Flow cytometric measurement of sperm nuclear DNA fragmentation in infertile men with normal standard sperm parameters

BackgroundMale-factor infertility plays a role in approximately 50% of infertile couples. In at least 30% of cases, repeated standard semen analyses of the male partner of an infertile couple reveal normal results. When diagnostic work-up of the female partner is also normal, they are classified as idiopathic. The objective of this study was to evaluate the levels of sperm nuclear DNA fragmentation in a population of infertile men with normal standard semen parameters and to compare their results with those from men who had abnormal semen parameters, as well as with a control group of fertile men.MethodsSemen samples were obtained from 202 infertile men and 30 fertile donors. Standard semen analysis was performed according to the World Health Organization guidelines. Flow cytometry has been extensively used to study sperm DNA fragmentation and the results are expressed as the percentage of sperm DNA fragmentation index (DFI).ResultsOf the 202 patients, 48 (23.8%) had normal standard sperm parameters, while 154 (76.2%) had an abnormality in one or more of these parameters. DFI in infertile men with normal sperm parameters was significantly higher than in fertile donors (p = 0.03), but not significantly different from infertile men with abnormal sperm parameters (p = 0.10). There were statistically significant negative correlations between DFI and the percentage of motile sperm from infertile men with abnormal and normal semen parameters, but not in fertile donors (r = ?0.26, p = 0.001 and r = ?0.48, p = 0.0001, respectively).ConclusionSperm from infertile men with normal standard sperm parameters may have significant levels of DNA fragmentation that are comparable to levels in infertile men with abnormal sperm parameters. Sperm DNA fragmentation analysis is an independent test of sperm quality and has an important diagnostic value in the evaluation of male infertility.     

Apoptotic sperm biomarkers and the correlation between conventional sperm parameters and clinical characteristics

The principal aim of this retrospective study was to examine the relationship between sperm apoptotic biomarkers and the patient's biclinical characteristics, the conventional sperm parameters and the results of assisted reproductive technology. Sperm analysis, activated caspases, annexin V staining for phosphatidylserine (PS) externalisation and labelling assay for DNA fragmentation were assessed in 122 males of infertile couples. Fifty‐seven couples were allocated to the natural conception group, and 65 couples underwent IVF or ICSI. Semen of IVF/ICSI patients showed a higher proportion of apoptotic spermatozoa in their spermatozoa when compared with a natural conception group (p < .05). Sperm apoptotic biomarkers correlated with age, FSH, and conventional sperm parameters. DNA fragmentation correlated positively with the percentage of semen having externalised PS (r = .78, p = 0) and activated caspases (r = .71, p = 0). Patients without clinical pregnancy had higher frequency of DNA fragmentation, externalised PS and activated caspases compared to patients with clinical pregnancy (p < .001). The best specificity and greater sensitivity were obtained with the test of the DNA fragmentation compared to the other biomarkers. Among the apoptotic biomarkers, only DNA fragmentation was found to predict natural or assisted pregnancy better than conventional sperm parameters.     

Effects of Chlamydia trachomatis infection on sperm chromatin condensation and DNA integrity

The present study was performed to investigate the relation of Chlamydia trachomatis infection to sperm chromatin/DNA integrity in a population of infertile men (male partner of infertile couples) from Iran. Blood, semen and first‐void urine samples were obtained from 250 infertile men. Data were analysed with regard to the results of (i) serological analysis for specific antibodies to C. trachomatis in serum; (ii) the presence of C. trachomatis and DNA in first‐void urine; and (iii) in the semen sample of the male partner, in addition to sperm analysis, four different tests (aniline blue, chromomycin A3, acridine orange and TUNEL) were used to detect sperm chromatin and DNA abnormalities. The main conclusions of the results were: (i) no evidence of C. trachomatis infection in semen samples was found; (ii) sperm DNA fragmentation and chromatin studies were not correlated with C. trachomatis diagnosis; (iii) the percentage of DNA fragmentation is positively correlated with the percentage of immotile sperm but negatively with semen volume, normal morphology; and (iv) in sperm chromatin evaluations, only the percentage of chromatin protamination was related to male age.     

