DNA methylation level of spermatozoa from subfertile and proven fertile and its relation to standard sperm parameters.

The epigenetic mechanism plays an important role in spermatogenesis such as DNA methylation where this episode is represented by either switching genes on or off. Twenty-eight samples (14 case and 14 controls) were subjected to Infinium 450K BeadChip arrays to identify genomic regions that differ in sperm DNA methylation patterns in the subfertile compared to the proven fertile group. Then two CpGs were validated by deep bisulphite sequencing on 82 sperm samples. The results screening study revealed eight CpGs were significantly different in their sperm DNA methylation levels between cases and control group. The results of the validation study for the two CpGs (cg19779893 and cg19406113) showed that a significant variation in the methylation level at 2 CpGs of 3 CpGs related to cg19779893 site amplicon in cases compared to the controls. Moreover, six CpGs related to the cg19406113 site amplicon showed significant differences in sperm DNA methylation between the cases and the control group. Furthermore, there was a significant decrease in the sperm parameters in the cases compared to the control group. This study found two CpGs altered in their sperm DNA methylation levels. In addition, a strong association was found between changes in the sperm DNA methylation levels in these CpGs sites and sperm parameters.

Impact of cigarette-smoking on sperm DNA methylation and its effect on sperm parameters.

DNA methylation is an epigenetic modification of the genome. The purpose of this study was to determine the influence of cigarette-smoking on sperm DNA methylation from a genomewide survey of sperm samples and to ascertain its effect on sperm parameters. Twenty-eight sperm DNA samples (from 14 fertile smokers as a case study and 14 proven fertile nonsmokers as controls) were subjected to Infinium 450K BeadChip arrays to identify the changes in the DNA methylation level between the two groups. Then, deep bisulphite sequencing was used to validate five CpGs on 78 samples. The results from the Infinium 450K found that only 11 CpGs showed a significant difference in DNA methylation between the case and the control groups. Five CpGs of the eleven (cg00648582, cg0932376, cg19169023, cg23841288 and cg27391564) underwent deep bisulphite sequencing where cg00648582, related to the PGAM5 gene, and the cg23841288 CpGs, related to the PTPRN2 gene amplicons, showed a significant increase in their DNA methylation level in more than one CpG in the case group. In contrast, a significant decrease was found at cg19169023 and at its various neighbouring CpGs in the TYRO3 gene-related amplicons. Furthermore, this study demonstrated a significant correlation between the variation in sperm DNA methylation level and standard sperm parameters in the case group.

Cigarette smoking induces only marginal changes in sperm DNA methylation levels of patients undergoing intracytoplasmic sperm injection treatment.

DNA methylation plays important roles in genome stability and regulation of gene expression. This study was designed to determine the influence of cigarette smoking on sperm DNA methylation. From a genome-wide survey on sperm samples, differentially methylated target CpGs should be selected and subjected to local deep bisulphite sequencing. Obtained methylation data are compared to sperm parameters and (ICSI) outcome. Similar to pilot study, samples were subjected to Infinium 450K BeadChip arrays to identify alterations in sperm DNA methylation between smokers and nonsmokers males. Routine testing on a significantly altered CpG site was performed on more samples using local deep bisulphite sequencing. Of approximately 485,000 CpG sites analysed, only seven CpGs were found to show a significant DNA methylation difference of >20% with the top six CpGs overlapping common SNP sites. The remaining CpG site (cg19455396) is located in intron 12 of the TAP2 gene. The results of deep bisulphite sequencing showed only a tendency towards hypomethylation in the smoking group. This study could not detect biologically relevant CpG positions that are altered in sperm DNA methylation on the influence of cigarette smoking beyond individual-specific effects that may be caused by other environmental factors.

Aberrant DNA methylation patterns of human spermatozoa in current smoker males.

