Genome-Wide Methylation Analyses of Human Sperm Unravel Novel Differentially Methylated Regions in Asthenozoospermia

Figures & data

Table 1. Semen parameters of different study groups.

Parameters	Normozoospermia (n = 5)		Asthenozoospermia (n = 7)
	Mean (± standard deviation)	Range	Mean (± standard deviation)	Range
Age (years)	34.00 (± 6.04)	27–42	35.43 (± 7.04)	30–50
Semen volume (ml)	3.16 (± 1.25)	1.8–5.0	3.71 (± 1.35)	2.4–5.3
Sperm concentration (×10^6/ml)	83.50 (± 31.04)	34.7–115.1	73.96 (± 40.30)	33.2–150.0
Motility (% progressive)	71.20 (± 10.55)	61–85	10.39 (± 6.92)	5–23
Percentage of normal sperm form (%)	9.48 (± 3.52)	5.9–13.8	5.04 (± 2.32)	2.8–8.7
Vitality (%)	91.00 (± 3.24)	88–95	53.57 (± 24.37)	15–84

Figure 1. Global DNA methylation profiles and asthenozoospermia-associated differentially methylated regions.

(A) Histogram of methylation values for all CpGs in one representative sample. As expected, it showed bimodal distributions, suggesting a high experimental quality. (B) Global methylation levels of all samples. The ast1-7 and nor1-5 are sample IDs of asthenozoospermia and normozoospermia, respectively. (C) No significant global DNA methylation differences were found between groups (p > 0.05). (D) Heatmap of methylation data z-scores of 238 significantly differentially methylated regions. (E) Distribution of candidate DMRs across chromosomes, where the dotted line represents a threshold of p < 0.05, and dots above the line were chosen as significant DMRs.

DMR: Differentially methylated region.

Figure 2. Distribution of the differentially methylated regions and their genomic locations.

(A) Count of hypermethylated DMRs and hypomethylated DMRs. (B) Histogram showing the distribution of degrees of methylation difference. (C) Percentage of DMRs in distinct genomic contexts. (D) Relative distances of DMRs to the TSS.

DMR: Differentially methylated region; TSS: Transcription start site.

Table 2. Summary of genes harboring differentially methylated regions in their promoter regions.

Gene symbol	Protein	Genomic location	Methylation direction^†	Methylation difference	Function summary
ACOT7	Acyl-CoA Thioesterase 7	Promoter	Hypomethylation	-15	Hydrolyze the CoA thioester of palmitoyl-CoA and other long-chain fatty acids
IK	IK Cytokine	Promoter	Hypomethylation	-13	Required for normal mitotic cell cycle progression
ZNF442	Zinc Finger Protein 442	Promoter	Hypomethylation	-19	May be involved in transcriptional regulation
MAP1S	Microtubule Associated Protein 1S	Promoter	Hypermethylation	16	Microtubule-associated protein that mediates aggregation of mitochondria, resulting in cell death and genomic destruction
TDRP	Testis Development Related Protein	Promoter	Hypomethylation	-14	Contributes to normal sperm motility
TPO	Thyroid Peroxidase	Promoter	Hypermethylation	11	Plays a central role in thyroid gland function
XRRA1	X-ray Radiation Resistance Associated 1	Promoter	Hypomethylation	-16	May be involved in the response of cells to x-ray radiation
ENO1	Enolase 1	Promoter	Hypomethylation	-14	Glycolytic enzyme that catalyzes the conversion of 2-phosphoglycerate to phosphoenolpyruvate
OR52B4	Olfactory Receptor Family 52 Subfamily B Member 4	Promoter	Hypermethylation	12	Odorant receptor
CHAMP1	Chromosome Alignment Maintaining Phosphoprotein 1	Promoter	Hypomethylation	-12	A zinc finger protein that functions as a regulator of chromosome segregation in mitosis
SMYD2	SET And MYND Domain Containing 2	Promoter	Hypomethylation	-11	Protein-lysine N-methyltransferase that methylates both histones and nonhistone proteins
TUBGCP5	Tubulin Gamma Complex Associated Protein 5	Promoter	Hypomethylation	-10	γ-Tubulin complex, necessary for microtubule nucleation at the centrosome
OR52K2	Olfactory Receptor Family 52 Subfamily K Member 2	Promoter	Hypermethylaton	12	Odorant receptor
AKIRIN1	Akirin 1	Promoter	Hypomethylation	-13	May regulate chemotaxis of both macrophages and myoblasts
PSTPIP1	Proline-Serine-Threonine Phosphatase Interacting Protein 1	Promoter	Hypermethylaton	16	A cytoskeletal protein involved in regulation of the actin cytoskeleton
RBM15B	RNA Binding Motif Protein 15B	Promoter	Hypomethylation	-13	An RNA-binding protein that regulates several different processes such as alternative splicing
RPS25	Ribosomal Protein S25	Promoter	Hypomethylation	-14	A ribosomal protein that is a component of the 40S subunit

† Methylation direction is the change of methylation in asthenozoospermia compared with normal controls.

Table 3. Differentially methylated region(s)-harboring genes involved in sperm functions or spermatogenesis.

Gene symbol	Protein	Genomic location	Methylation direction^†	Methylation difference	Function summary
LRGUK	Leucine-Rich Repeat and Guanylate Kinase Domain-Containing Protein	Exon	Hypermethylation	16	Involved in multiple aspects of sperm assembly, including acrosome attachment, shaping of the sperm head and early aspects of axoneme development
DZIP1	DAZ Interacting Zinc Finger Protein 1	Intron	Hypomethylation	-11	Participates in spermatogenesis and has a role in primary cilium formation
DNAAF5	Dynein Axonemal Assembly Factor 5	Intron	Hypomethylation	-16	Essential for the preassembly or stability of axonemal dynein arms and plays a crucial role in cilium motility
SRPK1	Serine/Arginine-Rich Splicing Factor Kinase 1	Distal Intergenic	Hypomethylation	-14	Can influence chromatin reorganization in somatic and sperm cells and cell cycle progression
TDRP	Testis Development Related Protein	Promoter	Hypomethylation	-14	Contributes to normal sperm motility

† Methylation direction is the change of methylation in asthenozoospermia compared with normal controls.

Figure 3. Methylation levels of selected representative differentially methylated regions and gene ontology analysis of annotated genes.

(A–E) Plots of five selected DMRs with regard to genes functioning in sperm motility or spermatogenesis. Each dot represents the methylation level of an individual CpG in a single sample, and the size of the dot is representative of coverage. The lines represent smoothed methylation levels for each sample, either normozoospermic controls (blue) or asthenozoospermic samples (red). CpG annotations are shown below the plot. If the DMR is located in the gene body of a gene, the exon of the gene is also shown below. (F) Gene ontology analysis of biological process, cellular component and molecular function of DMR-annotated genes.

DMR: Differentially methylated region.