In some men with germ cell maturation arrest, spermatogenesis can be resume
d during in-vitro culture of testicular biopsy samples. In this study, we e
xamined whether similar differentiation events can be induced in cultured g
erm cells from cryopreserved testicular biopsy specimens. Fresh and cryopre
served aliquots of the same testicular biopsy samples were cultured in medi
um supplemented with FSH and testosterone. After 24 and 48 h of culture, th
e progression of spermatogenesis and the percentage of Sertoli cells with D
NA damage, detected by terminal deoxynucleotidyl transferase-mediated dUTP
nick-end labelling (TUNEL), were evaluated. Spermatogenesis progressed in a
similar way id fresh and cryopreserved aliquots over the first 24 h of cul
ture. However, in contrast to fresh aliquots, no additional progress of spe
rmatogenesis was detected between the 24 and 48 h time points. The percenta
ge of TUNEL-positive Sertoli cells in fresh aliquots showed only a moderate
increase after 24 h of culture, whereas most Sertoli cells from cryopreser
ved aliquots became TUNEL-positive during the same culture period. These da
ta show that limited progression of spermatogenesis can be achieved by cult
uring cryopreserved testicular biopsy specimens for 24 h, but no additional
benefit can be expected from prolonging the culture beyond this time point
.