The purpose of this study was to investigate the impact of current cigarette smoking on sperm DNA methylation patterns. A total of 108 males (51 current smokers and 57 never smoked males) were included in the study. Using 450 BeadChip Arrays, the differentially methylated CpGs between current smokers (n=15) and never smoked males (n=15) were identified. Out of significantly 11 CpGs identified, 2 CpGs namely cg07869343 and cg19169023, which are located in the MAPK8IP3 and TKR genes were selected for further analysis. Using deep bisulfite sequencing in an independent cohort of current smokers (n=36) and never smoked males (n=42), 6 and 1 CpGs showed a significant difference in the MAPK8IP (CpG3, CpG5, CpG6, CpG7, CpG8, and CpG21) and in the TKR (CpG4) were identified, respectively (P≤0.05). Our results indicate that cigarette smoking causes biochemical changes in the sperm DNA methylation in many regions and could adversely affect semen parameters.

Spermatozoa from males with reduced fecundity exhibit differential DNA methylation patterns.

Infertility affects 10-15% of couples, and approximately 50% of cases are linked to male factor infertility. The purpose of this study was to evaluate the DNA methylation patterns in spermatozoa from males who are suffering from a reduction in fecundity. Thirty samples were subjected to 450K arrays as a screening study to evaluate the variation in sperm DNA methylation levels between cases and controls groups, and then four CpG sites (cg05799088, cg07227024, cg16338278, and cg08408433) underwent to deep bisulfite sequencing to validate the observed methylation differences in 111 samples (56 proven fertile males as 'controls' and 55 males suffering from a reduction in fecundity as 'cases'). A significant difference in the mean methylation level was found between cases and controls in the CpGs of PRICKLE2 gene-related amplicon (CpG1, p ≤ 0.002, and CpG2, p ≤ 0.004) and CpG of ALS2CR12 gene-related amplicon (CpG1, p ≤ 0.015, and CpG2, p ≤ 0.009). Besides, a significant difference was found at seven from thirteen CpGs tested in the ALDH3B2 gene amplicon CpG2, CpG6, CpG9, CpG10, CpG11, CpG12, and CpG13 (p ≤ 0.005, p ≤ 0.004, p ≤ 0.012, p ≤ 0.028, p ≤ 0.012, p ≤ 0.009, and p ≤ 0.001, respectively). In addition, the results showed that nine CpGs out of the twenty-six within the PTGIR gene-related amplicon (CpG4, CpG6, CpG8, CpG9, CpG11, CpG15, CpG19, CpG23, and CpG26) had a significant difference in their mean methylation level (p ≤ 0.006, p ≤ 0.009, p ≤ 0.003, p ≤ 0.003, p ≤ 0.007, p ≤ 0.002, p ≤ 0.018, p ≤ 0.018, and p ≤ 0.040, respectively) in the case vs. CONTROL GROUP: In conclusion, an alteration in the methylation levels of sperm DNA from males with reduced fecundity was observed. In addition, an association between changes in the methylation level for these CpGs and different semen parameters has been found.

Gut memories do not fade: epigenetic regulation of lasting gut homing receptor expression in CD4+ memory T cells.

The concept of a "topographical memory" in lymphocytes implies a stable expression of homing receptors mediating trafficking of lymphocytes back to the tissue of initial activation. However, a significant plasticity of the gut-homing receptor α4ß7 was found in CD8+ T cells, questioning the concept. We now demonstrate that α4ß7 expression in murine CD4+ memory T cells is, in contrast, imprinted and remains stable in the absence of the inducing factor retinoic acid (RA) or other stimuli from mucosal environments. Repetitive rounds of RA treatment enhanced the stability of de novo induced α4ß7. A novel enhancer element in the murine Itga4 locus was identified that showed, correlating to stability, selective DNA demethylation in mucosa-seeking memory cells and methylation-dependent transcriptional activity in a reporter gene assay. This implies that epigenetic mechanisms contribute to the stabilization of α4ß7 expression. Analogous DNA methylation patterns could be observed in the human ITGA4 locus, suggesting that its epigenetic regulation is conserved between mice and men. These data prove that mucosa-specific homing mediated by α4ß7 is imprinted in CD4+ memory T cells, reinstating the validity of the concept of "topographical memory" for mucosal tissues, and imply a critical role of epigenetic mechanisms.

