| Popis: |
There is considerable evidence to suggest that male fertility is decreasing worldwide, and it is thought that lifestyle and environmental factors could play a role. Lifestyle and environmental information was collected from men attending a fertility clinic by questionnaire and correlated with sperm parameters and the outcomes of assisted reproductive techniques (ART). Global and gene-specific DNA methylation in these men was measured by bisulphite pyrosequencing and compared with questionnaire results. Finally, an in vitro glycation assay was developed to investigate the effects of advanced glycation end products (AGEs) on sperm: AGE formation, oxidative DNA damage and reactive oxygen species (ROS) production were then measured using flow cytometry and immunocytochemical methods. BMI was the main determining factor for sperm quality and ART outcomes in this study: high BMI was associated with lower sperm concentration and lower fertilisation rates in in vitro cytoplasmic sperm injection (ICSI) patients. Surprisingly, having an unhealthy diet was positively associated with progressive sperm motility. DNA methylation analysis revealed that exposure to glues, adhesives and resins was associated with higher global DNA methylation. A major marker of low sperm concentration was promoter hypermethylation of the spermatogenesis-associated gene, DAZL. Furthermore, global DNA methylation and MEG3 methylation were higher in men with greater numbers of immotile sperm and those with low sperm motility, respectively. Overall, elevated DNA methylation was found to be the main feature of poor sperm quality, however prepared sperm showed higher methylation than whole sperm from neat semen. This large study indicates that DNA methylation is likely to be linked to sperm function, although the role of lifestyle-acquired epimutations in this may be minor. Treatment of sperm with glyoxal in vitro caused rapid formation of the major AGE-adduct, CML. This occurred alongside an increase in oxidative DNA damage but did not affect sperm hyaluronidase activity. This study shows that one of the mechanisms of AGE-related DNA damage is due to oxidative stress and this may present a risk to sperm DNA integrity in vivo. |
