressed low levels of integrin beta-3, vav3, and dicer, yet, their protein expression was up-regulated from 3 hours-post estradiol activation. Furthermore, addition of integrin beta-3 antibody suppressed attachment of trophoblast spheroids (blastocyst surrogate) onto endometrial epithelial cells in a co-culture model and the outgrowth of the spheroids on fibronectin-coated plates. Knockdown of Vav3 with siRNA decreased blastulation, hatching, and outgrowth rates of the embryos in vitro. Although the loss of vav3 activities did not affect embryo implantation, it suppressed trophoblast migration on fibronectin-coated plates and invasion into collagen matrix. In contrast, force-expression of vav3 enhanced blastocyst outgrowth, and promoted cell proliferation. Blocking integrin beta-3 activities in the vav3 knock-down embryos further suppressed blastocyst outgrowth, suggesting the intertwining effect of the integrins and vav3. Dicer knock-down in mouse blastocysts decreased mature Let-7a expression and suppressed blastulation and hatching in vitro and implantation in vivo. Dicer knock-down in estradiol activated mouse blastocysts decreased the epidermal growth factor receptor expression and lowered the affinity of the embryos to EGF, and suppressed the implantation potential to a level similar to that of dormant blastocysts. Taken together, the suppression of Let-7a by estradiol up-regulates integrin beta-3, vav3, and dicer. The increased Itgb3 expression promotes blastocyst attachment and intertwined with the up-regulated vav3 expression to enhance blastocyst outgrowth. The increased vav3 expression further stimulates cell proliferation and confers blastocyst invasion into the collagen matrix. Dicer, by altering microRNA processing, facilitates blastulation and thereby embryo implantation. Thus, the loss of Let-7a biological activities during blastulation is crucial to enhance blastulation and stimulate trophoblast activities for successful embryo implantation.
published_or_final_version
Obstetrics and Gynaecology
Doctoral
Doctor of Philosophy

One of the crucial events during mammalian embryogenesis is the process of implantation. Implantation enables the embryo to invade the uterine endometrium and to gain access to the maternal circulation. The attachment reaction requires a highly coordinated dialogue between the implanting blastocyst and the receptive uterus, known as the embryo-uterine cross-talk. Since the blastocyst expresses multiple Wnt genes, in this study, we have characterized the role and regulation of the Wnt/-catenin pathway in embryo-uterine communication. Using a transgenic mouse that reports Wnt signalling through the canonical pathway, we have demonstrated that the Wnt/-catenin pathway was transiently activated in the regions of the uterine luminal epithelium apposed to the blastocyst at the time of implantation. Activation of this pathway within the endometrium depended on the presence of the blastocyst and required the oestrogen surge. We further demonstrated that activation of the Wnt/-catenin pathway was essential for proper implantation to occur. One possible mechanism that regulates the responsiveness of the luminal epithelium to Wnt is the regulation of components of the Wnt pathway. We show that there is dynamic pattern of expression of the components of the Wnt pathway at the time of implantation. Furthermore, we demonstrate that LIF signalling is required for the expression of a subset of these Wnt components in luminal epithelial cells at the time of implantation. Our results demonstrate that the LIF and Wnt signalling pathway form a network involved in coordinating the process of implantation As Wnt/ß-catenin signalling is essential for embryo attachment, we proposed
Le processus de l'implantation embryonnaire joue un rôle crucial lors du développement des mammifères. L'implantation de l'embryon est un processus extrêmement complexe au cours duquel l'embryon va d'abord s'apposer, puis adhérer à l'endomètre pour ensuite y pénétrer. Ultérieurement, il a été démontré que le blastocyste exprime plusieurs gènes de la famille Wnt. C'est pour cette raison que dans cette thèse, j'ai décidé caractériser le rôle et la régulation de la voie de signalisation Wnt/-catenine lors du dialogue materno-fœtal. Nous avons utilisé une souris transgénique qui permet de contrôler la voie de signalisation Wnt/-catenine. Ainsi, nous avons démontré que la voie de signalisation Wnt/-catenine était activée de façon transitoire dans les cellules épithéliales du lumen de l'utérus, dans la région adjacente au blastocyte au moment de l'implantation. L'activation de cette voie de signalisation dépend de la présence du blastocyste et de la sécrétion d'oestradiol. Nous avons également démontré que l'activation de la voie de signalisation Wnt/-catenine était essentielle au processus d'implantation embryonnaire. Par la suite, nous avons examiné la possible interaction entre la voie de signalisation Wnt et LIF. En effet, dans les souris mutantes pour le gène LIF, la voie de signalisation Wnt/-catenine n'est pas activée. Grâce à la technique de rt-PCR, nous avons caractérisé le profil d'expression des récepteurs Frizzled, des co-récepteurs LRP, des antagonistes sFRP et DKK et des gènes Wnt dans les souris mutantes pour le gène LIF et dans les souris de type sauvage. Nous avons démontré que les composa

