Epigenetics of Male Infertility: The Role of DNA Methylation

1Male infertility affects about 15% of couples worldwide. In reproductive age, approximately 7% of males suffer from infertility. Male infertility is a multifactorial disease comprising a wide variety of disorders. Endocrine and immunological disorders, anatomical and genetic abnormalities as well as infections of the genital tract can affect the male reproductive potential. Several other factors including age, stress, and lifestyle, such as obesity, smoking, and alcohol, have been associated with male infertility.

2In recent years, a number of studies have focused on the role of epigenetics in spermatogenesis and male infertility. Epigenetics refers to the gene regulation process without changes in DNA sequence and includes DNA methylation, posttranslational histone modifications and microRNA (miRNA) regulation. Several specific epigenomic/epigenetic modifications are established during spermatogenesis to form highly specialized mature sperm cells, allowing significant reorganizations of sperm chromatin structure. Therefore, spermatogenesis is particularly vulnerable to epigenetic alterations. Dysregulations in the DNA methylation process during spermatogenesis can result in the abnormal expression of target genes, which may lead to infertility. While many epigenetic abnormalities causing male reproductive dysfunction are still unknown, it is likely that most cases of idiopathic infertility could be accounted for underlying DNA methylation mechanisms.

3This review provides an overview on gene and genome methylation and its regulation during spermatogenesis and the current knowledge of those DNA methylation defects potentially involved in the etiopathogenesis of male infertility.

4We performed an investigation of the scientific literature by searching PubMed (Medline) database until March 2021. All studies investigating the relationship between DNA methylation and male infertility published from 1987 up to March 2021 were reviewed for specific topic areas, and the most relevant reports were included. The literature search was performed using the following keywords (alone and/or in combination): epigenomics, epigenetics, gene, methylation, DNA methylation, genome, methylome, hypermethylation, hypomethylation, regulation, genomic imprinting, imprinted genes, sperm, sperm cells, spermatozoa, semen parameters, spermatogenesis, infertility, subfertility, sterility, and male infertility. The reference lists of all publications included in this review have also been considered for additional relevant works.

5DNA methylation is a biochemical process where a nucleotide is enzymatically methylated with a methyl group (–CH3) at the five-carbon position, usually cytosine. Cytosine methylation predominantly occurs at the CpG dinucleotide (Figure 1). Regions rich in CpGs are called CpG islands (CGI) and are usually found in gene promoters, where gene expression is regulated through methylation. CpG methylation in the promoters of genes typically leads to gene silencing. The suppression of gene expression can occur by DNA methylation itself that, in some cases, can prevent the binding of transcriptional factors. Methylated DNA could also be recognized and bound by the methyl-binding proteins methyl CpG binding protein 2 and methyl-CpG-binding domain protein-1, -2, and -4, which recruit the enzymes histone methyltransferases and histone deacetylases to trigger histone modifications and chromatin packing, leading to the repression of gene expression.

6DNA methylation plays a pivotal role in regulating germ cell development. During germ cell development, the genome is widely remodeled by waves of DNA demethylation and methylation (Figure 2;;;;;; ). The wave of DNA demethylation occurs in the primordial germ cells (PGCs), which are precursors of male and female germ cells. Initially, the content of DNA methylation is equally distributed between PGCs and embryo somatic cells. Upon migration of PGCs from the epiblast to the gonadal ridge, the DNA methylation marks are extensively erased, resulting in lower methylation levels in PGCs compared to those in embryo somatic cells. These epigenetic modifications are essential for the genome reprogramming of PGCs, allowing sex-specific germ cell development during embryo development. De novo methylation proceeds in prospermatogonia or gonocytes arrested in mitosis, and it is firstly established at the repeated DNA sequences, such as retrotransposons, and then at the imprinted genes, leading to appropriate sex-specific methylation patterns in the germ cells.

