Impact of air pollution from different sources on sperm DNA methylation.

Environmental exposure is associated with increased incidence of respiratory and cardiovascular diseases and reduced fertility. Exposure to air pollution can influence gene expression through epigenetic mechanisms. In this study, we analysed gene-specific CpG methylation in spermatozoa of city policemen occupationally exposed to air pollution in two Czech cities differing by sources and composition of the air pollution. In Prague, the pollution is mainly formed by NO2 from heavy traffic. Ostrava is a hotspot of industrial air pollution with high concentrations of particular matter (PM) and benzo[a]pyrene (B[a]P). We performed genome-wide methylation sequencing using the SureSelectXT Human Methyl-Seq system (Agilent Technologies) and next-generation sequencing to reveal differentially methylated CpG sites and regions. We identified differential methylation in the region chr5:662169 - 663376 annotated to genes CEP72 and TPPP. The region was then analysed in sperm DNA from 117 policemen using targeted methylation sequencing, which proved its hypermethylation in sperm of Ostrava policemen.

Association between sperm mitochondrial DNA copy number and deletion rate and industrial air pollution dynamics.

The effects of air pollution on men's reproductive health can be monitored by evaluating semen quality and sperm DNA damage. We used real-time PCR to analyse the effects of air pollution on sperm mitochondrial DNA copy number (mtDNAcn) and deletion (mtDNAdel) rates in semen samples collected from 54 men in two seasons with different levels of industrial and traffic air pollution. MtDNAdel rates were significantly higher following the high exposure period and were positively correlated with mtDNAcn. However, we did not find any difference in mtDNAcn between the two seasons. MtDNAcn was positively correlated with the DNA fragmentation index and the rates of sperm with chromatin condensation defects, previously assessed by sperm chromatin structure assay, and negatively correlated with sperm concentration, progressive motility, viability, and normal morphology. This indicates that mtDNAcn is more closely associated with male fertility than mtDNAdel rates. In contrast, mtDNAdel might be a more sensitive biomarker of air pollution exposure in urban industrial environments.

Effects of the air pollution dynamics on semen quality and sperm DNA methylation in men living in urban industrial agglomeration.

Human populations living in urban industrial regions of developed countries are exposed to high levels of environmental pollutants. The reproductive consequences of the exposure to air pollution can be monitored through semen analysis and molecular methods. In this study, we tested the possible impact of seasonal changes in the level of air pollution on the semen quality and sperm DNA methylation of 24 men living and working in the industrial agglomeration of Ostrava (Czech Republic). The study participants were healthy non-smokers. The study group was homogeneous regarding their profession, moderate alcohol consumption, no drug abuse and no additional exposure to chemical toxicants. We performed targeted methylation next generation sequencing (NGS) using Agilent SureSelect Human Methyl-Seq and Illumina NextSeq 500 platform to analyze semen samples collected repeatedly from the same men following the season of high (winter) and low (summer) air pollution exposure. We did not detect any adverse effects of the increased exposure on the semen quality; neither we found any difference in average sperm DNA methylation between the two sampling periods. Our search for differentially methylated CpG sites did not reveal any specific CpG methylation change. Our data indicate that the seasonal changes in the level of the air pollution probably do not have any substantial effect on sperm DNA methylation of men living in the highly polluted industrial agglomeration for a long period of time.

Semen quality and sperm DNA integrity in city policemen exposed to polluted air in an urban industrial agglomeration.