Investigating the relationship between BRCA1 and BRCA2 genes methylation profile and sperm DNA fragmentation in infertile men

The purpose of the study was to investigate whether the promoter methylation status of BRCA1 and BRCA2 DNA repair genes is associated with sperm DNA fragmentation (sDF) in infertile men with oligoasthenoteratozoospermia (OAT) which emerges due to various reasons and is effective in male infertility. Seventy‐three infertile men with OAT and 20 normozoospermic volunteers participated in the study. To investigate sDF and methylation patterns of BRCA1 and BRCA2 gene promoters, TUNEL assay and methylation‐specific PCR (MS‐PCR) were used. The mean sDF ratio for the patients was calculated as 22.50%. The calculated cut‐off value for sDF ratio was 17.0% in ROC curve analysis. Regarding sDF, a significant difference between the normozoospermic group and the OAT group with abnormal semen parameters (p < 0.001) was found. sDF demonstrated a significant effect on the semen parameters and negative correlations on sDF ratios and sperm motility, concentration and morphology. There was no statistically significant association between sDF and the methylation status of the promoter of either BRCA1 or BRCA2 genes. In routine clinical practice, sperm DNA integrity should be investigated before applying assisted reproductive techniques. To understand better the relationship between epigenetic regulation of DNA repair genes and male infertility, additional studies are required.     

Assessment of human sperm DNA integrity using two cytochemical tests: Acridine orange test and toluidine blue assay

Primary infertility affects approximately 15% of couples, with male factor infertility accounting for 50% of cases. Semen samples from 41 patients with asthenoteratospermia and 28 men with proven fertility were analysed according to World Health Organization guidelines. Abnormal sperm chromatin structure was assessed by toluidine blue assay (TBA), and DNA denaturation (DD) was detected by the acridine orange test (AOT). The mean (±SEM) rates of DD and abnormal chromatin structure were significantly higher in infertile subjects compared to fertile group respectively p = .003 and p < .001. A significant correlation was established between sperm DD and abnormal chromatin structure (R = .431, p < .001). Sperm DNA damage correlated significantly with abnormal morphology, sperm motility and necrozoospermia. Our study shows that men with increased levels of abnormal sperm chromatin structure have a high incidence of DNA denaturation and altered semen parameters. These findings suggest that male infertility has been linked to sperm DNA damage.     

The effect of cigarette smoking on human seminal parameters,sperm chromatin structure and condensation

Considerable debate still exists regarding the effects of cigarette smoking on male fertility. This work aimed to explore effects of cigarette smoking on semen parameters and DNA fragmentation on 95 infertile patients who were divided into infertile male nonsmokers (45) and infertile male smokers (50). Smokers were subdivided according to a number of cigarettes smoked per day into mild (≤10), moderate (11‐20) and heavy smokers (≥21). Semen analysis, sperm chromatin condensation integrity with aniline blue staining and sperm viability were compared between the study groups. A significant decrease has been shown in sperm count (p = .006), progressive motility (p = <.001), percentage of normal forms (p = <.001) and viability (p = .002) between infertile nonsmoker and infertile smokers. The percentage of abnormal sperm chromatin condensation was significantly higher in smokers compared to nonsmokers (p = <.001). A linear correlation was detected between the extent of cigarette smoking and the degree of worsening in progressive motility (p = .001), total motility (p < .001), viability (p < .001) and normal morphology (p < .001). These results indicate that cigarette smoking has detrimental effects on semen parameters. It negatively affected all conventional semen parameters in addition to sperm chromatin condensation and sperm viability. These abnormalities were also proportional to the number of cigarettes smoked per day and to the duration of smoking.     

Cystic fibrosis transmembrane conductance regulator is correlated closely with sperm progressive motility and normal morphology in healthy and fertile men with normal sperm parameters

Cystic fibrosis transmembrane conductance regulator (CFTR) has been demonstrated to be expressed in mature spermatozoa and correlated with sperm quality. Sperm CFTR expression in fertile men is higher than that in infertile men suffering from teratospermia, asthenoteratospermia, asthenospermia and oligospermia, but it is unknown whether CFTR is correlated with sperm parameters when sperm parameters are normal. In this study, 282 healthy and fertile men with normal semen parameters were classified into three age groups, group (I): age group of 20–29 years (98 cases, 27.1 ± 6.2), group (II): age group of 30–39 years (142 cases, 33.7 ± 2.6) and group (III): age group of more than or equal to 40 years (42 cases, 44.1 ± 4.6). Sperm concentration, total count and progressive motility were analysed by computer‐assisted sperm analysis. Sperm morphology was analysed by modified Papanicolaou staining. Sperm CFTR expression was conducted by indirect immunofluorescence staining. There was a significant positive correlation (P < 0.001) between CFTR expression and sperm progressive motility (r = 0.221) and normal morphology (r = 0.202), but there were no correlations between sperm CFTR expression and semen volume, sperm concentration, sperm total count as well as male age (P > 0.05). Our findings show that CFTR expression is associated with sperm progressive motility and normal morphology in healthy and fertile men with normal sperm parameters, but not associated with the number of spermatozoa and male age.     

