Pediatric cancer survivors experiencing gonadotoxic chemoradiation therapy may encounter subfertility or permanent infertility. However, previous studies of cryopreservation of immature testicular tissue (ITT) have mainly been limited to in vitro studies. In this study, we aim to evaluate in vitro and in vivo bioluminescence imaging (BLI) for solid surface-vitrified (SSV) ITT grafts until adulthood. The donors and recipients were transgenic and wild-type mice, respectively, with fresh ITT grafts used as the control group. In our study, the frozen ITT grafts remained intact as shown in the BLI, scanning electron microscopy (SEM) and immunohistochemistry (IHC) analyses. Graft survival was analyzed by BLI on days 1, 2, 5, 7, and 31 after transplantation. The signals decreased by quantum yield between days 2 and 5 in both groups, but gradually increased afterwards until day 31, which were significantly stronger than day 1 after transplantation (p = 0.008). The differences between the two groups were constantly insignificant, suggesting that both fresh and SSV ITT can survive, accompanied by spermatogenesis, until adulthood. The ITT in both groups presented similar BLI intensity and intact cells and ultrastructures for spermatogenesis. This translational model demonstrates the great potential of SSV for ITT in pre-pubertal male fertility preservation.

Background: The optimal method for cryopreserving immature testicular tissue (ITT) remains unknown and there is no standardized protocol. Controlled slow freezing remains the mainstream method of choice in human prepubertal male fertility preservation. Currently, the outcomes for ITT vitrification are conflicting, and most data are limited to in vitro animal studies.Methods: A total of 12 pairs of donor and recipient mice were included in our experiments. The donors were immature transgenic mice, and the recipients were wild-type male mice. In the vitrification group, ITT was vitrified and thawed before transplantation. In the control group, ITT was transplanted to the recipients immediately. After thawing, we measured the expression of apoptosis-related mRNA caspase-3. More importantly, we monitored to adulthood all the transplanted grafts in vivo using noninvasive bioluminescence imaging (BLI) technology. On day 31, we removed the grafts for evaluation via hematoxylin and eosin staining and immunohistochemistry (IHC).Results: We traced the survival of the grafts by in vivo BLI on days 1, 2, 5, 7, and 31 after transplantation. In both the vitrification and the control groups, bioluminescence decreased between days 2 and 5. Subsequently, the bioluminescence showed an upward trend until day 31. Compared with day 1, the bioluminescence was significantly stronger on day 31 after transplantation (P = 0.009). The differences between the two groups were constantly insignificant after analysis. These results indicate that both fresh and frozen–thawed testicular tissues can survive for at least 31 days after transplantation. Moreover, the vitrification group showed BLI signals comparable with those of fresh tissues. Compared with the control group, expression of the caspase-3 gene was significantly increased after vitrification (P = 0.04). Histology and IHC showed that both tissue structure and protein expression were intact in both groups.Conclusions: Transplanted vitrified ITT grafts could survive till adulthood with BLI intensity comparable to that of the fresh control. Intact cells and structures for spermatogenesis in vitrified ITT grafts were as well-preserved as those in the control group. This translational model of self-repairing vitrified ITT grafts in vivo, lends weight to the role of vitrification in prepubertal male fertility preservation.