BACKGROUND: We examined sperm quality in a cohort of city policemen in Ostrava at the end of a period with high concentrations of air pollutants (winter) and in the same cohort at the end of a relatively low exposure period (summer). METHODS: The study group was comprised of 54 nonsmoking city policemen living and working in Ostrava, Czech Republic. Average daily air-pollutant concentrations recorded by stationary monitoring for 90 days preceding the collection of semen samples were evaluated for different city districts of Ostrava. Standard semen parameters were assessed according to the guidelines of the World Health Organization (2010). The parameters were semen volume, sperm concentration, sperm morphology, sperm motility, acrosome reaction and sperm plasma membrane integrity. Sperm DNA damage was analysed by the sperm chromatin structure assay (SCSA). Sperm motion characteristics were determined by Computer Assisted Semen Analysis (CASA). RESULTS: The concentrations of all monitored pollutants (particulate matter, sulphur dioxide, nitrogen oxides, carbon monoxide, benzo[a]pyrene, benzene) were significantly increased during winter (p < 0.001), except for ozone, the concentration of which was significantly higher during summer. Sperm volume, concentration, % vitality, % sperm morphology (normal form) and % acrosome-intact sperm did not differ significantly between the monitoring periods. The percentages of total motility and progressive motility were significantly higher in March, i.e. at the end of winter (p = 0.001). However, CASA testing showed differences in sperm motion kinetics between spring and autumn samples. In the spring samples, we found a significantly lower % of straightness (p = 0.044) and the length of straight-line path (p = 0.01), while linearity and straight-line velocity were near the borderline value (p = 0.064; p = 0.054, respectively). As compared to summer, high exposure to air pollution during winter significantly increased the extent of sperm chromatin integrity damage (median 22.6 vs. 18.6%) (p = 0.003) and the proportion of immature spermatozoa (median 11.2 vs. 9.9%, p = 0.001). Sperm DNA damage negatively correlated with total motility and progressive motility (r = -0.611, -0.299; p < 0.001). The negative correlation with vitality, normal morphology and acrosome-intact sperm (r = -0.522, -0.550 and -0.511, respectively) was also significant (p < 0.001). CONCLUSION: The examination of the same cohort of city policemen at the end of a period of high air pollution and at the end of relatively low exposure reduced the effects of age, different lifestyles, different occupational exposures, localities and genetic polymorphism on sperm quality impairment associated with air pollution. This study did not demonstrate impaired standard semen parameters in association with exposure. It was shown that sperm chromatin damage and the percentage of immature sperm were highly sensitive to air pollution.

The effects of age on DNA fragmentation, the condensation of chromatin and conventional semen parameters in healthy nonsmoking men exposed to traffic air pollution.

BACKGROUND: Numerous studies have investigated age-based declines in semen traits, but the impact of paternal age on semen parameter values remains inconclusive. OBJECTIVES: The aim of this study was to detect an impact of age on semen quality was studied in healthy nonsmoking men exposed to traffic air pollution. METHODS: Semen samples from 150 Prague City policemen aged 23 to 63 years were examined for standard semen parameters, sperm DNA fragmentation and high DNA stainability. RESULTS: A significant positive correlation was found between age and %DFI (r = .359, P < .001), and negative correlations were found between age and sperm vitality (r = -.247, P < .001), the % acrosome-intact sperm (r = -.202, P = .013) and the % normal sperm heads (r = -.204, P = .012). A weak but significant negative correlation was found for high DNA stainability (% HDS) vs age (r = -.161, P = .050). No significant correlation was detected between male age and the other investigated semen quality parameters. At ages of 23 to 30, 31 to 40, 41 to 50, and 51 to 63 years, the mean %DFI values were 12.7 ± 7.18, 14.7 ± 7.42, 19.6 ± 11.25, and 34.2 ± 15.08, respectively. CONCLUSION: Our study shows a strong relationship (P < .001) between the age of men and sperm DNA fragmentation in an occupational cohort at risk of exposure to heavy traffic-related air pollution in a large city center.

Relationship between reactive oxygen species production in human semen and sperm DNA damage assessed by Sperm Chromatin Structure Assay.

AIM: The aim of this prospective study was to find possible relationship between ROS production measured by chemiluminescence and flow cytometry in human semen and sperm DNA damage estimated by Sperm Chromatin Structure Assay. METHODS: Study included 39 men from infertile couples and 23 fertile volunteers who served as a control group. Aliquot of neat semen was used for ROS detection by chemiluminescence. Aliquot of sperm suspension in phosphate buffered saline was used for the detection of ROS by flow cytometry. Another aliquot of sperm suspension was used for SCSA to measure DNA fragmentation index and High DNA stainability. RESULTS: DNA fragmentation index correlated negatively with sperm morphology and motility. High DNA stainability correlated positively with ROS production and negatively with sperm morphology and concentration. Although there were similar trends of rising DNA fragmentation index and ROS production among the three groups of men, the relationship did not reach statistical significance. CONCLUSIONS: Higher values of DNA fragmentation index and high DNA stainability may also reflect developmental and/or environmental problems and not only oxidative stress.