ABSTRACT Background. Sex-based differences, largely controlled by sex hormones, affect the natural and treated history of HIV infection and HIV-specific immune responses. Our previous work has shown that estrogen potently represses HIV transcription, thus decreasing cellular HIV RNA in women compared to men. Unexpectedly, women undergoing reproductive aging have a progressive increase in levels of inducible HIV reservoir, while estrogen declines. This observed expansion of the reservoir as women age is in sharp contrast to the steady decline in the reservoir size observed in men. Given the increasing number of women aging with HIV, it is critical to determine the interplay of HIV persistence and declining sex hormones during reproductive aging to design effective HIV cure strategies. Our goal. Our study is specifically designed with samples from both cisgender men and women across the reproductive aging spectrum. We will first define the impact of reproductive aging on multiple features of the reservoir including size, transcriptional activity, along with a novel exploration of clonal expansion (Aim 1). Next, we will precisely define the immunologic changes over the course of reproductive aging, using single cell sequencing combined with immunophenotyping using DNA-barcoded antibodies (Aim 2). The data will be integrated, and key features established using advanced statistical analyses. Study Cohort: Longitudinal samples (viable cells, plasma), collected, processed, and stored using standardized protocols in the multi-site Women's Interagency HIV Study (WIHS) and Multicenter AIDS Cohort Study (MACS) cohorts will be utilized for all proposed experiments. First, we will carefully select 25 cisgender WWH on suppressive combination antiretroviral therapy (ART) with biologic samples representing four reproductive stages (reproductive/pre-menopause, menopausal transition/early and late perimenopause, post-menopause). Then, we will identify 25 MWH as controls using a multivariate propensity score based matching algorithm. After matching, stored samples collected between 2009 and 2019 will be selected based on similar time-intervals across both groups. How will we advance the field? To date, the majority of HIV cure research has used male participants and therefore a significant knowledge gap exists between men and women. We do not know if the same immune- modulatory interventions will be effective in promoting HIV RNA transcription in men and women and how declining sex hormones will impact their efficacy. Agents that are designed for “kick and kill” strategies may be impacted by estradiol-mediated mechanisms as women undergo reproductive aging. A better understanding of these differences will assist in the design of future cure approaches that can be applied across sexes.