Evaluation of sperm DNA fragmentation and chromatin structure in infertile men with immotile short-tail sperm defect

Teratozoospermia is characterised by the presence of spermatozoa with abnormal morphology. One of the morphological disorders that lead to male infertility is immotile short-tail sperm (ISTS) defect. In this study, we evaluated the levels of chromatin packing and DNA fragmentation in patients with immotile short-tail sperm defect. Semen samples were obtained from 31 infertile men with ISTS as case group and 31 normozoospermic men as a control group. Protamine status was evaluated using chromomycin A3 (CMA3) staining and sperm DNA fragmentation assessed by sperm chromatin structure assay (SCSA) and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate biotin nick-end labelling (TUNEL). The percentage of positive CMA3 spermatozoa was significantly higher in patients’ samples (22.6 ± 6.9) compared with controls (16.3 ± 4.2) (p < .05) and also mean (±SD) of sperm DNA fragmentation was significantly higher in patients compared with controls, as measured by TUNEL assay (10.45 ± 4.60 vs. 7.03 ± 2.86, p < .05) and SCSA (24.80 ± 13.1 vs. 15.2 ± 7.2, p < .05). According to our study, sperm DNA fragmentation and chromatin packing abnormality are significantly higher in the ISTS samples compared with normal samples. A possible explanation for this relationship is that sperm chromatin condensation and sperm flagellum formation occur during the same phase of spermatogenesis.     

Effects of oral antioxidant treatment upon the dynamics of human sperm DNA fragmentation and subpopulations of sperm with highly degraded DNA

The primary aim of this study was to determine the effect of oral antioxidant treatment (1500 mg of l ‐Carnitine; 60 mg of vitamin C; 20 mg of coenzyme Q10; 10 mg of vitamin E; 10 mg of zinc; 200 μg of vitamin B9; 50 μg of selenium; 1 μg of vitamin B12) during a time period of 3 months upon the dynamics of sperm DNA fragmentation following varying periods of sperm storage (0 h, 2 h, 6 h, 8 h and 24 h) at 37 °C in a cohort of 20 infertile patients diagnosed with asthenoteratozoospermia. A secondary objective was to use the sperm chromatin dispersion test (SCD) to study antioxidant effects upon a specific subpopulation of highly DNA degraded sperm (DDS). Semen parameters and pregnancy rate (PR) were also determined. Results showed a significant improvement of DNA integrity at all incubation points (P < 0.01). The proportion of DDS was also significantly reduced (P < 0.05). Semen analysis data showed a significant increase in concentration, motility, vitality and morphology parameters. Our results suggest that antioxidant treatment improves sperm quality not only in terms of key seminal parameters and basal DNA damage, but also helps to maintain DNA integrity. Prior administration of antioxidants could therefore promote better outcomes following assisted reproductive techniques.     

Oxidative stress status and sperm DNA fragmentation in fertile and infertile men

Evidence suggests that disturbing the balance between reactive oxygen species levels and antioxidant contents in seminal plasma leads to oxidative stress resulting in male infertility. This study was carried out to identifying clinical significance of seminal oxidative stress and sperm DNA fragmentation in treatment strategies of male infertility in southwest Iran. Sperm parameters, lipid peroxidation and activity of antioxidant enzymes were assessed in fertile (n = 105) and infertile (n = 112) men. Malondialdehyde (MDA) levels in seminal plasma were found to be higher significantly (p < .001) in patients. Superoxide dismutase (SOD) and glutathione peroxidase (GPx) activities in seminal plasma were significantly (p < .001) lower in infertile men. Significant negative correlations were observed between MDA levels and sperm motility and normal morphology. Spermatozoa with fragmented DNA were higher (p < .001) in infertile men and significantly correlated with MDA levels and SOD and GPx activities. MDA of 4.2 nmol/ml, SOD of 4.89 U/ml and GPx of 329.6 mU/ml were optimum cut‐off limits to discriminate infertile patients from fertile men. The results show the leading role of oxidative stress in aetiology of male infertility in southwest Iran and indicate that evaluation of seminal antioxidant status and DNA integrity can be helpful in men attending infertility clinics during fertility assessment.     

