One ejaculate from each patient was independently assayed. The fractions obtained from each ejaculate were compared with sperm parameters, proportion of Lck Inhibitor with sDNAfrag and staining patterns. Statistical analysis was carried out with the software STATISTICA (Version 11 Statsoft, USA). As we could not homogenize data, a non-parametric approach was taken. To compare the percentages of TUNEL-positive sperm between the four fractions as well as to analyse the morphological patterns in the fractions the Kruskal-Wallis ANOVA (comparisons between independent groups) and the Friedman analysis of variance (ANOVA) (comparisons between dependent groups) tests were used. 1/square root and Acos transformations were used to grant homogeneity of variances before analysis. Post-hoc analysis (comparisons between independent groups) was made with the multiple comparison test. Additionally, an analysis with Mann–Whitney U-test was used. After the Friedman ANOVA, the Sign test was applied to determine differences between groups. Spearman rank correlation was used to measure the association of the sDNAfrag, staining patterns and semen parameters. P < 0.05 was considered to be statistically significant.