In-vitro differentiation of germ cells from frozen testicular biopsy specimens

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 .