Sperm DNA Denaturation Research Articles

Differences in generation of reactive oxygen species (ROS) in highly motile fractions of spermatozoa as a discriminating tool for semen samples from infertility patients

Objective: To assess the differentiating ability of ROS generated by highly motile fractions of spermatozoa in samples of donors and patients, and to determine interrelation among semen parameters, ROS, sperm DNA denaturation, fragmentation and phosphatidylserine externalization.

Effect of storage time and temperature on stallion sperm DNA and fertility

We used the sperm chromatin structure assay (SCSA) to study the change in stallion sperm DNA susceptibility to denaturation after exposure of extended semen to three different storage temperatures (5, 20, or 37 °C) at 7, 20, 31, and 46 h. In addition, we compared the rates of sperm DNA denaturation in fertile and subfertile stallions. Among fertile stallions, spermatozoa stored at 20 and 37 °C showed a significant ( P<0.05) rise in the SCSA measures (Mean( α t), S.D.( α t), and percent cells outside the main population—COMP( α t)) over time, with the degree of rise being more dramatic at 37 °C. Over all stallions, samples stored at 5 °C showed no significant ( P>0.05) changes in the SCSA values measured over time, indicating maintenance of chromatin quality for up to 46 h. The COMP( α t) from stallions classified as subfertile showed an increased susceptibility to denaturation or decline in chromatin quality between 20 and 31 h when stored at 5 °C; however, spermatozoa from fertile stallions did not change during the time intervals analyzed. These data suggest that sperm DNA from some subfertile stallions may decline at a greater rate than spermatozoa from fertile stallions when exposed to similar storage conditions.

The effect of semen processing on human sperm DNA integrity is not associated with increased levels of sperm free thiols.

Objective: Some forms of sperm processing may adversely affect sperm DNA integrity but the mechanism remains unknown. We monitored the levels of sperm free thiols (SH groups) and DNA denaturation (DD) before and after density gradient centrifugation in order to explore a possible mechanism (the role of SH groups in the stabilization of sperm nuclear protamines) for the observed augmented sperm DD post-processing. Design: Prospective laboratory study. Materials/Methods: Semen samples (n = 43) were obtained from consecutive non-azoospermic men presenting for evaluation at our institution. Samples were processed by 2-layer density gradient centrifugation. Sperm motility, sperm chromatin structure (evaluated by flow cytometry analysis of acridine orange-treated spermatozoa) and sperm SH groups were compared before and after semen processing. Sperm chromatin integrity was expressed as the percentage of spermatozoa demonstrating DD. Results: Following density-gradient centrifugation, mean sperm motility (±SEM) improved significantly compared to whole semen (54 ± 3 vs. 43 ± 3%, p < 0.01). As previously observed, following density-gradient centrifugation, the percentage of sperm with denatured DNA increased compared to whole semen (29 ± 4 vs. 18 ± 2%, p < 0.001). However, the level of sperm free thiol groups was lower in processed compared to whole semen (268 ± 26 vs. 534 ± 43 nmol/106 spermatozoa, p < 0.01). Conclusions: Our data demonstrate that density gradient centrifugation adversely affects sperm DNA integrity despite improved sperm motility and reduced levels of sperm SH groups in the recovered sample. Increased levels of sperm DD post-processing do not appear to be due to a significant degree of sperm-specific disulfides to SH group conversion. Supported by: Kidney Foundation of Canada.

