Recovery, survival and functional evaluation by transplantation of frozen-thawed mouse germ cells

Abstract

Background: Establishing a successful method for testicular stem cell transplantation of frozen-thawed testicular cells would be of immense benefit to boys with childhood cancer undergoing a sterilizing treatment. In this study, we evaluated different cryopreservation protocols in a mouse model by means of testicular germ cell transplantation (TGCT), in order to establish an optimal freezing protocol. Methods and results: In a first series of experiments, we compared an uncontrolled protocol with 1.5 mol/l dimethyl sulphoxide (DMSO) versus a controlled long protocol (cooling to -80°C) and observed a better viability with the latter protocol (36% versus 48%, P < 0.05). We then compared survival after two thawing methods (37°C water versus ice water) in either a DMSO- or an ethylene glycol (EG)-based protocol, and found no difference. In order to evaluate the functional capacity of the cryopreserved testicular suspension, TGCT was performed with both fresh and frozen-thawed suspensions. In 90% of the successfully injected testes, spermatogenesis was reinitiated using fresh suspensions. In contrast, this figure was only 12.5 and 22.7% after cryopreservation, for the short controlled EG protocol and the uncontrolled DMSO protocol, respectively. Conclusion: Reinitiation of spermatogenesis is possible after cryopreservation of testicular germ cell suspensions. Although cell survival was acceptable, our results after TGCT show that our protocols need further improvement. © European Society of Human Reproduction and Embryology 2004; all rights reserved.