Revealing the immune landscape of menstrual blood: unlocking insights into activation, exhaustion, and mitochondrial mass for reproductive health

Reproductive disorders such as endometriosis and polycystic ovary syndrome (PCOS) are increasingly recognized as immune-mediated conditions, yet their immunopathology remains poorly understood. Menstrual blood, a noninvasive and biologically relevant sample, offers a unique window into reproductive tract immunity but has been underutilized in this context. We optimized Cytek's® 25-color high-dimensional flow cytometry panel by incorporating a mitochondrial dye to investigate immune cell profiles in menstrual mononuclear cells (MMCs) from healthy individuals, and those with endometriosis or PCOS, in comparison with matched peripheral blood mononuclear cells (PBMCs). This enabled detailed assessment of 40 immune cell subsets and 546 immunological parameters, including markers of activation, exhaustion, migration, and mitochondrial content. MMCs displayed a distinct immune landscape compared to PBMCs, enriched with tissue-resident NK cells, macrophages, and dendritic cells, alongside changes in mitochondrial mass for various cell subsets and other markers such as PD-1. These findings support a metabolically active, tissue-adapted immune environment within menstrual fluid, representative of the reproductive tract. Exploratory analyses of MMCs from individuals with endometriosis or PCOS revealed disease-specific trends: for example, mitochondrial mass differed across Tregs, CD4 central memory cells, plasmablasts, and cDC1s, with endometriosis and PCOS exhibiting distinct patterns rather than a uniform "reproductive disorder" phenotype. Although these disease-associated findings did not consistently reach statistical significance due to the small cohort size, they demonstrate the potential of menstrual blood immunoprofiling to uncover biologically meaningful differences across diverse immune cell populations. Together, this study establishes menstrual fluid as a valuable, non-invasive sample for immunological assessment and a promising avenue for future biomarker discovery in reproductive disorders.