Establishment of a controlled slow freezing-based approach for experimental clinical cryopreservation of human prepubertal testicular tissues

ABSTRACT Objective To develop an efficient clinical-grade freezing protocol towards experimental clinical cryopreservation of testicular tissues in prepubertal boys suffering from cancer. Design Experimental cryopreservation of testicular tissue. Setting Hadassah Hebrew University Medical Center, Jerusalem, Israel. Patients Adult patients undergoing orchiectomy for various tumors, and prepubertal boys scheduled for gonadotoxic treatment. Intervention: None Main outcome measure(s) Histopathological analysis of tissue architecture, structural integrity, and cellular morphology, was performed for control and frozen-thawed cryopreserved tissues. The number of seminiferous tubules per testicular section was calculated. The survival of spermatogonial stem cells (SSCs) and Sertoli cells of control and frozen-thawed cryopreserved tissues was analyzed by immunofluoresence staining. Results Uncontrolled Slow-Freezing (USF), Controlled Slow-Freezing (CSF), and vitrification similarly preserved the integrity of the adult testicular tissues, and the survival of SSCs and Sertoli cells. CSF of prepubertal testicular tissues effectively preserved their architecture, the number of tubules, SSCs and Sertoli cells. In addition, we observed SSC loss following chemotherapy in prepubertal boys, reemphasizing the importance of fertility preservation prior to gonadotoxic treatment. Conclusion Future fertility restoration for male survivors of pediatric cancers depends on the development of an optimal prepubertal testicular tissue cryopreservation method. Our findings demonstrate the effectiveness of CSF for cryopreservation of human prepubertal testicular tissues, and may contribute to more effective banking of these tissues, and potential fertility restoration.