Cryopreservation of prepubertal testicular tissue : comparison of slow-freezing and vitrification methods
(2010)
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- Abstract
- (en) The growing success of oncological treatments and extension of gonadotoxic therapies to benign pathologies make implementation of techniques aimed at preserving reproductive function increasingly important for long-term quality of life. Although sperm banking is an highly efficient and proven fertility preservation option, this approach relies on the presence of spermatozoa and cannot be considered for prepubertal boys. Cryopreservation of prepubertal testicular tissue pieces has emerged as an ethically acceptable strategy to preserve fertility in young boys. Controlled slow-freezing has proved to be a promising method to preserve immature human testicular biopsies, but an efficient cryopreservation protocol has not yet been established. Vitrification might be an alternative approach, since it avoids ice crystal formation and ensuing freeze injuries. Our objective is to offer young patients at risk of testicular failure after gonadotoxic therapies realistic, safe and efficient fertility preservation options. This project therefore focuses on optimization of methods for immature testicular tissue cryopreservation and evaluates the potential of vitrification in this context. For this purpose, an organotypic culture system and vitrification protocol were set up using immature mouse testicular tissue, since immature human testicular tissue is difficult to obtain. The development of new evaluation tools to detect necrosis and apoptosis induced by cryopreservation procedures allowed us to demonstrate better preservation of membrane integrity after vitrification than slow-freezing, while the rate of apoptosis assessed by active caspase-3 immunostaining was higher after vitrification. These findings will be useful for further fine-tuning of protocols. However, normal development and integrity of immature mouse testicular tissue were seen after vitrification and short-term (3-day) organotypic culture, similar to results with slow-freezing, suggesting that cryoinjuries induced by either method are limited. Furthermore, spermatogonial and Leydig cell functionality appear to be maintained following long-term (10-day) organotypic culture after both vitrification and slow-freezing treatments. Indeed, spermatogonial cell survival, proliferation and normal differentiation to the spermatocyte stage, as well as Leydig cell expression of key steroidogenic enzymes, were observed. Finally, we showed that vitrification of immature human testicular tissue, according to the protocol developed in mice, is able to maintain survival and proliferation of human spermatogonial cells, as well as seminiferous tubule integrity, in long-term (10-day) organotypic culture, yielding similar results to fresh and slow-frozen tissue. Taken together, these results suggest that vitrification is able to cryopreserve immature testicular tissue as efficiently as established slow-freezing methods through a faster and more convenient process. This new cryopreservation method might therefore be considered an alternative strategy to slow-freezing and a promising approach to preserve the procreative ability of young boys before they start gonadotoxic therapy. Nevertheless, further studies are clearly required before this option can be offered to young patients undergoing gonadotoxic treatment.
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Citations
Curaba, M. (2010). Cryopreservation of prepubertal testicular tissue : comparison of slow-freezing and vitrification methods. https://hdl.handle.net/2078.5/148322