DNA Damage in Sperm: Should Semen or the 90% Density Gradient Layer be Used for Prospective Screening?

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Sperm chromatin damage may be related to pregnancy outcome in patients undergoing IVF. Therefore, many laboratories have initiated prospective screening of patients using assays to quantitate DNA breaks in sperm. Fresh semen is usually evaluated. The purpose of this study was to evaluate the clinical relevance of evaluating the fresh semen compared to the post-gradient 90% layer used for insemination. A prospective evaluation of chromatin damage in the fresh semen sample and 90% layer following density gradient centrifugation was followed by a retrospective chart review to evaluate semen quality factors which may influence or be related to changes observed in the amount of DNA damage, and to evaluate the results in regard to IVF outcome. Semen samples from 66 patients were evaluated for DNA damage using the TUNEL assay, as previously described. An aliquot of the fresh semen, and an aliquot of the sperm obtained from the 90% layer of the density gradient preparation were analyzed. The samples were classified as “normal” when <15% of the sperm stained positive in the TUNEL assay. Data were evaluated using ANOVA and Chi square analysis. Twenty-six fresh semen samples were classified as “abnormal” (39.4%). Of those patients, 16 samples (61.5%) demonstrated an improvement to the normal range following density gradient preparation. The mean TUNEL score was 24.6 ± 2.8 in the fresh sample and 8.3 ± 1.0 in the 90% layer (p < 0.001). Conversely, 8 patients classified as normal in the fresh semen aliquot (8/40, 20%), had abnormal TUNEL values for sperm in the 90% layer (4.9 ± 1.5 versus 27.8 ± 4.2, p < 0.01). When comparing patients with normal fresh TUNEL values who had abnormal values following density gradient isolation to those who remained in the normal category, significant differences were observed (p < 0.05) in the initial sperm count (38.4 ± 11.3 versus 118.4 ± 23.2), viability (30.9 ± 5.6 versus 45.8 ± 2.6), and HOS reactivity (35.7 ± 5.9 versus 48.8 ± 3.19), but not in white blood cells, morphology, or motility. No significant differences were observed in semen quality between patients who improved following density gradient separation and those who did not. IVF outcomes were more closely aligned to data observed from the 90% layer (p < 0.05). These data demonstrate that the results obtained using the TUNEL assay to evaluate DNA damage may vary in the same patient between the fresh semen and 90% layer used for actual insemination. Negative changes in the TUNEL results may be due to oxidative damage, or other inherent abnormalities in the sperm which result in damage during processing. Conversely, many samples with a high percentage of abnormal sperm seem to benefit from sperm selection during the gradient centrifugation. These results indicate a possible advantage of analyzing sperm actually used for insemination rather than the raw semen. Additionally, the results highlight the need for evaluation of other preparation techniques for sperm with an inherent susceptibility to damage during gradient centrifugation.