Primed Extracellular Vesicles as a Nanotherapeutic Strategy to Enhance Granulosa Cell Function in a Model of Premature Ovarian Insufficiency and Hormonal Decline

Premature ovarian insufficiency (POI) is characterized by granulosa cell (GC) dysfunction, reduced steroidogenesis, and hypoestrogenism, often secondary to chemotherapy. Despite advancements in regenerative medicine, cell-free nanotherapies remain underexplored for POI treatment. This study investigates the therapeutic efficacy of primed avian mesenchymal stem cell-derived small extracellular vesicles (primed AMSC-sEVs) in a cyclophosphamide (CTX)-induced GC injury model, mimicking POI-associated hormonal dysfunction. Primed AMSC-sEVs were characterized in terms of particle size, concentration, and zeta potential using the Exoid TRPS and electrophoretic mobility platforms. Functional comparisons between primed and naïve AMSC-sEVs were conducted in human granulosa cells (hGCs), evaluating cell viability, apoptosis, steroidogenesis, and ovarian function-associated gene expression. miRNA content was assessed by next-generation sequencing and qRT-PCR. Primed AMSC-sEVs displayed a smaller, more uniform size distribution, significantly enhanced particle concentration, and greater negative zeta potential (mean -24.4 mV vs -13.3 mV), suggesting improved colloidal stability. In vitro, primed sEVs dose-dependently enhanced hGC proliferation, restored CTX-induced suppression of AMH, FSHR, and LHCGR, and elevated estradiol and progesterone levels. Apoptotic markers (cleaved Caspase-3, BAX, PARP) were reduced, while BCL-2 was upregulated. Key miRNAs involved in steroidogenic and apoptotic regulation (miR-21, miR-22, miR-23b, miR-145, miR-199a) were enriched in primed sEVs. Primed AMSC-sEVs offer a promising nanotherapeutic strategy to restore ovarian cell function in POI-like conditions via enhanced anti-apoptotic and steroidogenic effects. These findings support the translational potential of Primed sEVs for fertility preservation and the management of menopausal hormone deficiency, including early-stage ovarian insufficiency and genitourinary syndrome of menopause.