BMSC-derived exosomal rno-miR-361-3p attenuates cyclophosphamide-induced azoospermia by suppressing STAT3-mediated spermatogonial apoptosis

Azoospermia, particularly non-obstructive azoospermia (NOA), represents a severe form of male infertility with limited therapeutic options. Increasing evidence suggests that mesenchymal stem cell–derived exosomes exert beneficial effects in male reproductive disorders; however, the underlying molecular mechanisms remain unclear. In this study, a cyclophosphamide (CTX)-induced azoospermia rat model was established to investigate the therapeutic potential and mechanism of bone marrow–derived mesenchymal stem cell exosomes (BMSCs-Exos). Systemic administration of BMSCs-Exos significantly improved sperm quality and restored sperm production in CTX-treated rats. To elucidate the molecular basis of this effect, RNA sequencing was performed on testicular tissues from normal controls (Control), CTX-treated rats (CTX), and CTX-treated rats receiving exosome intervention (CTX + BMSCs-Exos). Comparative transcriptomic analyses revealed that multiple apoptosis-related genes were markedly upregulated following CTX exposure but significantly downregulated after BMSCs-Exos treatment. Further analysis identified these genes as transcriptional targets of STAT3, whose expression was also suppressed in the CTX + BMSCs-Exos group. Mechanistically, BMSCs-Exos were found to contain rno-miR-361-3p, which directly targets the 3′-untranslated region (3′-UTR) of STAT3 . Upon exosome uptake by CTX-injured spermatogonial cells, exosomal rno-miR-361-3p inhibited STAT3 expression, thereby attenuating STAT3-mediated transcriptional activation of pro-apoptotic genes, including IFIT2 , CYBB , OAS1I , PLA2G4A , CTSS , FGR , FCGR2B , and MMP2 . Consequently, CTX-induced spermatogonial apoptosis was reduced, leading to enhanced spermatogenic recovery and increased sperm output. Collectively, these findings uncover a previously unrecognized BMSCs-Exos/rno-miR-361-3p/STAT3 signaling axis that mediates protection against chemotherapy-induced spermatogenic failure and highlight BMSC-derived exosomes as a promising cell-free therapeutic strategy for azoospermia.