SUMMARYTesticular 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 6.5 days 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 30 days 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 AE 0.53 vs. 10.1 AE 1.12 and 22.7 AE 2.83% vs. 37.3 AE 4.24% respectively). Moreover, the number of flagellated spermatozoa produced per mg of tissue was higher for mSSV 1 than for CSF (35 AE 3 vs. 9 AE 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.
