Observation of the Cryoprotective Effect of Chlorogenic Acid on Spermatogonial Stem Cells Transplanted into of Testicular Torsion-Induced Azoospermia Model.

Preserving spermatogonial stem cells (SSCs) in prepubertal males, followed by long-term cryostorage for future autologous transplantation, is a promising strategy for managing male infertility. This study investigated the effects of chlorogenic acid (CGA), an antioxidant agent, on SSCs maintained in a basal cryopreservation medium, aiming to evaluate its potential role in supporting cellular preservation under storage conditions. A torsion/detorsion protocol was applied to induce azoospermia in a murine model. Mice were randomized into five groups: fresh SSC transplantation, SSC transplantation with CGA, SSC transplantation using basal cryopreservation medium, a control group, and an untreated azoospermia group. Eight weeks post-transplantation, SSC proliferation and differentiation were assessed using specific markers. In addition, the expression of apoptosis-associated genes (Bax and Bcl-2), components of the PI3K/Akt/Nrf2 signaling pathway, as well as seminiferous tubule diameter and germinal epithelium thickness, were evaluated to assess both molecular and structural outcomes. Flow cytometry and morphological analysis confirmed SSC colonization capacity through expression of SALL4 and PAX7. A 100 µM concentration of CGA enhanced SSC proliferation and differentiation, increased seminiferous tubule diameter, and improved germinal epithelium thickness. CGA also increased PI3K/Akt/Nrf2 pathway gene expression and reduced apoptosis through its effects on Bax and Bcl-2. These findings suggest that supplementation of freezing medium with CGA may offer a promising approach to preserve and restore fertility in prepubertal males.