Doctor of Philosophy
Dr. Mellissa Mann
Infertile couples worldwide use assisted reproductive technologies (ARTs) to help conceive their own biological child. Due to the rising use of ARTs, there is continual emergence of new techniques implemented in human fertility clinics. When treatment is successful, there is an increased risk even within singletons for perinatal complications including preterm birth, intrauterine growth restriction, low and high birth weight and genomic imprinting disorders Beckwith Wiedemann Syndrome, Angelman Syndrome, and Silver-Russel Syndrome. Consequently, there is a need to investigate the effects of these treatments on the manipulated oocyte and preimplantation embryo. To address this, I first analyzed the combined effects of multiple ARTs on imprinted DNA methylation in human day 3 (6 to 8 cells) and blastocyst-stage embryos. As imprinted DNA methylation is acquired during gametogenesis and maintained throughout preimplantation development, I hypothesized that ARTs disrupt this regulation in donated, good quality, human preimplantation embryos. I observed that seventy-six percent of day 3 embryos and fifty percent of blastocysts exhibited perturbed imprinted methylation at the SNRPN, KCNQ1OT1 and/or H19 domains. This frequency was similar to that previously observed in the mouse, and importantly demonstrated that extended culture did not pose a greater risk for imprinting errors. Overall, human preimplantation embryos generated with ARTs possessed a high frequency of imprinted methylation errors. Next, I hypothesized that a single, indispensible ART treatment, ovarian stimulation, disrupts mitochondria in mouse oocytes and preimplantation embryos. Ovarian stimulation led to a decreased total and active mitochondrial pool in high hormone-treated oocytes, and an increase in the percentage of oocytes displaying mislocalization of active mitochondria. Although the total mitochondrial pool was unchanged in hormone-treated preimplantation embryos compared to controls, the active mitochondrial pool was decreased in hormone-treated 1-cell, 2-cell, morula and blastocysts. Ultimately, the lower active mitochondrial pool in treated embryos was associated with a decreased percentage of outer blastomeres containing high amounts of active mitochondria in morula and blastocysts. In blastocysts, this was associated with increased superoxide levels. Overall, my results provide novel insight onto ARTs-induced disruption of imprinted DNA methylation and mitochondria in human and mouse preimplantation embryos, respectively.
White, Carlee R., "Assisted Reproductive Technologies Disrupt Genomic Imprinting in Human and Mitochondria in Mouse Embryos" (2016). Electronic Thesis and Dissertation Repository. 3921.
Available for download on Wednesday, August 08, 2018