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Hum. Reprod. Advance Access published online on February 27, 2004
Human Reproduction, doi:10.1093/humrep/deh154
© 2004 by European Society of Human Reproduction and Embryology
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Received November 22, 2002
Revised October 14, 2003
Accepted November 11, 2003

Article

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

V. Frederickx 1*, A. Michiels 1, E. Goossens 1, G. De Block 1, A.C. Van Steirteghem 1, and H. Tournaye 1

1 Centre for Reproductive Medicine and Research Laboratories for Reproductive Medicine, University Hospital and Medical School, Vrije Universiteit Brussel, Laarbeeklaan 101, B-1090 Brussels, Belgium

* To whom correspondence should be addressed. E-mail: veerle.frederickx{at}az.vub.ac.be.


  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.

Key words: Key words: cryopreservation/mouse/spermatogonia/stem cell/transplantation


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