Widespread epigenetic abnormalities suggest a broad DNA methylation erasure defect in abnormal human sperm

Abstract

Background: Male-factor infertility is a common condition, and etiology is unknown for a high proportion of cases. Abnormal epigenetic programming of the germline is proposed as a possible mechanism compromising spermatogenesis of some men currently diagnosed with idiopathic infertility. During germ cell maturation and gametogenesis, cells of the germ line undergo extensive epigenetic reprogramming. This process involves widespread erasure of somatic-like patterns of DNA methylation followed by establishment of sex-specific patterns by de novo DNA methylation. Incomplete reprogramming of the male germ line could, in theory, result in both altered sperm DNA methylation and compromised spermatogenesis.

Methodology/principal finding: We determined concentration, motility and morphology of sperm in semen samples collected by male members of couples attending an infertility clinic. Using MethyLight and Illumina assays we measured methylation of DNA isolated from purified sperm from the same samples. Methylation at numerous sequences was elevated in DNA from poor quality sperm.

Conclusions: This is the first report of a broad epigenetic defect associated with abnormal semen parameters. Our results suggest that the underlying mechanism for these epigenetic changes may be improper erasure of DNA methylation during epigenetic reprogramming of the male germ line.

Figure 1. Box plots illustrating associations between semen parameters and level of methylation (PMR, on the natural-log scale) in DNA isolated from 65 study sperm samples.

DNA methylation was measured by MethyLight. Methylation targets were sequences specific to the genes HRAS, NTF3, MT1A, PAX8, PLAGL1, DIRAS3, MEST and SFN and the repetitive element Satellite 2 (SAT2CHRM1). P-value for trend over category of semen parameter is given for each plot. Rows: DNA methylation targets; columns: semen parameters.

Figure 2. Cluster analysis of 36 MethyLight targets in 65 study sperm DNA samples.

Left: dendrogram defining clusters; rows: 35 methylation targets; columns: 65 study samples ordered left to right on sperm concentration (samples A–G were also included in Illumina analyses (see Figure 3)) with poor to good concentration (blue), motility (purple), and morphology (green) represented by darkest to lightest hue; body of figure: standardized PMR values represented lowest to highest as yellow to red. X = missing.

Figure 3. Results of Illumina analysis of 1,421 autosomal sequences in DNA isolated from sperm and buffy coat.

Seven study sperm samples (A–G; with values of sperm concentration (10⁶ sperm/ml) A:20, B:56, C:62, D:67, E:75, F:94, G:95), screening sperm sample (S), two buffy coat samples (1–2). Level of DNA methylation scored as β-value. Color: β-value for column sample at row sequence (green: β<0.1; yellow: 0.1≤β≤0.25; orange 0.25<β≤0.5; red: β>0.5). MI and PI: maternally and paternally imprinted genes (black bar). Sequences assigned to tertile of median β-value among buffy coat DNA samples (I, II, III) and sorted within tertile on median β-value among sperm DNA samples. Box 1: sequences with sperm-specific DNA methylation; Box 2: sequences with buffy coat-specific DNA methylation.