Targeted activation of Nrf2 via sulforaphane-loaded exosomes attenuated azoospermic condition in the rat model

Azoospermia, the complete absence of sperm in the ejaculate, presents a major barrier to male fertility. Oxidative stress and impaired cellular homeostasis are key contributors to germ cell loss, particularly in chemotherapy-induced azoospermia. Sulforaphane (SFN), a potent activator of the Nrf2 pathway, offers antioxidant benefits, but its systemic delivery is limited by bioavailability and potential reductive stress. This study aimed to evaluate the regenerative potential of SFN-loaded exosomes (SFN + EXO) in a rat model of azoospermia. Human serum-derived exosomes were isolated, characterized and engineered to encapsulate SFN. Azoospermia was induced in Wistar rats via intratesticular busulfan injection. Animals were assigned to five groups: healthy control, azoospermic control, SFN, exosomes (EXO) and SFN + EXO. Spermatogenesis parameters, histopathology, testosterone levels, oxidative stress markers and gene expression of Nrf2, autophagy and germ cell markers were evaluated. SFN + EXO treatment significantly improved sperm count, motility, morphology and testis weight index compared to controls and monotherapy groups. Histological recovery of spermatogenic lineages was superior in SFN + EXO rats, accompanied by reduced fibrosis and normalized testicular architecture. Expression of DAZL and VASA was fully restored, while aberrant upregulation of Nrf2 and autophagy genes (LC3, Beclin1, p62) in azoospermic testes was normalized only by SFN + EXO. Antioxidant enzyme activity (GPx, TAC) was significantly enhanced, suggesting redox balance recovery. Local delivery of sulforaphane via exosomes effectively reverses chemotherapy-induced spermatogenic failure through modulation of oxidative stress and autophagy, promoting germ cell regeneration. This exosome-based platform offers a promising therapeutic avenue for male infertility.