Sperm DNA damage in neat and density gradient fractions of patient and donor semen samples by different chromatin evaluation assays

Abstract

Interest in evaluating chromatin quality has increased since DNA damage in sperm from infertile men has been associated with infertility and poor embryo quality. We compared the neat semen and density gradient isolated sperm fractions for DNA damage with different assays. Fresh semen samples from 54 patients undergoing IVF with a wide range of semen quality and 7 fertile donors were collected under IRB approval and evaluated for sperm DNA damage. Fresh ejaculates with 2–3 days of sexual abstinence were collected by masturbation and evaluated according to WHO criteria (1999). Briefly, an aliquot of neat semen was frozen for sperm chromatin structure assay (SCSA). The remaining sample was subjected to a discontinuous ISolate density gradient (Irvine) at 300 × g for 15 minutes. The supernatant, 35% and 90% fractions were washed in mHTF (Irvine) at 300 × g for 10 minutes. The sperm from the neat sample, supernatant, 35% and 90% fractions were evaluated for DNA damage using TdT-mediated-dUTP nick end labeling (TUNEL), sperm chromatin dispersion (SCD) test and acridine orange staining technique (AOT). All the assays were performed following the established protocols. The SCSA evaluated more than 5000 sperm and the results are expressed as percent DNA fragmentation index (DFI). A total of 500 sperm from neat and gradient fractions were evaluated in each assay under a fluorescent microscope. The data was analyzed with ANOVA, repeated measures analysis and Pearson correlation tests. Sperm DNA damage in neat semen under SCSA, TUNEL and SCD test correlated (r=0.86: P>0.05) but no correlation was found for AOT. Differences were observed for DNA damage within each assay for sperm in neat, supernatant, 35% and 90% fractions. The results for sperm DNA damage were different (p<0.05) between patients and donors under each assay except for AOT, which showed significantly (p<0.05) higher DNA damage compared to SCSA, TUNEL and SCD. The AOT showed extreme variations for sperm DNA damage within gradient fractions for the same individual and the problems of indistinct colors, rapid fading and heterogeneous staining of the slides were encountered. The DNA damage was significantly (p<0.05) lower in patient and donor sperm isolated from the 90% pellets under TUNEL and SCD test. The values in the figure are the average percent damage ± SEM. These data demonstrate that the SCSA, TUNEL and SCD test show similar predictive values for the assessment of sperm DNA damage. The AOT shows high DNA damage in sperm and the variations in results make AOT of questionable clinical value. Density gradient centrifugation significantly improves the chromatin quality of sperm in the 90% pellet. Lastly, the data demonstrate a lower sperm DNA damage in donors compared to infertile patients.