7DNA methylation establishment in the germline is of particular importance, as failure to establish correct methylation in retrotransposons and imprinted genes has serious consequences for embryo development. Indeed, retrotransposons, such as long interspersed nuclear elements 1 (LINE1), are interspersed repeated DNA sequences which are able to propagate throughout the genome. Lack of methylation in retrotransposons could allow their propagation throughout the genome, causing insertional mutagenesis and, in turn, several diseases, including male infertility. Regarding the imprinted genes, methylation regulates the expression through an important process termed “genomic imprinting,” which leads to the expression of either the maternal or paternal allele. When maternal/paternal alleles undergo global demethylation upon fertilization, the imprinted genes preserve the methyl marks of the parental genome, leading to parental-origin patterns of mono-allelic gene expression. This process occurs in specific sequences called differentially methylated regions (DMRs), also known as imprinting control regions (ICRs). For instance, the imprinted genes IGF2-H19 share common enhancers and ICR located downstream and upstream of the H19 gene, respectively. Under normal circumstances, ICR and H19 are methylated in sperm cells and unmethylated in oocytes, leading to the reciprocal expression of the maternal H19 allele and paternal IGF2 allele in somatic cells (Figure 3;; ). Other paternally imprinted genes have been found to be similarly regulated in sperm cells, such as Ras Protein Specific Guanine Nucleotide Releasing Factor 1, Delta Like Non-Canonical Notch Ligand 1, and Zinc Finger DBF-Type Containing 2. Contrariwise, maternally imprinted genes are methylated in female germ cells, while they are expressed in male germ cells, including mesoderm-specific transcript (MEST), also known as paternally expressed gene 1 (PEG1), paternally expressed 3 (PEG3), and small nuclear ribonucleoprotein polypeptide N (SNRPN). However, failure in the maintenance of imprinted gene methylation patterns in the germline might occur, and this has been associated with low sperm quality and pregnancy rate and impaired post-fertilization development.

8DNA methylation is carefully coordinated by a family of enzymes named DNA methyltransferases (DNMTs). Four members of this family, DNMT1, DNMT3A, DNMT3B, and DNMT3C, are endowed with catalytic activity. DNMT1, also known as the maintenance DNMT, copies pre-existing methylation marks onto the new strands following DNA replication. DNMT3A and DNMT3B are de novo methyltransferases, as they are able to methylate previously unmethylated DNA sequences. DNMT3C, along with P-element induced wimpy testis/interacting RNA (PIWI/piRNA), is responsible for the promoter methylation of retrotransposons in the male germline. piRNAs are small non-coding RNAs that form RNA–protein complexes through interaction with PIWI proteins, a gonad-specific class of Argonaute proteins. These RNA–protein complexes are essential for the LINE1 methylation processes in male germ cells. DNA methylation is a reversable chemical modification, and active demethylation processes are mediated by erasing DNA methylation mechanisms, mainly controlled by Ten-Eleven Translocation (TET) enzymes, such as TET1, -2, and -3,. TET enzymes specifically convert 5mC to 5-hydroxymethylcytosine (5hmC). The TET-mediated oxidation of 5mC induces DNA demethylation by passive or active processing. As 5hmC is not recognized by DNMT1 during DNA replication, passive DNA demethylation can occur in proliferating cells. In the active process, TETs convert 5hmC to 5-formylcytosine (5fC) and then 5fC into 5-carboxylcytosine (5cC). 5fC and 5cC are both recognized and excised by the base-excision DNA repair machinery and subsequently replaced with cytosine.

9DNA methylation plays a critical role during spermatogenesis. The correct methylation of DNA ensures proper chromatin condensation in the sperm head, enabling sperm maturation and its ability in fertilization and post-fertilization events. Contrariwise, incomplete or abnormal condensation of the sperm chromatin results in damaged DNA, leading to the impairment of egg cell fertilization and/or reduction in pregnancy rates. Considering these aspects, several studies have analyzed the gene and genome methylation levels of sperm DNA in association with male reproductive dysfunctions. These studies have mainly, but not only, investigated associations between improper DNA methylation in spermatozoa genome and negative variations in semen parameters, such as concentration (oligozoospermia), morphology (teratozoospermia), and progressive motility (asthenozoospermia), alone or in combinations, i.e., oligoasthenozoospermia (OA), oligoteratozoospermia, asthenoteratozoospermia (AT), and oligoasthenoteratozoospermia (OAT). Semen samples are considered normal when the concentration, motility, and morphology are ≥15 × 106 sperm/ml, ≥32% (sperm progressive motility), and ≥4% normal, respectively.

