The synergic impact of decellularized testis scaffold and extracellular vesicles derived from human semen on spermatogonial stem cell survival and differentiation

Introduction : Decellularized scaffolds create a biomimetic niche to support spermatogonial stem cell (SSC) function and engraftment. Semen-derived extracellular vesicles (SEVs), containing proteins, lipids, and microRNAs with various functions, facilitate intercellular communication, enhance sperm maturation, and regulate the testicular microenvironment. This study explored the combined effect of rat decellularized testicular scaffolds and human SEVs on SSC survival and differentiation. Materials and methods : The experimental approach involved decellularizing rat testes using detergents, followed by histological, immunohistochemical, and scanning electron microscopy analyses to confirm extracellular matrix (ECM) preservation and cellular removal. SEVs were isolated from human seminal plasma via ultracentrifugation and characterized for size, morphology, and uptake by testicular cells. Whole testicular cells, including Dolichos Biflorus Agglutinin (DBA)-positive SSCs, were seeded onto scaffolds with or without SEVs and evaluated in vitro and in vivo in castrated neonatal rats. Results : In vitro results demonstrated that SEV-enriched scaffolds significantly enhanced cell viability and upregulated DAZL and PIWI expression, indicating improved SSC survival and functionality, though meiosis (SCP1 expression) was not achieved. In vivo, scaffolds were largely absorbed within two months, replaced by connective tissue, with no surviving SSCs or restoration of sex hormone levels, likely due to inadequate angiogenesis. Conclusion : The findings underscore the potential of integrating SEV-laden decellularized scaffolds to partially promote SSC differentiation for fertility restoration in spermatogenic failure. However, challenges such as scaffold resorption and limited in vivo endocrine function highlight the need for further refinement, including angiogenic factor incorporation or optimized implantation strategies, to enhance clinical applicability for male infertility treatment.