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


CONCLUSIONS
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.
Keros
Victoria
V
Karolinska Institute, Division of Obstetrics and Gynaecology, Department of Clinical Science, Karolinska University Hospital, Huddinge, SE 141 86 Stockholm, Sweden. Victoria.Keros@klinvet.ki.se
Rosenlund
Björn
B
Hultenby
Kjell
K
Aghajanova
Lusine
L
Levkov
Lev
L
Hovatta
Outi
O
D000328:Adult; D002478:Cells, Cultured; D015925:Cryopreservation / Q000379:methods*; D003451:Cryoprotective Agents / Q000494:pharmacology*; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D005990:Glycerol / Q000494:pharmacology; D006801:Humans; D007985:Leydig Cells / Q000378:metabolism; D008297:Male; D008853:Microscopy / Q000379:methods; D009926:Organ Preservation / Q000379:methods*; D011409:Propylene Glycols / Q000494:pharmacology; D013093:Spermatogonia / Q000502:physiology; D013737:Testis / Q000166:cytology / Q000502:physiology*; D013739:Testosterone / Q000378:metabolism
D003451:Cryoprotective Agents; D011409:Propylene Glycols; D013739:Testosterone; D005990:Glycerol; D004121:Dimethyl Sulfoxide