First successful transplant of human immature testicular tissue after gonadotoxic therapy during childhood: complete spermatogenesis in intra-testicular grafts

ABSTRACT Background Approximately one-third of men having undergone gonadotoxic treatment in their childhood experience impaired testicular function for whom autologous transplantation of cryopreserved immature testicular tissue may represent the only opportunity to restore their fertility. Pre-clinical studies have demonstrated successful restoration of spermatogenesis following grafting of immature testicular tissue in various species, including non-human primates. In 2002, our institution pioneered with clinical testicular tissue banking for fertility preservation in boys and adolescents. Over time, this strategy has been increasingly implemented by numerous fertility centres worldwide for patients at high risk of treatment-induced sterility. Here, we report the first human case of autologous transplantation of frozen-thawed immature testicular tissue. Patient In 2008, testicular tissue was cryopreserved from a pre-pubertal boy diagnosed with sickle cell disease. The procedure was performed after a three-year hydroxyurea treatment and prior to receiving conditioning therapy with busulfan and cyclophosphamide for haematopoietic stem cell transplantation. One testis was surgically removed, sectioned into small fragments, and cryopreserved. Histological analysis confirmed preserved tubular architecture and the presence of spermatogonia. During the period from 2022 to 2024, the patient consistently presented with azoospermia. In December 2024, at the time of transplantation, two abnormal sperm cells were detected after enzymatic digestion. Method Eleven testicular tissue fragments (4–21 mm 3 ) were thawed and autologously grafted to four intra-testicular and four subcutaneous scrotal sites. Over a one-year follow-up period, graft survival, vascularization, hormone profiles, and semen parameters were monitored. One year after transplantation, all grafts were surgically retrieved. Results Post-operative recovery was uneventful. No significant changes in endocrine or semen parameters were observed during follow-up. Whereas the intra-testicular grafts exhibited a compact parenchyma that was distinct from the looser surrounding adult parenchyma and remained readily identifiable as graft tissue, the scrotal grafts appeared more fibrotic. Enzymatic digestion of the grafts was required to recover spermatozoa, with one spermatozoon obtained from one of the four intra-testicular grafts. Histological evaluation revealed intact tubular architecture and maturation of somatic cells across all grafts. Spermatogonial stem cells, together with evidence of active spermatogenesis, were identified in two of the four intra-testicular grafts, whereas no germ cells were detected in the subcutaneous scrotal grafts. Conclusion These findings demonstrate that human immature testicular tissue can survive long-term cryostorage, revascularize after transplantation and establish spermatogenesis in vivo . This study provides essential proof-of-concept for fertility restoration in individuals who banked testicular tissue before puberty. Funding This study was supported by the Research Programme of FWO Vlaanderen (Research Foundation-Flanders; G0A6U25N) and VUB strategic research program (SRP89). Trial Registration: NCT05414045