DNA fragmentation in concert with the simultaneous assessment of cell viability in a subfertile population: establishing thresholds of normality both before and after density gradient centrifugation

Abstract

Purpose TUNEL assay is the most common, direct test for sperm chromatin integrity assessment. But, lack of standardized protocols makes interlaboratory comparisons impossible. Consequently, clinical thresholds to predict the chance of a clinical pregnancy also vary with the technique adopted. This prospective study was undertaken to assess the incidence of sperm DNA fragmentation in a subfertile population and to establish threshold values of normality as compared to a fertile cohort, both before and after density gradient centrifugation in the total and vital fractions. Method Men presenting at a university hospital setup for infertility treatment. DNA damage via TUNEL assay was validated on fresh semen samples, as conventional semen parameters, to reduce variability of results. Results Total DNA fragmentation in the neat semen was significantly higher in the subfertile group, but the vital fraction was not significantly different between the two cohorts. After gradient centrifugation, DNA fragmentation increased significantly in the total fraction of the subfertile group but decreased significantly in the vital fraction. In the fertile cohort, there was a nonsignificant increase in total fragmentation and in the vital fraction the trend was unclear. Conclusions Estimating total and vital sperm DNA fragmentation, after density gradient centrifugation, increased both the sensitivity and the specificity, thereby lowering the number of false negatives and false positives encountered. These findings provide opportunities to investigate the significance of the total and the vital fractions after different assisted reproductive technologies.

Diagnostic performance of discontinuous density gradient centrifugation for estimating human semen quality

“…However, our study also showed that there was no significant difference in the sperm DNA fragmentation rate between infertile men and fertile men before and after DDGC, which indicated that DDGC might not increase the diagnostic accuracy of sperm DNA fragmentation detection. However, a recent study also estimated sperm DNA fragmentation after DDGC and concluded that DDGC could increase both sensitivity and specificity 30 . This is not consistent with our results.…”

“…However, a recent study also estimated sperm DNA fragmentation after DDGC and concluded that DDGC could increase both sensitivity and specificity. 30 This is not consistent with our results. We believe the reason for this discrepancy is that the previous study did not analyze its data according to the reference values derived from DDGC-processed semen samples.…”

Sperm as a Carrier of Genome Instability in Relation to Paternal Lifestyle and Nutritional Conditions

“…In 47.4% of the semen samples total SDF was high (>13%) before intervention [ 54 ]. Only one in three benefited from this oral supplementation.…”

“…DNA fragmentation was analyzed in the total sperm sample (total SDF) comprising viable and nonviable sperms, as well as in the vital fraction (vital SDF), thereby analyzing only viable sperm. The method was standardized and cut-off values were defined [ 53 , 54 ].…”

New device for sperm preparation involving migration-gravity sedimentation without centrifugation compared with density-gradient centrifugation for normozoospermic intrauterine insemination

“…However, sperm preparation methods have changed little, and remain reliant on density-gradient centrifugation (DGC). Although centrifugation can recover many sperm with good morphology and motility (1), various adverse effects of centrifugation have also been reported (2,3).…”