Evaluation of sperm DNA fragmentation using multiple methods: a comparison of their predictive power for male infertility

Abstract

ObjectiveThe usual seminal profile has been customarily used for diagnosing male infertility based on an examination of semen samples. However, sperm DNA fragmentation has also been causally linked to reproductive failure, suggesting that it should be evaluated as part of male infertility assessments. To compare the ability of the five most widely utilized methodologies of measuring DNA fragmentation to predict male infertility and reactive oxygen species by Oxisperm kit assay.MethodsIn this case-control study, which received ethical committee approval, the participants were divided into fertile and infertile groups (50 patients in each group).ResultsThe alkaline comet test showed the best ability to predict male infertility, followed by the terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay, the sperm chromatin dispersion (SCD) test, and the sperm chromatin structure assay (SCSA), while the neutral comet test had no predictive power. For our patient population, the projected cut-off point for the DNA fragmentation index was 22.08% using the TUNEL assay, 19.90% using SCSA, 24.74% using the SCD test, 48.47% using the alkaline comet test, and 36.37% using the neutral comet test. Significant correlations were found between the results of the SCD test and those obtained using SCSA and TUNEL (r =0.70 and r =0.68, respectively; p<0.001), and a statistically significant correlation was also found between the results of SCSA and the TUNEL assay (r =0.77, p<0.001). Likewise, the results of the alkaline comet test showed significant correlations with those of the SCD, SCSA, and TUNEL tests (r =0.59, r =0.57, and r =0.72, respectively; p<0.001).ConclusionThe TUNEL assay, SCSA, SCD, and the alkaline comet test were effective for distinguishing between fertile and infertile patients, and the alkaline comet test was the best predictor of male infertility.

Relationship of Seminal Oxidation-Reduction Potential with Sperm DNA Integrity and pH in Idiopathic Infertile Patients

“…The results for DNA integrity suggest no direct correlation between seminal oxidation-reduction potential and fragmentation of sperm DNA, as assessed by either TUNEL or Comet assays (Table 4). Previous studies have shown a direct relationship between ROS levels and single-strand SDF [10,33,34]. The lack of a similar relationship between sORP and SDF suggests that, similarly to what has been commented on the previous paragraph, the incorporation of antioxidants in seminal plasma compensates for testicular ROS levels.…”

“…Even so, TUNEL and Comet alkaline assays negatively correlate with sperm motility and, in the case of computational analysis of alkaline comet assay, with morphology (Table 4), an association with seminal quality which has been previously described [25][26][27][28]30,33].…”

“…Different studies have shown that SDF values are inversely related to sperm motility and other reproductive health indicators, such as fertilization rates, implantation rates, or recurrent miscarriages [25][26][27][28][29][30][31], especially for in vitro fertilization. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and Comet assays display a very accurate sensitivity and specificity for predicting fertility rates [32,33]. The TUNEL assay is based on the detection of fluorescent labeling of the 3 free ends resulting from DNA breaks.…”

“…Additionally, the Comet assay is the only method that allows the quantitative measurement of DNA damage individually for each cell and the only one that detects the incidence of single-strand and double-strand breaks separately, depending on alkaline or neutral pH conditions, respectively [29]. Previous studies have demonstrated a direct correlation between seminal ROS and single-strand SDF [10,33,34].…”

The higher risk for sperm DNA damage in infertile men

“…Another known limitation is associated with SCD method which was perform to evaluation of sperm DNA integrity. SCD is not a direct method of DNA breaks assessment in contrast to comet assay, TUNEL or SCSA [6,[29][30][31], but yet this method has some significant diagnostic and clinical advantages. Halosperm G2 is ready to use kit which minimizes the risk of mistakes and positive or negative false results.…”