Apoptotic gene expression in potentially fertile and subfertile men

Human sperm contain similar amounts of protamine-1 (P1) and protamine-2 (P2). Although an aberrant protamine ratio have been observed in subfertile men, functional evidence is provided by protamine knockout mice exhibiting male infertility. As sperm DNA integrity is known to be linked with DNA fragmentation and apoptosis, we investigated whether the DNA fragmentation factor 40 (DFF40) ratio or caspase (Casp4, Casp6) and tumor necrosis factor superfamily member 10 (TNFSF10) ratio together with the P1/P2 ratio represents a reliable biomarker to discriminate between fertile and subfertile men. Real-time quantitative RT-PCR was used for amplification of P1, P2 and DFF40 in 49 testicular biopsies. Casp4, Casp6 and TNFSF10 have been selected from a PCR apoptosis array and were further investigated in another group of testicular biopsies (22 subfertile men versus 11 potentially fertile men). Using Spearman's rank correlation coefficient analysis, we did not find a correlation between DFF40 and P1, P2, P1/P2, score, fertilization rate and age. In addition, logistic regression analysis demonstrated no statistically significant effect of the analyzed variables on pregnancy. A two-way analysis of variance with repeated measures of relative expression of Casp4, Casp6 and TNFSF10 versus P1 or P2 in potentially fertile men and subfertile patients demonstrated statistically significant differences between both groups, all tested gene combinations and the interaction between two genes and both groups in all cases analyzed. Furthermore, significant differences in the expression of Casp4 and TNFSF10 between the groups of potentially fertile and subfertile men could be demonstrated. In addition, the means of differences of selected gene combinations revealed that the protamine to apoptotic gene ratio is statistically different between both groups. Our data suggest that Casp4, Casp 6 and TNFSF10 are differentially expressed in potentially fertile and subfertile men and represent useful biomarkers for predicting male fertility in combination with P1 and P2.