L’implantation embryonnaire repose sur une synchronisation spatio-temporelle entre un placenta fonctionnel et un endomètre réceptif. La réceptivité endométriale requiert la différenciation des cellules stromales en cellules déciduales sous l’effet des hormones ovariennes (œstrogènes et progestérone). Le placenta est un organe transitoire constitué de deux types cellulaires. Le cytotrophoblaste villeux est responsable des échanges fœtaux maternels et de la fonction endocrine du placenta. Le cytotrophoblaste extravilleux présente des propriétés invasives et assure ainsi l’ancrage du placenta dans l’endomètre maternel. Un dialogue paracrine complexe entre les cellules placentaires et endométriales s’établit au cours des premières étapes de l’implantation.L’adiponectine est une adipokine produite majoritairement par le tissu adipeux. Elle contrôle le métabolisme glucido-lipidique et joue le rôle d’hormone insulino-sensibilisatrice. Dans de nombreux tissus, l’adiponectine exerce des effets anti-prolifératifs, pro-invasifs et pro-différenciants. L’adiponectine et ses récepteurs ADIPOR1 et ADIPOR2 sont présents à l’interface fœto-maternelle. Le placenta et l’endomètre sont donc des tissus cibles de l’adiponectine.Au cours de ce travail, nous nous sommes intéressés aux effets directs de l’adiponectine à l’interface foeto-maternelle au cours du premier trimestre de grossesse.Dans une première partie, nous avons observé que l’adiponectine exerce des effets anti-différenciants et anti-invasifs dans les cellules stromales endométriales.Dans un second temps, nous avons démontré que l’adiponectine favorise la production de glycogène dans les cellules déciduales. Inversement, l’adiponectine semble limiter l’entrée du glycogène dans les cellules placentaires. Ces résultats démontrent que l’adiponectine pourrait contrôler la nutrition histiotrophe du foetus.Dans une dernière partie, nous avons observé que l’adiponectine diminue l’expression des transporteurs de nutriments et exerce une action pro-apoptotique dans le trophoblaste villeux. Ces derniers résultats pourraient permettre de mieux comprendre le rôle de l’adiponectine dans les pathologies placentaires telles que le retard de croissance intra-utérin qui se caractérise par une diminution du poids foetal et une augmentation de l’apoptose des cellules trophoblastiques.L’ensemble de ces résultats montre que l’adiponectine est un acteur clé du dialogue foeto-maternel au cours de la grossesse précoce en contrôlant la maturation d’un endomètre fonctionnel et en régulant les échanges nutritifs transplacentaires
Embryo implantation requires a spatiotemporal synchronization between a functional placenta and a receptive endometrium. Endometrium receptivity based on the differentiation of stromal cells into decidual cells, under the influence of ovarian hormones (estrogens and progesterone). The placenta is a transient organ composed of two cell types. Villous trophoblast ensures fetal-maternal exchanges and the endocrine functions. Extravillous trophoblast acquire an invasive phenotype resulting in the placenta anchoring in the endometrium. Then, a complexe paracrine dialog between placental cells and endometrial cells is established during the first stages of the embryo implantation.Adiponectin is an adipokine predominantly produced by the adipose tissue. This cytokine has an important role in the control of energy metabolism and displays an insulin-sensitizing action. In some cell types, adiponectin limits proliferation, but promotes invasion and differentiation. Adiponectin and its receptors ADIPOR1 and ADIPOR2, are expressed at the fetal-maternal interface. Thus, endometrium and placenta are adiponectin targets.In this work, we aimed to determine adiponectin direct effects at the human fetal maternal interface during the first trimester of pregnancy.In a first part, we observed that adiponectin limits differentiation and invasion in endometrial stromal cells.In a second part, we showed that adiponectin promotes glycogen production by decidual cells. Conversely, adiponectin seems to limit glycogen uptake by placental cells. These results demonstrate that adiponectin could regulate histiotrophic nutrition to the fetus.In a last part, we demonstrated that adiponectin down-regulates the expression of nutrient transporters and promotes apoptosis in villous trophoblast. These last results could help to better understand the adiponectin roles in some placental pathologies, as intrauterine growth restriction, characterized by a decreased fetal weight and a enhanced trophoblastic apoptosis. Altogether, these results demonstrate that adiponectin is a key regulator of the fetal-maternal dialog by controlling the differentiation of a functional endometrium and by regulating transplacental nutrient exchanges