Combination of knockout serum replacement and plasma rich in growth factors does not support in vitro spermatogenesis in mice.

Cancer treatments can lead to infertility, particularly in prepubertal boys who cannot preserve sperm before therapy. In vitro spermatogenesis offers a promising strategy for fertility preservation in this population by enabling the development of sperm from immature testicular tissue under controlled conditions. This study investigates the effects of a novel culture medium containing plasma rich in growth factors (PRGF) and knockout serum replacement (KSR) on in vitro spermatogenesis. Testicular tissues from five-day-old male NMRI mice were cultured in either a medium containing 5% KSR + 5% PRGF or a control medium containing 10% KSR for 42 days. Histological analysis revealed significant degeneration of peripheral seminiferous tubules in the 5% KSR + 5% PRGF group compared to the control. Gene expression analysis showed reduced levels of spermatogenesis markers (Plzf, Tekt1, Tnp1) and the proliferation marker Ki67, alongside elevated expression of the pro-apoptotic marker Bax. Immunofluorescence confirmed fewer spermatogonial stem cells (PLZF), spermatocytes (SYCP3), and proliferating cells (Ki67), with complete absence of post-meiotic marker ACRBP in the 5% KSR + 5% PRGF group. Additionally, higher Bax and lower Bcl-2 fluorescence intensities were observed in this group. These findings indicate that a medium supplemented with 5% KSR and 5% PRGF is ineffective for supporting complete in vitro spermatogenesis and may promote apoptosis. Importantly, these results provide insights into culture system design for future applications in fertility preservation strategies for prepubertal cancer patients at risk of gonadotoxicity.