Biologic variability of sperm DNA denaturation in infertile men

Objectives. To examine sperm DNA denaturation (DD) in fertile and infertile men and assess the variability of conventional semen parameters and sperm DD in repeated semen samples from infertile men. Methods. Twenty-one consecutive nonazoospermic, infertile men each submitted two semen samples, 2 to 6 weeks apart. We examined semen samples from consecutive fertile men (n = 10) presenting for vasectomy as controls. Standard semen parameters (World Health Organization criteria) and sperm chromatin structure (evaluated by flow cytometry analysis of acridine orange-treated spermatozoa and expressed as the percentage of spermatozoa with denatured DNA) were monitored. Results. Fertile men had a significantly higher sperm concentration and percentage of sperm motility and a significantly lower percentage of sperm with DD than did infertile men (36 ± 5.2 × 10 6/mL versus 12.5 ± 2.2 × 10 6/mL, 60.0% ± 5.2% versus 30.1% ± 4.1%, and 8.9% ± 1.9% versus 20.3% ± 2.5%, respectively, P <0.05). The sperm concentration, sperm motility, and percentage of spermatozoa with DD were not significantly different between the first and second semen samples from the infertile men. Sperm DD showed the lowest average within-subject coefficient of variation (SD/mean), followed by motility and concentration (coefficient of variation 21%, 24%, and 35%, respectively). Conclusions. Our data demonstrate that infertile men have significantly higher sperm DD compared with fertile men and that sperm DD exhibits a low coefficient of variation (∼20%) on repeated assessment. These data suggest that sperm DD has a relatively low degree of biologic variability.

Free thiols in human spermatozoa: correlation with sperm DNA integrity

Objectives. To examine the levels of sperm protamine free sulfhydryl or thiol (SH) groups and DNA denaturation (DD) in semen from fertile and infertile men. Methods. Semen samples were obtained from consecutive nonazoospermic men presenting for infertility evaluation (n = 66) and fertile men presenting for vasectomy (n = 10). The standard semen parameters (sperm concentration, motility, and morphology), sperm DD, and SH content were monitored. Sperm DNA integrity was evaluated by flow cytometry analysis of acridine orange–treated spermatozoa and expressed as the percentage of spermatozoa demonstrating denatured DNA. Results. The mean (± SE) levels of sperm SH groups and the percentage of spermatozoa with DD were significantly higher in the infertile men than in the fertile men (SH groups 1083 ± 97 and 570 ± 101 nmol/10 6 spermatozoa, respectively; and DD 21% ± 1.7% and 8.9% ± 1.9%, respectively). The levels of sperm SH groups and DD correlated positively ( r = 0.53). Also, the levels of sperm SH groups and DD correlated negatively with the standard semen parameters. Conclusions. Our data demonstrate that sperm SH content correlates positively with sperm DD and that significantly higher levels of sperm DD and SH content are found in infertile men than are found in fertile men. These data suggest that the enhanced susceptibility of sperm DNA to denaturation in infertile compared with fertile men may be associated with incomplete oxidation of the sperm SH groups.

Correlations between two markers of sperm DNA integrity, DNA denaturation and DNA fragmentation, in fertile and infertile men

Objective: To evaluate two different assays of human sperm DNA integrity, DNA denaturation (DD) and DNA fragmentation (DF), and to correlate these with standard semen parameters. Design: Prospective, observational study. Setting: University infertility clinic. Patient(s): Forty consecutive semen samples from 33 nonazoospermic men presenting for infertility evaluation and 7 fertile men presenting for vasectomy. Intervention(s): Assessment of sperm concentration, motility, morphology, DD and DF. Main Outcome Measure(s): Sperm DD and DF in fertile and infertile men. Result(s): The mean (±SE) rates of DD and DF were significantly higher in infertile subjects compared to fertile controls, respectively: 25.4 ± 3.0 vs. 10.2 ± 2.3 ( P=.028) and 27.6 ± 2.5 vs. 13.3 ± 2.5% ( P=.016). DF and DD correlated strongly (r = 0.71, P<.0001). Also, DD and DF correlated negatively with standard semen parameters (concentration, motility, and morphology), the strongest correlation being with sperm motility. Conclusion(s): The strong correlation between sperm DD and DF, and the higher levels of sperm DNA damage in infertile compared with fertile men, indicate that male infertility is associated with poor sperm DNA integrity. Although infertile men may father children with assisted conception, fertilization with DNA-damaged spermatozoa may increase the risk of genetic disease in the offspring.

Semen granulocyte elastase: its relevance for the diagnosis and prognosis of silent genital tract inflammation.

