Methods of cryopreservation of testicular tissue with viable spermatogonia in pre-pubertal boys undergoing gonadotoxic cancer treatment

TLDR

Banking testicular tissue from pre‑pubertal boys before gonadotoxic therapy is essential for future fertility preservation. The study aimed to identify optimal cryopreservation methods for pre‑pubertal testicular tissue by adapting fetal and adult protocols. Five pre‑pubertal boys’ testicular tissue was cryopreserved using two protocols (adult and fetal) with 5 % DMSO, then evaluated by culture, immunohistochemistry, light microscopy and TEM to assess structural integrity. The adult protocol preserved tissue structure, whereas the fetal protocol caused more damage; overall, slow programmed freezing with 5 % DMSO maintained spermatogonia, Sertoli cells and stromal compartments, demonstrating that pre‑pubertal testicular tissue can be cryobanked for fertility preservation.

Abstract

Banking of testicular tissue from pre-pubertal boys before gonadotoxic treatment is a crucial step in fertility preservation. We wanted to find optimal methods for cryopreservation of testicular tissue from pre-pubertal boys, modifying techniques developed for fetal and adult human testicular tissue cryopreservation. Testicular tissue was collected from five pre-pubertal boys undergoing gonadotoxic treatment in a clinical programme. Two freezing protocols, originally developed for fetal and adult human testicular tissue, were applied for pre-pubertal testicular tissue cryopreservation. In both methods, 5% dimethyl sulphoxide (DMSO) was used as a cryoprotectant. The integrity of the tissue was investigated in non-frozen tissue cultured for 24 h and in cryopreserved-thawed tissue, using two different programmes. We also analysed frozen–thawed samples cultured for 24 h in comparison with untreated fresh fixed control tissue. Immunohistochemical analysis using anti-MAGE-A4, vimentin and CD34 monoclonal antibodies was performed in order to visualize and characterize the cryodamage of the different testicular cells and compartments. The structure of the tissue was evaluated using light microscopy. Qualitative control analysis was performed using transmission electron microscopy. No clear structural changes were observed in the fresh, fresh cultured and cryopreserved testicular tissue after using the protocol developed for adult testicular tissue. The programme earlier successfully used for human fetal testicular tissue cryopreservation caused more tissue damage. Pre-pubertal testicular tissue from boys facing gonadotoxic treatment survives cryopreservation, can be cryobanked and hopefully used for fertility preservation. Slow programmed freezing with DMSO as a cryoprotectant is efficient in maintaining the spermatogonia, Sertoli cells and stromal compartment during freezing, thawing and tissue culture.