This project is designed to obtain the first comprehensive assessment of epigenetic defects (epimutations) induced by the use of assisted reproductive technologies (ART) in the offspring produced. The genome-wide occurrence of abnormalities in DNA methylation patterns and gene expression patterns will be assessed in mice produced by intracytoplasmic sperm injection (ICSI) using cutting-edge, high-throughput epigenomic and genomic technologies. In addition, the etiology of ART-induced epimutations will be investigated by determining the precise timing of appearance of epimutations in mice produced by ART, as well as by discerning the relative contribution of each of three specific aspects of the ART process - gonadotropin stimulation of folliculogenesis, prolonged culture of preimplantation embryos, and embryo transfer - to the induction of epimutations. Finally, the fate of ART-induced epimutations will be examined to determine if these are consistently corrected by epigenetic reprogramming during either embryogenesis or gametogenesis. The significance of the research proposed in this application is that it will include high (single-base) resolution, comprehensive analyses of the disruptive effects of ART on epigenetic programming genome-wide, and it will facilitate controlled, prospective studies of the genesis, etiology, extent and fte of epimutations induced by ART, including parallel, simultaneous studies of the relative contribution(s) of multiple different aspects of the ART process to the induction of epimutations in the offspring produced. The proposed research is innovative and timely because we are now able to generate datasets of unprecedented scale and resolution describing abnormal epigenetic programming and gene expression in ART offspring, and we can use these to understand the genesis, consequences and fate of ART-induced epimutations in ways that will have significant implications for basic biology and human health. The resulting enhanced understanding of the etiology of ART-induced epimutations will inform future modifications of clinical ART protocols to minimize the induction of epimutations in ART-derived human offspring and thereby reduce the occurrence of epigenetic disorders in individuals produced by this technology.
Multiple studies have shown an association between assisted reproductive technology (ART) methodologies and disruption of epigenetic programming in the embryos or offspring produced, however, the scope of abnormalities examined in these studies has typically been quite limited, being focused on small numbers of exemplary gene loci (e.g. specific imprinted genes), or on very specific abnormal phenotypes (e.g. imprinting disorders), or on a single potential contributing factor in any one study. Thus, to date, we have only examined 'the tip of the iceberg'of potential epigenetic defects in ART offspring. We will employ recently developed, powerful epigenomics methods that will allow us to perform the first high-resolution, genome-wide assessments of the extent of disruptions of the epigenome induced by ART, which will, in turn, allow us to answer questions that have not previously been addressable regarding the genesis, etiology, extent and fate of epimutations induced by ART.