Doctor of Philosophy
Physiology and Pharmacology
Betts, Dean H.
Watson, Andrew J.
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.
Summary for Lay Audience
For couples undergoing fertility treatment that require in vitro fertilization, in order to improve the likelihood of having a healthy newborn, a single early embryo must first be chosen several days after fertilization to be transferred back to the recipient. To make this selection, embryos are evaluated in the clinic by a number of methods – often based solely on their appearance – which are currently insufficient to identify an embryo that will develop into a healthy newborn with a high degree of certainty. In this thesis, in order to improve this selection process, small RNA sequences known as microRNAs that are left behind by the developing embryo in the surrounding culture media were studied and used to gain insights into the embryo’s health. To do this, early mouse embryos were cultured in vitro and these microRNAs were allowed to accumulate in the surrounding culture media. By studying this media afterwards, several dozen microRNA sequences were discovered and, by investigating a subset of these further, it was realized the amounts of these microRNAs are consistent with the amounts in the embryo itself. This is diagnostically important as it means that if any of these are altered as a result of poor embryonic health, this alteration may be detected non-invasively within the media as well. Next, we compared the microRNAs derived from poor quality mouse embryos to those released by healthy embryos and noticed that failing mouse embryos were releasing greater amounts of microRNAs. This is diagnostically important as well as it suggests embryos with greater amounts of microRNAs left behind in their microdroplets may be less inclined to develop successfully into a healthy newborn. Finally, we applied these new insights to the study of human embryo media kindly made available to us by our local fertility clinic and similarly saw the same association between elevated microRNAs and poor quality embryos. Excitingly, these results presented in this thesis indicate microRNAs may be a valuable, non-invasive means of selecting healthy embryos to improve the likelihood of couples conceiving a healthy newborn.
Hawke, David C., "MicroRNAs as Non-invasive Predictors of Preimplantation Embryo Developmental Competence" (2022). Electronic Thesis and Dissertation Repository. 9087.
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Available for download on Wednesday, January 01, 2025