10Several genes carrying defective methylation have been associated with abnormalities in semen parameters (Table 1). One of the most highly studied genes is methylenetetrahydrofolate reductase (MTHFR), which is a key regulatory enzyme involved in folate metabolism and in DNA synthesis and methylation. In male mice testes, the activity of MTHFR is five times higher than in other organs, whereas its inactivation results in hyperhomocysteinemia, decreased methylation capacity, and the arrest of spermatogenesis due to sperm DNA hypomethylation. In humans, a number of studies have pointed out that impairment of the MTHFR gene can contribute to disease, including male infertility. Mutations/polymorphisms in MTHFR gene are broadly known causes of reduced MTHFR enzyme activity in sperm cells, resulting in reduced methionine availability and decreased DNA methylation. The improper methylation of MTHFR gene in sperm germ cells may likewise result in decreased MTHFR enzyme activity, leading to aberrant methylation of sperm DNA. Hypermethylation of MTHFR has been found in testes from males affected by non-obstructive azoospermia and in sperm cells from males affected by oligozoospermia and teratozoospermia as well as in idiopathic infertile males. In our previous studies, hypermethylation at the MTHFR promoter has been found to be associated with aberrant concentration, motility, and morphology in males from infertile couples affected by recurrent spontaneous abortion. In addition, we have found a correlation between MTHFR gene promoter hypermethylation and extensive methylation defects at the paternal imprinted gene H19 in sperm DNAs from infertile males with both normal and abnormal semen parameters. These results have suggested that MTHFR hypermethylation-induced inactivation may affect methylation at the imprinted loci of sperm cells, leading to impaired fertilization. Contrariwise, no correlation was found between methylation errors at H19 and MEST genes and inactivating MTHFR C677T single-nucleotide polymorphism (SNP) [a cytosine (C) to a thymine (T) substitution at position 677] in sperm DNA from infertile patients with defective semen parameters. However, a higher incidence trend of aberrant DNA methylation among severe oligozoospermic infertile males carrying C677T genotype was observed.

11Germline mutations arise from replication errors, potentially resulting in reproductive health impairment. Accordingly, the genetic defects of DNA mismatch repair mechanisms have been related to impaired spermatogenesis and male infertility. A recent study has investigated the methylation profile of the DNA mismatch repair gene MutL alpha (MLH1) in sperm DNA from oligozoospermic patients and normozoospermic males enrolled as the control group. The results indicated an association between MLH1 hypermethylation and male infertility, suggesting that this gene might be under epigenetic regulation during sperm cell development. High methylation levels of the DNA repair X-ray repair cross-complementing protein 1 gene have been found in OAT males, thereby suggesting that the improper methylation of this gene may have a role in sperm chromatin condensation and OAT. Contrariwise, two additional well-known DNA repair genes, i.e., breast cancer type 1 susceptibility protein (BRCA1) and BRCA2, did not show defective methylation in infertile men with OAT.

12Several other genes found aberrantly methylated in altered sperm samples from infertile males have been investigated at a single-study level (Table 1). Overall, several abnormally methylated genes have been found to be associated to altered sperm parameters, mainly oligozoospermia, indicating a potential involvement of DNA methylation in male infertility. All, but MTHFR, gene investigations have been carried out at a single-study level and need further studies to be confirmed. On the contrary, improper MTHFR promoter methylation, such as hypermethylation, has been repeatedly found in aberrant sperm samples, suggesting the methylation-induced MTHFR dysregulation as a potential causal factor of abnormal sperm changes. Interestingly, MTHFR hypermethylation has been found in normal sperm samples of males from idiopathic infertile couples. This may indicate that MTHFR dysfunction could play a role in changing sperm function and health, possibly by lowering CpG methylation content in imprinted genes, which, in turn, may affect embryo development and pregnancy outcome. In view of finding gene methylation hallmarks in male infertility, these data are interesting and will deserve further investigations. For instance, large studies of different infertile populations along with a functional analysis of hypermethylation are needed in order to elucidate the role and the mechanisms of the MTHFR gene in human spermatogenesis and the related pathologies.

