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Hum. Reprod. Advance Access published online on April 28, 2005
Human Reproduction, doi:10.1093/humrep/deh797
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© The Author 2005. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oupjournals.org
Received June 4, 2004
Revised December 1, 2004
Accepted January 20, 2005

Article

Optimizing cryopreservation of human testicular tissue: comparison of protocols with glycerol, propanediol and dimethylsulphoxide as cryoprotectants

Victoria Keros 1*, Björn Rosenlund 2, Kjell Hultenby 3, Lusine Aghajanova 2, Lev Levkov 2, and Outi Hovatta 2

1 Karolinska Institute, Division of Obstetrics and Gynaecology, Department of Clinical Science, Sweden, 3Department of Reproductive Systems Cryobiology, Institute for Problems of Cryobiology and Cryomedicine of the National Academy of Sciences of the Ukraine, 61 015 Kharkov, Ukraine
2 Karolinska Institute, Division of Obstetrics and Gynaecology, Department of Clinical Science, Sweden,
3 Clinical Research Centre, Karolinska University Hospital, Huddinge, SE 141 86 Stockholm, Sweden and

* To whom correspondence should be addressed.
Victoria Keros, E-mail: Victoria.Keros{at}klinvet.ki.se


  Abstract

BACKGROUND: Cryopreservation of testicular tissue is an option in fertility preservation for pre-pubertal boys who will lose spermatogenic cells as a result of chemotherapy. We compared three different protocols and cryoprotectants in cryopreservation of testicular tissue. METHODS: Testicular tissue obtained from 16 infertile men was evaluated by light microscopy(LM), immunostaining against MAGE-A4, transmission electron microscopy (TEM) and organ culture. Seminiferous tubules (1312) from non-frozen (n=16) and frozen-thawed samples (n=34) were studied following cryopreservation using protocols with either 1,2-propanediol (PrOH), glycerol or dimethylsulphoxide (DMSO) as cryoprotectants. RESULTS: Normal structure was seen in 86±6% (mean ±SD) of the fresh tissue. After freezing with DMSO, 70±6% and after PrOH, 37±3% of the tubules were judged to be good. When glycerol was used, the structure of the basal compartment of the tubules was severely damaged. The ultrastructure of the cryopreserved samples as revealed by TEM and MAGE-positive spermatogonia confirmed the findings. Cryopreserved Leydig cells maintained their morphology and ability to release testosterone in culture. CONCLUSION: DMSO as a cryoprotectant (at a 0.7 mol/l concentration) proved to maintain the structure of testicular tissue, especially spermatogonia, after cryopreservation better than PrOH or glycerol.

Keywords: cryopreservation; culture; electron microscopy; spermatogonia; testicular tissue.
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