Pyroptotic Macrophage Extracellular Vesicles Drive miR-103a-3p Mediated Mitochondrial Dysfunction in Granulosa Cells in Diminished Ovarian Reserve.

To define the role of pyroptotic macrophage-derived extracellular vesicles (EVs) in diminished ovarian reserve (DOR) and to elucidate whether EVs-mediated transfer of miR-103a-3p drives mitochondrial dysfunction in granulosa cells. Single-cell RNA sequencing data from public GEO datasets were analyzed to characterize pyroptosis-associated features in ovarian macrophages across age groups. EVs were isolated from pyroptotic macrophages and applied to granulosa cells (KGN) to assess mitochondrial function, oxidative stress, and apoptosis. miRNA sequencing was performed to identify EVs-enriched miRNAs. miR-103a-3p expression was further evaluated in follicular fluid EVs from DOR patients and correlated with ovarian reserve markers. Mechanistic studies were conducted using in vitro assays and an in vivo mouse model. Pyroptosis signatures were enriched in ovarian macrophages with aging. EVs from pyroptotic macrophages were enriched in miR-103a-3p and induced mitochondrial dysfunction in granulosa cells, characterized by reduced ATP, increased ROS, and enhanced apoptosis; these effects were alleviated by miR-103a-3p inhibition. Clinically, miR-103a-3p was elevated in follicular fluid EVs from DOR patients and negatively correlated with AMH and AFC. Mechanistically, miR-103a-3p targeted MFN2 and BCL2L13, disrupting mitochondrial homeostasis in KGN cells. Restoration of these targets mitigated dysfunction in KGN cells. In vivo, AAV-mediated inhibition of miR-103a-3p improved follicular development and ovarian function. Pyroptotic macrophages may contribute to DOR progression through EVs-mediated delivery of miR-103a-3p, which impairs mitochondrial homeostasis in granulosa cells by targeting MFN2 and BCL2L13.