Age-related methylation changes in the human sperm epigenome

Abstract

Advanced paternal age is associated with increased risks for reproductive and offspring medical problems. Accumulating evidence suggests age-related changes in the sperm epigenome as one underlying mechanism. Using reduced representation bisulfite sequencing on 73 sperm samples of males attending a fertility center, we identified 1,162 (74%) regions which were significantly (FDR-adjusted) hypomethylated and 403 regions (26%) being hypermethylated with age. There were no significant correlations with paternal BMI, semen quality, or ART outcome. The majority (1,152 of 1,565; 74%) of age-related differentially methylated regions (ageDMRs) were located within genic regions, including 1,002 genes with symbols. Hypomethylated ageDMRs were closer to transcription start sites than hypermethylated DMRs, half of which reside in gene-distal regions. In this and conceptually related genome-wide studies, so far 2,355 genes have been reported with significant sperm ageDMRs, however most (90%) of them in only one study. The 241 genes which have been replicated at least once showed significant functional enrichments in 41 biological processes associated with development and the nervous system and in 10 cellular components associated with synapses and neurons. This supports the hypothesis that paternal age effects on the sperm methylome affect offspring behaviour and neurodevelopment. It is interesting to note that sperm ageDMRs were not randomly distributed throughout the human genome; chromosome 19 showed a highly significant twofold enrichment with sperm ageDMRs. Although the high gene density and CpG content have been conserved, the orthologous marmoset chromosome 22 did not appear to exhibit an increased regulatory potential by age-related DNA methylation changes.

Keywords: ART outcome; DNA methylation; human sperm epigenome; male germ cells; paternal age effect.

Figure 1

Region characteristics. (A) Distribution of methylation levels of ageDMRs versus other (non-significant) regions. The average methylation levels of ageDMRs (orange line) are predominantly in the mid-range (20-80%), whereas other regions (blue line) are either in the low range (< 20%) or high range (> 80%) of methylation. (B) Box plots showing the distance of analyzed regions to the nearest transcription start site (TSS). The median is represented by a horizontal line. The bottom of the box indicates the 25^th percentile, the top the 75^th percentile. The blue box represents non-significant regions, the orange box ageDMRs which lose methylation with age and the red box ageDMRs which gain methylation with age. Please note that hypomethylated ageDMRs are significantly (*** P < 0.001) closer to the TSS, whereas hypermethylated DMRs are more distant from the TSS than other regions. (C) Localization of hypomethlyated ageDMRs (orange bars) and hypermethylated DMRs (red bars) in different genic and intergenic regions, compared to non-significant regions (blue bars). Please note that some regions may be assigned to several gene parts and, therefore, the percentages of all bars (of one color) total > 100%.

Figure 2

Validation of sperm ageDMRs by bisulfite pyrosequencing. Scatter plots showing the correlations between average regional methylation (y-axis in %), determined by bisulfite pyrosequencing, and donor age (x-axis in years) in 94 human sperm samples. The blue dots represent pyrosequencing measurements for an ageDMR (identified by RRBS) in EEF1A2, the red dots for an ageDMR in MBD3, the green dots for an ageDMR in PRAM1, and the yellow dots for an ageDMR in PRKAR2A. Consistent with the results of RRBS, the regression lines of all analyzed regions indicate a significant (also see Supplementary Table 3) loss of methylation with age. The correlations remain virtually unchanged when excluding the 72-year-old sample from analysis.

Figure 3

Chromosomal distribution of human sperm ageDMRs. The upper panel shows a Manhattan plot of 360,264 regions analysed by RRBS. 1,565 (0.4%) regions above the red line are endowed with genome-wide significant ageDMRs. The bottom plot shows the chromosomal distribution of the 1,565 ageDMRs (orange bars), compared to the 358,699 other (non-significant) regions (blue bars). The y-axis represents the percentage of ageDMRs and other regions, respectively, on each chromosome. AgeDMRs are significantly overrepresented on chromosome 19, and depleted on chromosomes 6, 10, and 21.