Aims: This study aimed to explore the therapeutic effects of amniotic fluid-derived extracellular vesicles including exosomes (AF-Exos) on the recovery of sperm production capacity in a rat model of azoospermia. Main methods: The non-obstructive azoospermia (NOA) was induced in rats using intratesticular administration of Busulfan. Azoospermia was confirmed by testis histology. AF-Exos samples containing 10 or 40 μg exosomal proteins were injected into testicular tissue of NOA rats. After two months, the recovery of spermatogenesis was monitored via histopathological staining, spermiogram, and hormonal analysis. Immunohistochemistry staining for OCT-3/4 was used to identify of spermatogonial progenitors. The expression of DAZL and VASA, was also measured. Key findings: AF-Exos exhibited sphere-shaped morphology with the mean diameter and zeta potential of 50 ± 7.521 nm and ???? 7.16 mV. Immunoblots revealed that isolated nanoparticles were CD63, CD9, and CD81 positive. Histopathological evaluation revealed that spermatogenesis was improved significantly in NOA rats after AFExos injection. Data showed that the sperm parameters and spermatogenesis index were significantly improved after AF-Exos injection compared to azoospermic groups. OCT-3/4+ cells were increased in NOA rats after AF-Exos injection, showing the restoration of spermatogenesis. In the present study, both doses of exosome (10 and 40 μg) restored the testicular function of NOA rats. DAZL and VASA were increased significantly in animals who received 40 μg exosomal protein compared to azoospermic rats. Except in a high dose of AF-Exos (40 μg) for Testosterone and FSH, no statistically significant differences were found regarding hormones postexosome injection. Significance: Our study demonstrated that AF-Exos regenerated spermatogenesis and improved sperm quality in NOA rats.

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