MicroRNAs as Non-invasive Predictors of Preimplantation Embryo Developmental Competence
Abstract
Elective single embryo transfer (eSET) has become the new standard of care for good prognosis patients undergoing in vitro fertilization owing to the avoidance of multiples pregnancies and associated complications. As such, clinical pregnancy outcomes have become dependent on the selection of a single viable human blastocyst for transfer however the diagnostic techniques used to make this selection continue to yield marginal pregnancy rates, creating a need for new, non-invasive methods. During in vitro culture, developing preimplantation embryos release factors into the surrounding culture media including short non-coding RNA sequences known as microRNAs. Extracellular microRNAs are considered ideal biomarkers of cell state owing to their accessibility, high stability, extensive variety, ubiquitous expression and ease of detection. Within this thesis, embryo-derived microRNAs are explored for their links to major clinically relevant factors indicative of embryo developmental competence in order to assess their diagnostic feasibility. Using a diverse panel of microRNAs, a highly concordant extracellular microRNA signature was identified in microdroplets conditioned with murine preimplantation embryos and, as a simple proof-of-concept, used to distinguish between different stages of developing murine preimplantation embryos. Further investigation suggested embryo degeneration intensified the media signature and its ablation by centrifugal force indicated microRNA encapsulation within extracellular vesicles was likely. Translation of these results to clinical spent human blastocyst media revealed similar correlation of homologous extracellular microRNAs with blastocyst morphological grade. Of high diagnostic significance, low-grade grade Day 6 human blastocysts exhibited a more pronounced media signature than mid- and high-grade blastocysts. Further investigation of these extracellular miRNAs in fractionated media conditioned with naïve-like H9 hESCs suggested these microRNAs are encapsulated primarily within dead cells and cell debris, rather than the small extracellular vesicle fraction, such as exosomes, suggesting a common release mechanism such as cell death or apoptosis. Together, this thesis supports the use of embryo-derived microRNAs as a means of assessing embryonic health and enabling development of a non-invasive viability diagnostic tool for clinical use.