Investigation of serum vitamin D levels in Chinese infertile men

Recently, the question of whether vitamin D exerts an effect on the pathogenic process of infertility has become the centre of attention. There are some controversial conclusions on this issue. Based on previous studies, we sought to explore the difference of serum 25‐hydroxyvitamin D₃, 1,25‐dihydroxyvitamin D₃ levels between infertile patients and fertile men, and to find the influence on semen quality. The analysis of serum 25‐hydroxyvitamin D₃ level showed no significant difference between infertile patients and fertile men. However, the levels of serum 1,25‐dihydroxyvitamin D₃ in oligospermia (P < 0.05), asthenospermia (P < 0.01), oligoasthenospermia (P < 0.05) and azoospermia (P < 0.01) patients were significantly lower than those in fertile men. Moreover, serum 1,25‐dihydroxyvitamin D₃ level was positively correlated with progressive motility and total sperm number in infertile patients. In addition, a positive correlation between serum prolactin and 1,25‐dihydroxyvitamin D₃ was observed in fertile men. Our results indicated that lower vitamin D could be a risk factor for poor semen quality in infertile men. The 1,25‐dihydroxyvitamin D₃, as the biologically active form of vitamin D, may be more significant.     

Male age is more critical to sperm DNA integrity than routine semen parameters in Chinese infertile males

This retrospective study evaluated the correlation between the sperm DNA integrity results and infertile male age or sperm motility in 654 infertile men undergoing infertility evaluations from 2013 to 2016. The correlation between the results of sperm DNA integrity and male age was positive, while a negative correlation was detected between sperm DNA integrity and sperm motility in all subjects. According to age (≤30, 30–35 and ≥35), men with normozoospermia or abnormal semen parameters were, respectively, divided into groups 1, 2 and 3, or groups A, B and C. The sperm DNA fragmentation index (DFI) and DFI abnormality rates in groups 3 and C were highest among their respective cohorts. But they were not significantly different between groups within the same age range. Statistically significant differences were found in male age, progressive motility, as well as total motility between patients with normal DFIs and those with abnormal DFIs in group C, but not in group 3. Older (≥35 years) infertile men have increased sperm DNA fragmentation, independent of conventional semen parameters. Male age is more critical to sperm DNA integrity than routine semen parameters.     

Hypoxia pathway has more impact than inflammation pathway on etiology of infertile men with varicocele

This study aimed to compare main molecular markers of hypoxia (HIF1‐α and P53) and inflammation (TLR‐2, TLR‐4 and TNF‐α) pathways between infertile men with varicocele and fertile individuals. Sperm parameters such as sperm concentration, motility and morphology were assessed according to World Health Organization (Laboratory manual for the examination and processing of human semen. Geneva, Switzerland, 2010) guideline in 20 infertile men with grade II or III varicocele, and 20 fertile men candidate of family balancing. In addition, sperm DNA fragmentation and molecular markers involved in hypoxia and inflammation pathways were evaluated by terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay and real‐time PCR respectively. Mean of sperm parameters (concentration, motility and morphology) and DNA integrity were significantly lower in infertile men with varicocele compared to fertile individuals. Unlike markers involved in inflammation pathway, mean expression of markers of hypoxia pathway (HIF1‐α and P53) was significantly higher in infertile men with varicocele compared to fertile individuals (p < 0.05), and also a significant correlation was observed between expression of HIF1‐α and P53 (r = 0.461; p = 0.003). Overall, the result of this study suggests higher likelihood of involvement of hypoxia pathway, in comparison with inflammation pathway, in pathogenesis varicocele associated with male infertility.     

Randomised clinical trial of comparing effects of acupuncture and varicocelectomy on sperm parameters in infertile varicocele patients

The aim of the study was to evaluate the effect of the acupuncture treatment on sperm parameters and pregnancy rates in patients with primary infertility. Between January 2008 and May 2010, 30 men with the primary infertility (one year of unprotected intercourse, healthy wife) and varicocele with normal hormone levels and abnormal semen analysis were randomised into two groups. Group 1 underwent subinguinal microscopic varicocelectomy, and Group 2 underwent acupuncture treatment twice a week for 2 months. Both groups were evaluated with semen analysis at 6 months after the treatment. Patients in both groups evaluated with telephone calls and e‐mail in terms of pregnancy. The mean age of the patients was 27.2, and groups were comparable regarding the age (P = 0.542). The pre‐treatment sperm concentration, motility and morphological characteristics were similar in both groups. Sperm concentration and motility improved significantly in both groups after the treatment. Increase in sperm concentration was higher in the acupuncture group compared to the varicocelectomy group (P = 0.039). The average follow‐up was 42 months, and pregnancy rates were emphasised 33% in both groups. Acupuncture treatment in primary infertile varicocele patients with semen abnormalities seems to be effective and has comparable results with the varicocelectomy treatment.     

