PROJECT SUMMARY Fertility preservation is a significant concern of pediatric cancer patients undergoing gonadotoxic therapies. The rate of premature menopause is significantly increased in childhood cancer survivors when compared to their siblings without a history of cancer, and this has important implications for quality of life as well as overall health. With increased awareness of oncofertility, more patients are seeking fertility preservation as part of their cancer care. Currently, the only available option for fertility preservation for female pre-pubertal patients is ovarian tissue cryopreservation (OTC), yet OTC has been optimized for adult ovaries. Our main goal is to elucidate mechanisms that lead to the improvement and expansion of fertility preservation options for pediatric cancer survivors. Ex vivo in vitro maturation studies have shown that follicles isolated and matured from pre- pubertal ovaries exhibit reduced developmental competence than their post-pubertal counterparts. This suggests that progression through puberty is important in determining reproductive potential. Previous work has elucidated the contribution of the hormonal environment in puberty, however, microenvironment changes occurring with puberty have yet to be defined. Our preliminary studies demonstrate that pre-pubertal ovaries display fundamental differences when compared to post-pubertal ovaries. In terms of structure, pre-pubertal ovaries are less likely to display compartmentalization, which may explain recent observations of primordial follicles present in tissue fragments discarded from pediatric OTC. Stromal cell populations may also be influenced by the pubertal transition, as specific cellular populations are recruited by late stage follicles in post-pubertal, cycling ovaries. In mice, this heterogenous and incompletely characterized population has been shown to impact follicle growth and maturation by molecular factors that remain undefined. These observations lead to my hypothesis that structural, molecular, and cellular changes in the pediatric ovarian microenvironment across the human pubertal transition contributes to reproductive potential. This hypothesis will be tested across three aims. In aim 1, I will establish the progression of structural compartmentalization of the ovary across human puberty using analysis of donated punch biopsies. In aim 2, I will use a multi-omics approach to establish a spatially resolved, single cell atlas of the pediatric ovary. In aim 3, I will elucidate contributions of stromal cell paracrine signaling to follicle maturation using an in vitro co- culture system. Taken together, these aims will provide foundational knowledge regarding the biology of the pediatric ovarian microenvironment, informing clinical practice and future work that maximizes fertility preservation options for pediatric cancer survivors.