Testicular Tissue Cryopreservation in Prepubertal Boys: Current Advances, Clinical Feasibility, and Future Directions — A Comprehensive Literature Review

This literature review aims to evaluate the current scientific and clinical advancements in testicular tissue cryopreservation (TTC) as a fertility preservation strategy for prepubertal boys who are at risk of infertility due to gonadotoxic treatments. The objective is to summarize available protocols, assess tissue viability outcomes, and examine future translational potential, including autotransplantation and in-vitro maturation.

A structured literature search was conducted across PubMed, Embase, and Google Scholar for studies published between 2015 and 2025. Inclusion criteria were preclinical or clinical studies involving human or mammalian testicular tissue cryopreservation, spermatogonial stem cell (SSC) survival analysis, cryoprotectant optimization, and post-thaw functional assessment. Review articles, translational studies, and early-phase clinical protocols were included. Data were extracted on cryopreservation techniques (slow freezing, vitrification), cell viability assays, transplantation outcomes, and ethical considerations. Findings were synthesized descriptively due to heterogeneity of methods.

Recent literature demonstrates significant progress in preserving SSC viability using optimized cryoprotectant mixtures and controlled freezing protocols. Studies report 60–90% post-thaw survival of SSCs in optimized slow-freezing methods, with improved preservation of testicular architecture. Early animal models show successful restoration of spermatogenesis following autotransplantation of cryopreserved tissue. Human clinical data remain limited but indicate that TTC is safe, feasible, and increasingly offered in specialized fertility-preservation programs worldwide. No cases of successful human sperm generation or live births have yet been reported. Ethical concerns—including consent, long-term storage, and risk of reintroducing malignant cells—remain central challenges. Overall, advancements suggest a growing translational potential for fertility restoration in survivors of childhood cancer.

Testicular tissue cryopreservation represents the most promising fertility-preservation option for prepubertal boys. Although still experimental, accumulating evidence demonstrates meaningful improvements in tissue viability, safety, and feasibility. As cancer survival rates rise, TTC is becoming increasingly relevant in clinical practice. Continued research in SSC transplantation, in-vitro spermatogenesis, and organoid technologies will be essential to achieve functional fertility restoration. This review highlights the need for standardized clinical protocols and long-term outcome studies.