Assessment of the optimal vitrification protocol for pre-pubertal mice testes leading to successful in vitro production of flagellated spermatozoa

Testicular tissue cryopreservation offers the hope of preserved future fertility to pre-pubertal boys with cancer before exposition to gonadotoxic treatments. The objective of this study was to compare controlled slow freezing (CSF) with five vitrification techniques for cryopreservation of murine pre-pubertal testicular tissue and to evaluate the best protocol that could provide a successful completion of spermatogenesis after in vitro maturation. Testicular tissue from 24 mice at 6.5days post-partum (dpp) was used to compare several vitrification protocols with one another, as well as with a CSF protocol. Toxicity test using additional 12 mice was performed for all cryopreservation solutions. Fresh tissue (FT) from six mice was used as a control. Once the optimal vitrification protocol was selected [the modified solid surface vitrification No. 1 (mSSV(1))], testes from 18 mice were cultured in vitro for 30days with (i) fresh, (ii) slow-frozen/thawed and (iii) vitrified/warmed tissues. Testes from six mice at 36.5 dpp were used as controls. At day 30 of in vitro culture, germ cells of the seminiferous tubules showed a high ability to proliferate and elongated spermatids were observed after both freezing techniques, confirming the successful completion of in vitro spermatogenesis. However, after mSSV(1), the morphological alterations and the percentage of pyknotic seminiferous tubules were lower than CSF (4.67 +/- 0.53 vs. 10.1 +/- 1.12 and 22.7 +/- 2.83% vs. 37.3 +/- 4.24% respectively). Moreover, the number of flagellated spermatozoa produced per mg of tissue was higher for mSSV(1) than for CSF (35 +/- 3 vs. 9 +/- 4 cells), with amounts of secreted testosterone during the culture close to those of FT. The mSSV(1) protocol resulted in success rates better than CSF in maintaining testicular tissue structure, tubular morphology and tissue functions not solely for immediate frozen/thawed tissues but also after a long-term in vitro culture.