Regional distribution of body fat in relation to DNA methylation within the LPL, ADIPOQ and PPARγ promoters in subcutaneous adipose tissue.

Obesity may be related to differential DNA methylation and thus to differential expression of key genes in adipose tissue metabolism, such as LPL, ADIPOQ and PPARγ. Using subcutaneous adipose tissue (SAT) from 59 individuals of the European Prospective Investigation into Cancer and Nutrition-Potsdam study, we performed quantitative DNA methylation analysis within the promoters of LPL (LPL-CG1 and -CG2), ADIPOQ (ADIPOQ-CG1 and-CG2) and PPARγ (PPARγ-CG1). We then studied DNA methylation in relation to SAT gene expression, body composition measured using whole-body magnetic resonance imaging, body mass index (BMI), waist circumference (WC) and long-term changes in BMI and WC. For LPL-CG1 and LPL-CG2, higher methylation levels were associated with lower LPL expression, but with higher past WC gain. LPL-CG1 was also positively associated with BMI, WC, and visceral and subcutaneous fat mass. ADIPOQ-CG1 or -CG2 methylation exhibited no association with ADIPOQ expression or with anthropometric parameters. PPARγ-CG1 methylation was significantly higher in individuals with higher visceral fat mass. Among the investigated sites, LPL-CG1 methylation showed the strongest association with gene expression and regional body fat distribution, thereby possibly linking the degree of obesity with major metabolic processes in SAT.

Next-generation sequencing reveals regional differences of the α-synuclein methylation state independent of Lewy body disease.

The α-synuclein gene (SNCA) plays a major role in the aetiology of Lewy body disease (LBD) including Parkinson's disease (PD). Point mutations and genetic alterations causing elevated gene expression are causally linked to familial PD. To what extent epigenetic changes play a role in the regulation of α-synuclein expression and may contribute to the aetiology of sporadic LBD is a matter of debate. We analysed the methylation state of the promoter region and a CpG-rich region of intron 1 of α-synuclein in several brain regions in sporadic LBD and controls using 454 GS-FLX-based high-resolution bisulphite sequencing. Our results indicate that there are significant differences in the level of methylation between different brain areas. The overall methylation levels in the promoter and intron 1 of α-synuclein are rather low in controls and-in contrast to previously reported findings-are not significantly different from LBD. However, single CpG analysis revealed significant hyper- and hypomethylation at different positions in various brain regions and LBD stages. A slight overall increase in methylation related to LBD patients' age was detected.

DNA methylation studies on imprinted loci in a male monozygotic twin pair discordant for Beckwith-Wiedemann syndrome.

Beckwith­Wiedemann syndrome (BWS) is one of the most prevalent congenital disorders predominantly caused by epigenetic alterations. Here we present an extensive case study of a monozygotic monochorionic male twin pair discordant for BWS. Our analysis allows to correlate BWS symptoms, like a protruding tongue, indented ears and transient neonatal hypoglycaemia, to an abnormal methylation at the KvDMR1. DNAs extracted from peripheral blood, skin fibroblasts, saliva and buccal swab of both twins, their sister and parents were analysed at 11 differentially methylated regions (DMRs) including all four relevant DMRs of the BWS region. The KvDMR1 was exclusively found to be hypomethylated in all cell types of the affected BWS twin, while the unaffected twin and the relatives showed normal methylation in fibroblasts, buccal swab and saliva DNA. Interestingly, the twins share a common blood-specific hypomethylation phenotype most probably caused by a feto-fetal transfusion between both twins. Because microsatellite analysis furthermore revealed a normal biparental karyotype for chromosome 11, our results point to an exclusive correlation of the observed BWS symptoms to locally restricted epimutations at the KvDMR1 of the maternal chromosome.