Methods of cryopreservation of testicular tissue with viable spermatogonia in pre-pubertal boys undergoing gonadotoxic cancer treatment.
BACKGROUND
Banking of testicular tissue from pre-pubertal boys before gonadotoxic treatment is a crucial step in fertility preservation. We wanted to find optimal methods for cryopreservation of testicular tissue from pre-pubertal boys, modifying techniques developed for fetal and adult human testicular tissue cryopreservation.


METHODS
Testicular tissue was collected from five pre-pubertal boys undergoing gonadotoxic treatment in a clinical programme. Two freezing protocols, originally developed for fetal and adult human testicular tissue, were applied for pre-pubertal testicular tissue cryopreservation. In both methods, 5% dimethyl sulphoxide (DMSO) was used as a cryoprotectant. The integrity of the tissue was investigated in non-frozen tissue cultured for 24 h and in cryopreserved-thawed tissue, using two different programmes. We also analysed frozen-thawed samples cultured for 24 h in comparison with untreated fresh fixed control tissue. Immunohistochemical analysis using anti-MAGE-A4, vimentin and CD34 monoclonal antibodies was performed in order to visualize and characterize the cryodamage of the different testicular cells and compartments. The structure of the tissue was evaluated using light microscopy. Qualitative control analysis was performed using transmission electron microscopy.


RESULTS
No clear structural changes were observed in the fresh, fresh cultured and cryopreserved testicular tissue after using the protocol developed for adult testicular tissue. The programme earlier successfully used for human fetal testicular tissue cryopreservation caused more tissue damage.


CONCLUSIONS
Pre-pubertal testicular tissue from boys facing gonadotoxic treatment survives cryopreservation, can be cryobanked and hopefully used for fertility preservation. Slow programmed freezing with DMSO as a cryoprotectant is efficient in maintaining the spermatogonia, Sertoli cells and stromal compartment during freezing, thawing and tissue culture.
Keros
Victoria
V
Karolinska Institute, Division of Obstetrics and Gynaecology, Department of Clinical Science, Technology and Intervention, Karolinska University Hospital, Stockholm, Sweden. Victoria.Keros@ki.se
Hultenby
Kjell
K
Borgström
Birgit
B
Fridström
Margareta
M
Jahnukainen
Kirsi
K
Hovatta
Outi
O
D000293:Adolescent; D002478:Cells, Cultured; D002648:Child; D002675:Child, Preschool; D015925:Cryopreservation / Q000379:methods*; D003451:Cryoprotective Agents; D004121:Dimethyl Sulfoxide; D006801:Humans; D007150:Immunohistochemistry; D015470:Leukemia, Myeloid, Acute / Q000532:radiotherapy; D015479:Leukemia, Myelomonocytic, Acute / Q000532:radiotherapy; D008297:Male; D008853:Microscopy; D046529:Microscopy, Electron, Transmission; D009926:Organ Preservation / Q000379:methods*; D054198:Precursor Cell Lymphoblastic Leukemia-Lymphoma / Q000532:radiotherapy; D011627:Puberty; D012208:Rhabdomyosarcoma / Q000532:radiotherapy; D012708:Sertoli Cells / Q000502:physiology; D013093:Spermatogonia / Q000502:physiology*; D013737:Testis / Q000166:cytology / Q000502:physiology*; D017086:beta-Thalassemia / Q000532:radiotherapy
D003451:Cryoprotective Agents; D004121:Dimethyl Sulfoxide