Total antioxidant status and lipid peroxidation with and without in vitro zinc supplementation in infertile men

The aim of this study was to assess the total antioxidant capacity (TAC) and malondialdehyde (MDA) level in infertile men with asthenozoospermia and asthenoteratozoospermia compared to fertile donors, and to examine the effect of zinc on sperm lipid peroxidation and antioxidant status in infertile and fertile men. Semen samples provided by infertile men (n = 38) and fertile donors (controls; n = 12) were exposed to 6 mmol/L of zinc for 2 hr at 37°C. After semen analysis, lipid peroxidation was detected by MDA assay and seminal TAC was assessed by colorimetric method using TAS (total antioxidant status) Kit. TAC was significantly lower in infertile group compared to controls (p = .037). However, lipid peroxidation did not alter in infertile patients compared to controls (p > .05). After in vitro incubation of samples with zinc, a significant increase in TAC level was found only in infertile men (p < .001). Meanwhile, zinc had no effect on sperm lipid peroxidation in both fertile and infertile men (p > .05). Our data indicate that antioxidant treatment based on zinc in vitro supplementation may be helpful to enhance the rate of seminal antioxidant status in infertile men; however, it does not prevent sperm lipid peroxidation.     

Efficacy of the density gradient centrifugation method in eliminating sperm with aneuploidy

The aim of this study was to evaluate the efficacy of the PureSperm density gradient centrifugation on the selecting sperm with less chromosomal aneuploidy. Semen samples were obtained from 30 infertile men with teratozoospermia and 15 fertile men with normal semen parameters. The frequencies of numerical chromosomes aberrations were simultaneously identified in neat semen and in the different fractions of the density gradient centrifugation from the same samples. Using a triple colour FISH, we show that patients with severe teratozoospermia have a significantly increased frequency of chromosomal abnormalities in their neat semen compared with normozoospermic men (P < 0.001). The mean sperm motility and sperm morphology were improved significantly after semen processing with three layers PureSperm gradient compared with whole semen (P < 0.001). In addition, aneuploidy frequencies were lower in specimens enriched by the gradient centrifugation compared with unprocessed semen. Significant differences were observed in the disomy rates for the autosome and for either sex chromosome between the neat semen and the different PureSperm fractions (P < 0.001). In conclusion, our study shows that semen processing by density gradient centrifugation is very efficient in reducing sperm with aneuploidy and diploidy.     

Relationship of semen hyperviscosity with IL‐6, TNF‐α, IL‐10 and ROS production in seminal plasma of infertile patients with prostatitis and prostato‐vesiculitis

Changes in levels of oxidative damage products in semen and their relationship to seminal fluid viscosity (SFV) have recently received increasing research interest. We analysed whether SFV was associated with ROS generation, levels of cytokines TNF‐alpha (TNF‐α), IL‐6 and IL‐10 and seminal leucocyte concentration, and whether ROS production was related to the extent of infections/inflammations at one (prostatitis) or two (prostato‐vesiculitis) male accessory glands. We studied 169 infertile patients, with chronic bacterial prostatitis (PR, n = 74) and/or bilateral prostato‐vesiculitis (PV, n = 95), as diagnosed by the ultrasound (US) criteria. Healthy fertile men (n = 42) served as controls. In the PV patient group, SFV, semen characteristics and ROS production had median values that were significantly higher than those found in PR patients and controls, although other sperm variables had values significantly lower than those found in PR patients or controls. In PV infertile patients, ROS generation and pro‐inflammatory cytokines levels were higher than those found in PR infertile patients and controls, although seminal IL‐10 levels in PV and PR patients were lower than those found in the controls. In PR patients, the levels of SFV were positively related to TNF‐α (r = 0.67; P < 0.01), fMLP‐stimulated ROS production in the 45% Percoll fraction (r = 0.687, P < 0.01) and the 90% Percoll fraction in basal condition (r = 0.695, P < 0.01), and after fMLP‐stimulation (r = 0.688, P < 0.01). Thus, our data indicated that seminal hyperviscosity is associated with increased oxidative stress in infertile men and increased pro‐inflammatory interleukins in patients with male accessory gland infection, more when the infection was extended to the seminal vesicles.     

