PROJECT SUMMARY Overall tissue function deteriorates with age, but the female reproductive system is the first to age. Female reproductive aging is characterized by a decline in egg quantity and quality which contributes to miscarriages, infertility, and birth defects. Cessation of reproductive function at menopause also accelerates overall aging because the gonadal hormone, estrogen, regulates numerous tissues (e.g., brain, heart, bone, immune cells, reproductive tract). The consequences of female reproductive aging are significant because women are delaying childbearing, and medical interventions have increased the gap between menopause and lifespan. Thus there is a critical need to discover the molecular mechanisms underpinning female reproductive aging. A hallmark of aging tissues is “inflammaging” or chronic physiologic stimulation of the innate immune system leading to low levels of sterile inflammation with age. The Duncan and Gerton laboratories recently discovered a prominent inflammatory signature in the aging ovary, both within the somatic compartment of the follicle (granulosa cells) and in the stroma or tissue microenvironment. However, the mechanism by which this age-related ovarian inflammation is generated, sustained, and propagated across cell types is not known and must be addressed to advance the field. Our long-term goal is to discover the molecular regulators of female reproductive aging from perspectives of the gamete, follicle, and ovarian microenvironment. Thus, our application is aligned with the NICHD’s Fertility and Infertility Branch high-priority research area of reproductive transitions. The major objective of this grant is to discover signals exchanged between oocytes and their surrounding granulosa cells, and how intercellular communication drives the broader spatiotemporal pattern of ovarian aging. Our overarching hypothesis is that, with advanced reproductive age, cytosolic DNA originating from loss of genomic stability in the oocyte stimulates the innate immune response and inflammatory pathways in ovarian granulosa cells which are then further amplified by the tissue microenvironment. Central to our model is the cGAS-STING pathway which links genomic instability and inflammatory responses across cells within a tissue. This pathway has never been examined in the ovary, nor within the context of ovarian aging, but our preliminary data strongly support a fundamental role. To address our overarching hypothesis, we will identify age-associated genomic instability signatures in the mouse oocyte that serve as trigger signals (Aim 1). We will then determine how granulosa cells integrate oocyte-derived signals to initiate an age-associated innate immune response (Aim 2). Finally, we will discover how the spatio-temporal architecture of ovarian fibrosis and inflammaging govern the follicular response and vice versa through spatial transcriptomics (Aim 3). These aims will provide a comprehensive and integrated molecular mechanism of inflammaging at high spatial temporal resolution that considers the gamete, follicle, and the ovary. The positive impact will be the discovery of novel molecular pathways that could be targeted in cell type-specific manners to improve gamete quality, reproductive longevity, and healthspan.

PROJECT NARRATIVE The proposed research addresses the molecular origins of female reproductive aging, which impacts fertility, endocrine function, and overall health. We will test the hypothesis that ovarian aging is fueled by damaged DNA in the oocyte which stimulates an inflammatory response in the surrounding somatic follicular cells that is further amplified by spatial cues within the broader microenvironment. This project provides a novel paradigm for female reproductive aging, a major reproductive transition, and thus aligns with NICHD priorities.