Human Umbilical Cord Mesenchymal Stem Cell–Derived Extracellular Vesicles Attenuate Oxidative Stress in an In Vitro Ovarian Aging Model

Ovarian aging is characterized by progressive granulosa cell dysfunction driven by oxidative stress, leading to impaired follicular development and reduced fertility. Human umbilical cord mesenchymal stem cell–derived extracellular vesicles (UC-MSC-EVs) have emerged as promising cell-free therapeutic candidates capable of mitigating oxidative damage through their anti-apoptotic and anti-senescence properties. The present study aimed to evaluate the protective effects of UC-MSC-EVs in an in vitro model of granulosa cell aging induced by hydrogen peroxide (H₂O₂).

Human granulosa cells (HUGC) were exposed to escalating concentrations of H₂O₂ (25–200 µM) for 24 h and 48 h to establish oxidative stress–induced aging. Cells were treated concurrently with UC-MSC-EVs at concentrations ranging from 2 µg/mL to 20 µg/mL. Exosomes were isolated and characterized using nanoparticle tracking analysis (NTA), which revealed a yield of approximately 4.2 × 10¹⁰ EVs/mL. Morphological assessment, cell proliferation, viability, and senescence staining assays were performed to evaluate the cytoprotective effects of EVs across treatment groups. The relative gene expression was performed by Q.RT-PCR and the protein expression was analyzed by western blot for oxidative stress and senescence markers. Annexin V staining and TUNEL assay were performed using flow cytometry performed and immunofluorescence to differentiate between apoptosis and senescence. Mitophagy and autophagy analysis were also included to assess the role of mitochondrial dynamics.

Exposure to H₂O₂ resulted in marked morphological degeneration, decreased cell viability, and increased senescence. Co-treatment with UC-MSC-EVs restored normal cellular morphology and significantly improved viability compared to cells treated with H₂O₂ alone. EV treatment reduced senescent cell counts and preserved proliferative morphology in a dose-dependent manner, with the most pronounced protection observed at 20 µg/mL (p<0.01). Furthermore, EV-treated cells exhibited enhanced survival compared with untreated controls, suggesting a regenerative effect beyond baseline. The mitochondrial regeneration was evident in UC-MSC-EV treated groups.

UC-MSC-EVs effectively protect granulosa cells from oxidative stress–induced apoptosis and senescence, highlighting their therapeutic potential as a cell-free strategy to counter ovarian aging and improve fertility outcomes. Future work will extend these findings in vivo using aging mouse models and engineered EVs for enhanced functional delivery.