The correlation between mammalian target of rapamycin (mTOR) gene expression and sperm DNA damage among infertile patients with and without varicocele

This study aimed to assess the possible correlation between mammalian target of rapamycin (mTOR) gene expression and sperm DNA damage among infertile patients with and without varicocele. The study included sixty infertile males and fifty fertile males as controls. The infertile group was subdivided into the following subgroups: thirty males with varicocele and thirty males without varicocele. All subjects underwent medical history collection, clinical examination, semen analysis, sperm DNA integrity assessment, mTOR gene expression assessment and scrotal colour Doppler ultrasound. The mean mTOR gene expression in infertile patients with varicocele (23.52 ± 14.65) was significantly higher than that in infertile patients without varicocele (12.24 ± 12.44) and fertile control subjects (3.92 ± 3.26; p = 0.003 and p < 0.001 respectively). In the infertile varicocele‐positive group, mTOR gene expression showed a significant negative correlation with sperm count (p = 0.028, r = ?0.400) and progressive sperm motility (p = 0.038, r = ?0.381), as well as a significant positive correlation with the sperm DNA fragmentation index (DFI; p = 0.001, r = 0.578). In the infertile varicocele‐negative group, mTOR gene expression showed a significant negative correlation with progressive sperm motility (p = 0.018, r = ?0.429) and a significant positive correlation with sperm DFI (p < 0.001, r = 0.673). In conclusion, according to these results, there is a significant positive correlation between mTOR gene expression and sperm DFI among infertile patients with and without varicocele.     

A randomised trial comparing conventional semen parameters,sperm DNA fragmentation levels and satisfaction levels between semen collection at home and at the clinic

The aim of the randomised trial was to compare conventional semen parameters, sperm DNA fragmentation levels and satisfaction levels between semen samples collected at home and at the clinic. We recruited 110 men with a history of infertility for at least 1 year from the outpatient andrology clinic. Each man collected two semen samples, one at home and one at the clinic. Men were randomly assigned into the home first (n = 55) or clinic first (n = 55) groups. The primary outcome was sperm concentration. There was no significant difference in sperm concentration, sperm DNA fragmentation levels or other conventional semen parameters between home first and clinic first samples (p > .05), while satisfaction levels were significantly higher for home first samples (p < .01). Consistent results were obtained when comparing home-collected and clinic-collected samples within individuals. Men can be offered the option to collect semen samples at home for examination or assisted reproduction without compromising semen quality, especially for those with difficulty in producing semen samples at the clinic.     

Semiquantitative promoter methylation of MLH1 and MSH2 genes and their impact on sperm DNA fragmentation and chromatin condensation in infertile men

To investigate the semiquantitative methylation alterations of MLH1 and MSH2 and the possible association among methylation of MLH1 and MSH2, sperm DNA fragmentation and sperm chromatin condensation in idiopathic oligoasthenoteratozoospermic men. Seventy-five idiopathic infertile men and 52 fertile and/or normozoospermic men were included in the study. SDF was analysed using the TUNEL assay in semen samples of 100 men. Promoter methylation of MLH1 and MSH2 genes was assessed by semiquantitative methylight analysis in semen samples of 39 and 40 men respectively. Sperm chromatin condensation was evaluated using aniline blue staining in 114 men. MLH1 promoter methylation was positively correlated with the percentage of aniline blue positive spermatozoa (r = 0.401, p = 0.0188). On the other hand, MSH2 promoter methylation was negatively correlated with sperm concentration and total sperm count (r = −0.421, p = 0.0068 and r = 0.4408, p = 0.009 respectively). The percentage of aniline blue positive spermatozoa in the control group was significantly lower than in the OAT group (p < 0.0001) and negatively correlated with total sperm count (r = −0.683, p < 0.0001), progressive sperm motility (r = −0.628, p < 0.0001), total motility (r = −0.639, p < 0.0001) and normal morphology (r = −0.668, p < 0.0001). Promoter methylation profile of MLH1 and MSH2 genes may play role on sperm DNA packaging and conventional semen parameters respectively.