PROJECT SUMMARY/ABSTRACT Premature ovarian insufficiency (POI) is characterized by a decline in ovarian function resulting in infertility and reduced ovarian hormone production. Globally, the rate of POI is approximately 3.5% resulting in ~138,250,000 women experiencing subfertility or infertility. Consequently, fertility preservation and restoration are a priority among patients with POI and individuals who desire children later in their reproductive lifespan. However, many limitations to the current fertility preservation and restoration options result in short hormonal functionality of transplanted ovarian tissue, low live birth rates, and low numbers of good-quality oocytes for in vitro maturation. Therefore, the development of novel technologies and methodologies for the advancement of reproductive science is imperative. Our main goal through this work is to elucidate matrisome-dependent mechanisms that contribute to folliculogenesis while also designing a defined biomaterial that improves follicle growth and oocyte quality in vitro. We will accomplish this through the interrogation of Elastin Microfibril Interface-located protein 1’s (EMILIN1) role in folliculogenesis (Aim 1) and by engineering a biomaterial that increases follicle growth and oocyte quality (Aim 2). Our preliminary studies demonstrate our ability to investigate matrisome-dependent cues in vitro using decellularized extracellular matrix (dECM) hydrogels derived from bovine ovarian tissue. We have shown that hydrogels with a high quantity of EMILIN1 increase murine follicle diameter in vitro compared to hydrogels with a low quantity of EMILIN1. This effect can be rescued with the addition of rhEMILIN1. Importantly, this effect can only be observed when follicles are cultured in dECM and EMILIN1 had no effect on growth when added to a biologically inert hydrogel, alginate. Therefore, we will elucidate the mechanism by which EMILIN1 increases follicle growth by performing bulk-RNA sequencing on murine follicles grown in the presence or absence of EMILIN1 in dECM and interrogating the signaling pathways associated with EMILIN1. Then, we will engineer a hydrogel of defined matrisome composition to recapitulate the follicle growth dynamics observed in dECM hydrogels with an abundance of EMILIN1. To do this, we will perform a co-immunoprecipitation (co-IP) and subsequent unbiased proteomics to define matrisome proteins that EMILIN1 associates with. We will then use this list of proteins to design an ECM hydrogel for ovarian follicle culture that increases follicle growth and oocyte quality via an EMILIN1-driven mechanism. Taken together, these aims will expand our understanding of the role of EMILIN1 in folliculogenesis, advise future work in designing advanced biomaterials for follicle culture, and lead to future fertility preservation and restoration options. Finally, this research will be influential to the greater scientific community by expanding our technical repertoire for s...