Extracellular Vesicles Present in Bone Marrow Mesenchymal Stromal/Stem Cell Conditioned Media Restore Spermatogenesis in Azoospermic Mice.

 Background: We aimed to examine the therapeutic efficacy of exosome-enriched conditioned media (CM), known for its high concentration of extracellular vesicles (EVs), in comparision with mesenchymal stromal/stem cells (MSCs) in treating non-obstructive azoospermia.

Materials and methods: In this experimental study, we used adipose tissue-derived MSCs (AT-MSCs), bone marrowderived MSCs (BM-MSCs), and BMCM containing EVs to treat busulfan-induced azoospermia in animal models. The study included thirty adult male Balb/C mice and two female enhanced green fluorescent protein-positive (eGFP+/+) Balb/C mice for experimental groups and stem cell culturing. Groups consisted of an intact control, an azoospermia group, an AT-MSC therapy group, a BM-MSC therapy group, a BMCM therapy group, and a spontaneous healing group. Testes were removed from all mice, and histomorphometry and flow cytometry analyses were performed 60 days post-treatment. Additionally, protein structure extraction, protein-protein docking analysis, and data visualization were conducted.

Results: Histomorphometry and flow cytometry showed that most seminiferous tubules in the therapy groups exhibited normal morphology and restored spermatogenesis, unlike the azoospermia group. In silico protein docking analysis revealed that exosome factors in BM-MSCs positively impacted spermatogenesis. The BM-MSC and BMCM therapy groups showed more favorable outcomes compared to other groups. Key exosome factors like Basigin, E3 ubiquitinprotein ligase (UBR2), transforming growth factor beta (TGF-β), hepatocyte growth factor (HGF), and programmed death-ligand 1 (PD-L1) interacted with receptors critical to this process.

Conclusion: Our findings indicate that both BMCM enriched with EVs and the administration of AT-MSCs and BM-MSCs effectively induced spermatogenesis in mice with busulfan-induced azoospermia. Specifically, BM-MSC therapy exhibited superior outcomes compared to AT-MSCs and BMCM alone. This study highlights the potential of EV-based therapies, particularly BMCM, as a promising strategy for treating non-obstructive azoospermia. Furthermore, the interaction of key exosome factors with critical receptors enhances our understanding of the underlying molecular mechanisms involved in restoring reproductive function in testes.