The methylation level in each cytosine in sperm DNA was analyzed by pyrosequencing. (A) DNA methylation in 12 cytosines (CpG) in H4K12ac-associated NSD1 promoter in fertile donors and subfertile patients. (B) Averaged DNA methylation in 1 to 4 CpGs and 5 to 12 CpGs of NSD1 promoter in fertile and subfertile men. (C) DNA hypermethylation in 12 CpGs in sperm-specific protamine promoter. (D) Averaged methylation from 12 CpGs in protamine promoters showing no significant difference between fertile and subfertile sperm.