Developmental Potential of Vitrified Mouse Testicular Tissue after Ectopic Transplantation

Objective: Cryopreservation of immature testicular tissue should be considered as an important factor for fertility preservation in young boys with cancer. The objective of this study is to investigate whether immature testicular tissue of mice can be successfully cryopreserved using a simple vitrification procedure to maintain testicular cell viability, proliferation, and differentiation capacity. Materials and Methods: In this experimental study, immature mice testicular tissue fragments (0.5-1 mm²) were vitrified-warmed in order to assess the effect of vitrification on testicular tissue cell viability. Trypan blue staining was used to evaluate developmental capacity. Vitrified tissue (n=42) and fresh (control, n=42) were ectopically transplanted into the same strain of mature mice (n=14) with normal immunity. After 4 weeks, the graft recovery rate was determined. Hematoxylin and eosin (H&E) staining was used to evaluate germ cell differentiation, immunohistochemistry staining by proliferating cell nuclear antigen (PCNA) antibody, and terminal deoxynucleotidyl transferase (TdT) dUTP Nick- End Labeling (TUNEL) assay for proliferation and apoptosis frequency. Results: Vitrification did not affect the percentage of cell viability. Vascular anastomoses was seen at the graft site. The recovery rate of the vitrified graft did not significantly differ with the fresh graft. In the vitrified graft, germ cell differentiation developed up to the secondary spermatocyte, which was similar to fresh tissue. Proliferation and apoptosis in the vitrified tissue was comparable to the fresh graft. Conclusion: Vitrification resulted in a success rates similar to fresh tissue (control) in maintaining testicular cell viability and tissue function. These data provided further evidence that vitrification could be considered an alternative for cryopreservation of immature testicular tissue.