Decellularized Testicular Extracellular Matrix Scaffolds Support Mature Spermatogenesis: Impact of Donor Age and Transplantation Microenvironment

Immature testicular tissue transplantation is a promising strategy for restoring fertility in prepubertal boys undergoing gonadotoxic treatments, yet it faces challenges such as significant germ cell loss and poor graft survival due to the initial period without a blood supply. This study utilized an in vivo tissue-engineering platform involving decellularized testicular extracellular matrix to enhance graft stability and maturation. Immature testicular tissue from three-week-old transgenic mice was transplanted into age-matched recipient mice across four experimental groups: tissue co-transplanted with young decellularized matrix into a cleared testicular cavity, tissue with adult matrix in a cleared cavity, tissue transplanted alone in a cleared cavity, and tissue injected directly into an intact recipient testis. Graft growth was monitored longitudinally using bioluminescence imaging, and spermatogenesis was evaluated via histology and immunohistochemistry sixty-five days post-transplantation. The decellularization protocol successfully removed more than 98 percent of host deoxyribonucleic acid while preserving key matrix components. Grafts injected into intact testes showed the earliest bioluminescence peak but subsequently declined. Conversely, tissue co-transplanted with young decellularized matrix in a cleared cavity exhibited a delayed but significantly higher and more sustained peak compared to the group without a scaffold. The young matrix group appeared to provide more sustained support for spermatogenic progression, as reflected by a more stable increase and a prolonged spermatogenic peak over time. Evidence of advanced spermatogenic stages, including spermatids and sperm-like cells, was observed in all groups by day sixty-five. In conclusion, decellularized testicular matrix serves as a supportive bioactive scaffold that improves long-term graft stability, with outcomes significantly influenced by the age of the scaffold’s donor and the transplantation microenvironment.