Cellular and molecular functions in hormonally-controlled tissues like the uterus are coordinately controlled by both the endocrine system and the immune system. Immune cells of the female reproductive tract (FRT) must maintain a microenvironment that is permissible to fertilization but resistant to foreign pathogens and infection, resulting in a complex milieu of cell types and states. Cyclic changes in steroid sex hormone levels within the FRT across the menstrual cycle, coupled with heterogeneous physical compartments, necessitate a systems-level approach to evaluating the fates and functions of these cells. To date, epithelial and stromal responses to hormones in reproductive tissues like the uterus have been well-characterized, but there is a gap in our understanding of how immune cells are impacted, including macrophages (MØs). MØs perform diverse functions in immune defense and tissue maintenance. These cells are also highly responsive to their environment, and rapidly adapt to shifting tissue milieus. In this regard, MØs are important in the pathology of endometriosis, a prevalent, painful, and hormone-dependent uterine disease in which endometrial-like tissue grows outside of the uterus. The long-term goal of the Research Project Leader’s lab is to delineate the role of sex hormones in modulating MØ functions to better understand their role in health and disease. This project will specifically address this outstanding knowledge gap by employing a systems-biology approach to characterize the impact of sex hormones on macrophage fates and functions. First, the team will deeply profile how sex hormones impact MØ differentiation and response to immune stimuli (Aim 1). They will then use longitudinal single-cell RNA-sequencing (scRNA-seq) and assay for transposase-accessible chromatin followed by sequencing (scATAC-seq) to fully map the transcriptional and regulatory impact of sex hormones on MØ activation and differentiation. Second, the team will use spatial transcriptomics to identify ligands and receptors that drive key MØ interactions in healthy and endometriotic tissues (Aim 2). This proposed Research Project will advance the Leader’s research goals and independent career significantly by allowing her to generate foundational and comprehensive datasets to determine how sex hormones alter MØ differentiation and activation. This COBRE and its Single-Cell Genomics Core and Genomic Data Science Core will allow the Project Leader to begin building a systems-level understanding of the cellular programs that are modified by sex hormones to drive specialized MØ fates and functions. This research has practical implications for understanding the diverse roles of MØ populations in hormonally-controlled tissues in pregnancy, menstruation, and disease and will enable approaches for designing macrophage-targeted therapeutics for reproductive tissue diseases.