Abstract: Cancer therapy-induced ovarian insufficiency is a major public health problem affecting millions of women over their reproductive life span. Our overarching aim is to understand and prevent the damage caused by cancer treatments and to extend that knowledge to prevent ovarian aging. In the prior period, we successfully achieved all specific aims and showed: i) Breast cancer chemotherapy but not tamoxifen alone induces a significant decline of ovarian reserve; ii) Women with BRCA mutations have fewer primordial follicles in their ovaries, which accumulate more DNA damage with age compared to controls, and, iii) they experience greater ovarian reserve loss after chemotherapy; iv) Selective knock-down of BRCA1 renders oocytes more liable to chemotherapy-induced death; v) Gonadotoxic chemotherapy causes primordial follicle death by inducing DNA double strand breaks (DSBs), which prompts apoptotic follicle death in primordial follicles but not by activation of their growth in the acute phase. In addition, vi) A ceramide-induced death pathway inhibitor Sphingosine-1- Phosphate (S1P) prevents chemotherapy-induced human ovarian follicle death, likely by enhancing the ATM- Pathway. Given the shared mechanisms through DNA damage and repair, these findings opened new avenues in the prevention of chemotherapy-induced damage to gonadal function, and, to reverse, retard or prevent reproductive aging. In the current renewal, we aim to further these studies with the following translational aims: 1. To determine whether the mutations in BRCA1 vs. BRCA2 or other key members of the ATM-mediated DNA DSB Repair Pathway result in the increased susceptibility to chemotherapy-induced ovarian reserve loss and oocyte death in women with breast cancer; 2. To determine differences in the molecular mechanisms of acute and delayed chemotherapy-induced human primordial follicle loss; 3. To test the efficacy and mechanism of an FDA-approved S1P analogue, FTY-720, in the prevention of chemotherapy-induced human ovarian reserve loss. To achieve these translational aims we will combine clinical studies with studies of basic, translational mechanisms using in vivo human ovarian xenograft models, laser-captured individual-oocyte RNA sequencing and single-cell quantitative real-time PCR approaches which evolved in our laboratory during the past funding periods. These translational studies with human material will be mechanistically strengthened with an in vitro mouse oocyte gene-interference and genotoxicity assay (GTA), which we also developed and validated in the past funding periods. This translational proposal will not only expand knowledge of the mechanisms and prevention of chemotherapy- induced ovarian damage but also result in a paradigm shift in our understanding of how ovarian germ cells mitigate genotoxic stress in general. The exploitation of such mechanisms, in addition to leading to new fertility preservation strategies, will provide new insights into future ...