Non-Invasive Molecular Assessment to Determine Embryonic Viability and Reproductive Success

The success of human IVF is essentially determined by the quality of the embryo (itself a function of the quality of the oocyte and sperm), the endometrium and of the interaction between them. While there are established means of establishing the quality of each individually, some of these are limited and, in particular, the embryo-endometrial dialogue is difficult to capture and observe. As a result, it is under-studied and, with this in mind, the aims of this thesis were threefold:

First, to investigate the corona cell transcriptome of euploid oocytes, RNA sequencing of corona cells from individual cumulus oocyte complexes that developed into euploid blastocysts was employed. Alongside bioinformatic and statistical analysis to compare IVF outcomes, a mean number of sequence reads of 21.2 million were produced. Differentially expressed gene analysis revealed 343 statistically significant transcripts and enriched pathway analysis showed WNT signaling, MAPK signaling, focal adhesion and TCA cycle to be related to IVF outcome. Specifically, key genes within the WNT/beta-catenin signaling pathway, including AXIN, were associated with oocyte competence. Furthermore, key genes and signaling pathways were identified in corona cell profiles in association with IVF outcome following the transfer of euploid blastocysts previously vitrified in a frozen embryo transfer.

A second study investigated an association between advanced maternal age (AMA) and endometriosis on the embryo-endometrial molecular dialogue before implantation. Co-culture experiments were performed with endometrial epithelial cells (EEC) and cryopreserved day 5 blastocysts from AMA or endometriosis patients. Extracellular vesicles isolated from the co-culture supernatant were analyzed for miRNA expression and revealed significant alterations correlating to AMA or endometriosis. Functional annotation analysis of miRNA-target genes revealed enriched pathways for both infertility etiologies, including disrupted cell cycle regulation and proliferation. These extracellular vesicle-bound secreted miRNAs are key transcriptional regulators in embryo-endometrial dialogue.

The third study was concerned with the placental epigenome, which plays a critical role in regulating mammalian growth and development. Alterations to placental methylation, often observed at imprinted genes, can lead to adverse pregnancy complications such as intrauterine growth restriction and preterm birth. Similar associations have been observed in offspring derived from advanced paternal age fathers. As parental age at time of conception continues to rise, the impact of advanced paternal age on these reproductive outcomes is a growing concern, but limited information is available on the molecular mechanisms affected in utero. This longitudinal murine research study investigated the impact of paternal aging on genomic imprinting. Paternal age significantly impacted embryonic placental weight, fetal weight and length, significant hypermethylation was observed upon natural paternal aging and several transcript level alterations attributable to advanced paternal age were identified.

This thesis also presents a series of co-authored studies on related subjects such as embryonic epigenetic dysregulation, unexplained male factor and the sperm epigenome, the dynamic transcriptome of IVF blastocysts in association with infertility, redox balance and aging-related changes in the mouse ovary/oocyte, obesity and the human sperm proteome.

Collectively, these data could inform the development novel biomarkers for non-invasive assessment of oocyte competence and implantation success. They also demonstrate a paternal age effect with dysregulation at numerous imprinted loci, providing a mechanism for future adverse placental and offspring health conditions.