Elastase-inhibitor complex was assessed by immunoassay in the seminal plasma of 312 men attending the outpatient infertility clinic. Using receiver operating characteristic (ROC) curve analysis, elastase at the cut-off value of > or =290 ng/ml was shown to be efficient (sensitivity 79.5%, specificity 74.4%) in the detection of genital tract inflammation as defined by leukocytospermia (>1x10(6) leukocytes/ml). The prevalence of increased elastase in 292 infertile men was significantly higher (34%) as compared with that (5%) observed in 20 fertile men (P: = 0.02). Moreover, high elastase concentration (> or =290 ng/ml) was observed in 66 of the 264 men (25%) without leukocytospermia. A significant positive correlation was found between elastase concentration and patient age (r = 0.202, P: < 0.0001) and the number of leukocytes (r = 0.330, P: < 0.0001). A negative correlation was found between elastase concentration and semen volume (r = -0.146, P: = 0.01) and the percentage of spermatozoa with single-stranded DNA (r = -0.194, P: = 0.024), but there was no correlation between elastase and sperm reactive oxygen species production. A higher seminal elastase concentration was significantly associated with tubal damage in female partners (P: < 0.001). After norfloxacine antibiotic therapy, decrease in elastase concentration was observed in 15 (25%) of the 60 treated patients. Tubal damage in the partner negatively affected the response to antibiotic therapy. In conclusion, granulocyte elastase is a reliable screening test for silent genital tract inflammation of the couple. The elastase-inhibitor complex may have a protective effect in reducing sperm DNA denaturation.

Potential adverse effect of semen processing on human sperm deoxyribonucleic acid integrity

Objective: To examine the effect of standard Percoll density-gradient centrifugation on human sperm DNA denaturation. Design: Prospective, observational study. Setting: University-based infertility clinic. Patient(s): Twenty-five nonazoospermic men. Intervention(s): Semen samples (n = 25) were obtained from consecutively seen nonazoospermic men presenting for infertility evaluation. Samples were processed by two-layer and four-layer Percoll density gradients. Sperm motility and sperm chromatin structure (evaluated by flow cytometry analysis of acridine orange–treated spermatozoa) were monitored before and after semen processing. Sperm chromatin integrity was expressed as the percentage of spermatozoa that demonstrated denatured DNA. Main Outcome Measure(s): Sperm motility and DNA integrity. Result(s): Mean sperm motility improved significantly after processing with two-layer and four-layer Percoll gradients compared with whole semen (54% and 57% motility versus 44% motility, respectively). In contrast, the percentage of sperm with denatured DNA increased after processing with two-layer and four-layer Percoll gradients compared with whole semen (34% and 32% versus 18%, respectively). Conclusion(s): Our data demonstrate that the improvement seen in sperm motility after Percoll processing is not associated with a similar improvement in sperm DNA integrity. These data suggest that we reexamine current sperm processing techniques to minimize sperm DNA damage and the potential transmission of genetic mutations in assisted reproductive cycles.

Change in semen quality and sperm chromatin structure following occupational styrene exposure

Organic solvents have been suspected to exert detrimental effects on human spermiogenesis. Styrene, which is both mutagenic and neurotoxic, was selected as a suitable organic solvent for further assessment of a possible effect on semen quality and sperm DNA damage. Semen samples were collected from 23 reinforced plastics workers at the time of employment and after 6 months of styrene exposure and from 21 nonexposed farmers. Intra-individual changes in conventional semen parameters and sperm-DNA denaturation patterns were related to the internal dose of styrene exposure as measured by postshift urinary mandelic acid. A statistically significant decline in sperm density was seen during styrene exposure from 63.5 to 46.0 million sperm/ml, whereas no decline was seen in the nonexposed subjects. The total sperm count was almost halved from an initial value of 175 million sperm/ejaculate. However, no relationship was apparent when the sperm parameters were related to internal levels of exposure. However, an exposure-response relationship was shown for DNA-denaturation patterns, but the numbers were small. A declining sperm count following styrene exposure is suggested. However, the findings of the internal and external comparisons are inconsistent, and this may be due to the high intraindividual variability of semen parameters and the limited study size but may also be attributable to a weak internal exposure gradient. Spermatogenesis may be vulnerable to styrene exposure. However, due to the small numbers these findings are only preliminary.

O-067. Sperm DNA denaturation as a possible indicator for the outcome of ICSI

O-067. Sperm DNA denaturation as a possible indicator for the outcome of ICSI