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

Abstract

Background: 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. Purpose: 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. Methods: 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. Results: 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. Conclusion: 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.