Abstract: Surviving childhood cancer treatment often results in long-term health complications. Male survivors suffer a significantly higher risk of infertility, due to the gonadotoxic side effects of these cancer regimens. Although in vitro cultivation of prepubertal testis tissues has been previously proposed as an option to preserve fertility, clinical application of this method remains challenging, primarily because producing large amounts of sperm in vitro remains unfeasible. To overcome these limitations, we developed a hydrogel microneedle-based culture system. We then used this system to culture mouse testes from 5 days postpartum (dpp) in vitro, and establish a model of ‘whole testicular spermatogonia pool’ (WTSP). We found that undifferentiated spermatogonia in WTSP proliferated more than fourfold compared to 5 dpp mouse testis. In contrast, we found that testes showed a declining trend in the number of undifferentiated spermatogonia during in vivo development. Transplantation of WTSP into nude mice resulted in a twofold increase in spermatids count per tubule compared to transplantation of conventional whole testes. Furthermore, in vitro meiosis induction of WTSP significantly enhanced spermatid proportion, thus generating fertile offspring. Lastly, we showed that the cellular states of our WTSP closely resemble those of 5 dpp mouse testes in vivo, and the role of X in promoting spermatogonia proliferation by activating the PI3K-AKT-mTOR pathway. In conclusion, our WTSP offers a promising method for preserving fertility in prepubertal male cancer patients by maintaining and expanding spermatogonia extracted before treatment.
