Table 1.

Summary of studies investigating the relationship between spermatozoal DNA methylation and male infertility

Gene	Study group (sample size)	Summary of findings	Reference
Genome-wide DNA methylation
Global	Normospermic fertile men (17) and normospermic infertile patients (29)	• Aberrant methylation regions are locus-specific • Genome of spermatozoa is hypomethylated	[17]
Global	Male (20) “High quality sperm” vs. “Poor quality sperm”	• No global methylation • No differential methylation in CGIs 772 significant regional methylation alternations	[46]
H19 and DAZL LINE1	Oligozoospermic men (20) Asthenozoospermic men (20) Normozospermic men (20)	• No association between asthenozoospermia and normozospermia in H19-DMR • Severe hypomethylation pattern at H19-DMR CTCF-binding site 6 DAZL promoter methylated only in infertile patents	[47]
Global	Infertile males (38)	• Hypermethylation in spermatogenesis- related genes • Loss of methylation in inflammation and immune response-related genes	[18]
Global	NOA (65) and oligoastenozoospermic (29)	• 78 hypomethylated sites • 143 hypermethylated sites in NOA compared oligoastenozoospermia	[19]
Global	Cell-free seminal DNA of Normozospermic men (12) and post vasectomy (11)	• 367 testis and epididymis specific hypomethylated genes • 134 hypermethylated	[11]
Global	OAT (69)	• Broad epigenetic defects associated with abnormal semen parameters	[20]
Global	Infertile males (7)	• 5/7 infertile men had non-programmatic histone • No difference in localization of H3 lysine 4 methylation (H3K4me) or H3 lysine 27 methylation (H3K27me) in the gametes of infertile men compared with fertile men • No single locus displays a complete change in chromatin packaging or DNA modification • Reduction in the amount of H3K4me or H3K27me retained at developmental transcription factors and certain imprinted genes	[21]
Gene-specific DNA methylation
DDR1	NOA (16) and fertile normospermic men (5)	• Aberrant DNA methylation and expression of DDR1	[48]
MTHFR, SNRPN	OAT (27) and control (11)	• Low motility • Poor morphology	[49]
MTHFR H19	Infertile males (20)	• Methylation defects at the H19 locus associated with hypermethylation at MTHFR promoter	[22]
GTF2A1L	NOA (86): Normozospermia (26) Hypospermatogenesis (17) SCO (26)	• Aberrant TDMR methylation at the GTF2A1L promoter associated with hypospermatogenesis • Testicular sperm extraction (TESE) technique may be used to overcome male infertility due to aberrant TDMR methylation	[50]
MEST	212 consecutive infertile patients: Normozoospermic (31) volunteers (single samples) and Normozoospermic volunteers (10)	• 23 % of patient cohort displayed an aberrant MEST DNA methylation • MEST DNA methylation associated with oligozoospermia, decreased bi-testicular volume and increased FSH levels • DNA methylation in normozoospermic volunteers was stable over a time period of up to 951 days in contrast to classical semen parameters	[51]
RHOX homeobox genes	Infertile males (140)	• Aberrantly regulated in infertile patients	[52]
PEG1/MEST and H19	Normospermic (119) and azoospermic 175	• PEG1/MEST methylation in 20 % and H19-DMR in 3 % of oligozoospermic men	[53]
MTHFR	NOA (50) and fertile control (50) peripheral blood: NOA (32) obstructive azoospermia (5) TESE	• No difference in peripheral blood • Hypermethylation of MTHFR in testis biopsies (53 %), 0 % in obstructive azoospermia	[54]
MTHFR	RSA couples (20) Non-RSA couples (147) Fertile men (20)	• Methylated MTHFR epigenotype detected in 75 % of RSA men, 54 % of NRSA men and 15 % of fertile men	[55]
MTHFR	Idiopathic infertile (94): Idiopathic infertile men with normozoospermia (30) Idiopathic infertile men with oligozoospermia (64) Fertile controls (54)	• Hypermethylation of MTHFR in 45 % of idiopathic infertile males, 15 % of fertile males • Higher methylation pattern was found in the group with oligozoospermia	[11]
H19-ICR, KvDMR, SNRPN-ICR, IG-DMR and MEG3-DMR	Infertile men (107): Normozoospermic (15) Oligozoospermic (1) Astenozoospermic (8) Teratozoospermic (30) Oligoastenozoospermic (1) Oligoteratozoospermic (5) Astenoteratozoospermic (31) OAT (16) Fertile men (30)	• Altered methylation patterns associated with H19-ICR, SNRPN-ICR and MEG3 • Methylation anomalies in at least 20 % of the CGIs • Significant inverse relation between the percent of altered CGIs and the number of affected individuals decreased	[38]
CREM	Abnormal protamine 1/protamine 2 (P1/P2) ratio (60) Oligozoospermia (32) Fertile controls (40)	• Significantly higher rate of methylation of CREM in patients with abnormal protamination and oligozoospermia • Sperm concentration, sperm motility, and normal head morphology negatively correlated with the amount of CGI methylation	[56]
H19, IG-GTL2 and MEST	Oligoastenozoospermic (10), NOA (5), and unknown pathology (3) vasectomy reversal (17)	• A significant decrease in DNA methylation at the H19 DMR in testicular sperm of azoospermic (NOA and oligoastenozoospermic) men and vasectomy reversal compared with fertile men, suggesting that aberrant DNA methylation may be associated with obstruction • No association between G-GTL2 and MEST DMRs and studied groups	[57]
H19, GTL2 LIT1, MEST, NESPAS, PEG3 and SNRPN ALU and LINE1	Couples with strictly male-factor infertility (106)	• Significant association between aberrant methylation imprints and abnormal semen parameters, but not with ART outcome • Significant repeat methylation difference between sperm samples from infertile and presumably fertile males	[58]
OCT4, SOX2, NANOG, HOXC11, miR-17 and CREM	Patients with a high P1/P2 ratio (10) and patients with a low P1/P2 ratio (10) Normozoospermic controls with normal P1/P2 ratio (10)	• No significant quantitative differences between groups of patients with either an abnormally high or low P1/P2 ratio compared to normal controls • No extreme methylation defects in severely infertile men • Two patients exhibited altered methylation of the CREM gene	[59]
DAZL	OAT men (5) and Normozoospermic (5)	• Increased methylation defects in the DAZL promoter CGI in OAT patients compared with NZ controls	[60]
DAZ	Idiopathic infertile patients (174) and fertile controls (58)	• No differences between DAZ gene methylation patterns among groups with different spermatogenic status and somatic cells, completely methylated except for the group with AZ	[61]
H19, MEST	Normozoospermic controls (27) Oligozoospermic patients (96)	• No aberration in MEST DNA methylation • Aberrant DNA methylation in H19 in oligozoospermic patients	[62]
H19, MEST and LINE1	Normozoospermic (5) and OAT (20)	• Hypomethylation of H19 • Hypermethylation of MEST • High level of global methylation	[63]
H19 and MEST	Anejaculation (5) Secondary (5) Primary obstructive azoospermia (5) Secretory azoospermia (9)	• Hypomethylation of MEST in all patients • Significantly reduced DNA methylation of H19 in secretory azoospermia patients	[64]
H19, GTL2, PEG1 (MEST), LIT1 (KCNQ1OT1), ZAC (PLAGL1), PEG3and SNRPN	Normozoospermic (79) and oligozoospermic patients (18)	• H19 unmethylation • MEST methylation	[65]

NOA non-obstructive azoospermia, OAT oligoastenoteratozoospermic, TESE testicular sperm extraction