13The methylation status of LINE1 in sperm DNA from infertile males was initially evaluated by who reported no correlation between LINE1 methylation and male reproductive function. No correlation has also been found in sperm DNA from idiopathic infertile males belonging to recurrent spontaneous miscarriage couples. Low methylation levels at LINE1 have been shown in asthenospermic and OAT patients in an additional study, although no statistical significance was found between cases and controls. A meta-analysis in 291 infertile patients and 198 fertile individuals from seven published works found no significant correlation with LINE1 methylation levels. Conversely, DNA methylation marks at LINE1 and the repetitive sequences ALU YB8, NBL2, and D4Z4 have been found to be significantly lower in spermatozoa from idiopathic infertile males than in somatic cells. A significant decrease in global 5mC levels and LINE1 promoter methylation in spermatozoa of males from couples experiencing recurrent pregnancy loss has also been described. Similarly, LINE1 hypomethylation along with promoter hypermethylation-associated silencing of PIWIL2 and Tudor Domain Containing 1 genes, two PIWI/piRNA pathway-related genes, has been found in sperm DNA from infertile males affected by spermatogenic disorders. Animal model studies have reported similar results with other PIWI/piRNA pathway-related genes and satellite DNA regions.

14LINE1 methylation levels evaluated through sperm genome methylation analyses might be a good indicator of genome-wide alterations. Indeed, these repetitive sequences have frequently been considered as surrogate markers for global methylation changes. However, the role of LINE1 methylation in male infertility is far to be elucidated. Few studies are currently available and often reporting contradictory data, probably due to the lack of homogeneity in experimental design and data processing among studies.

15Over the years, with the development of molecular technology/methods, many studies have investigated the relationship between DNA methylation and male infertility on a genome-wide scale by employing high-throughput techniques. The first array-wide analysis was performed by highlighting improper DNA methylation at repetitive element satellite 2, H-RAS, neurotrophin 3, paired-box gene 8, and Stratifin, 3′-nucleotidase, and metallothionein 1A genes in semen samples with abnormal sperm concentration, motility, and morphology. DNA methylation variations at over 9K CpGs spread throughout the testicular genome have been detected between obstructive and non-obstructive azoospermic patients. The human methylation 450k array analysis in semen samples from couples who conceived and couples who did not showed significantly decreased and increased methylation in two and three genomic regions, respectively, in the “failure-to-conceive” group. Interestingly, the two sites with low methylation content were at closely related genes known to be expressed in the sperm, i.e., HSPA1L and HSPA1B. A recent methylome and transcriptome study in euploid blastocysts derived from couples with OAT males reported significant alterations in approximately 1.1K CpGs compared to the cryopreserved euploid blastocysts used as controls. A pathway analysis elucidated the genes involved in the regulation of cellular metabolic process as universally affected. This epigenetic dysregulation provided an explanation for the reduced reproductive potential in OAT patients despite euploid blastocyst transfers.

16Additional array-wide studies have been reported to show the following: (i) changes in the methylation of testis/epididymis-specific genes in the testis and epididymis of vasectomized males, (ii) loss of methylation in inflammation- and immune response-related genes in sperm samples from males belonging to infertile couples, and (iii) gain of methylation in spermatogenesis-related genes in sperm samples derived from individuals undergoing assisted reproduction.

17In normospermic males, the DNA methylome of the sperm cell differs between infertile and fertile, and this difference may be predictive of embryo quality during in vitro fertilization. Nevertheless, differences in sperm epigenome within healthy subjects have been also detected, indicating intra-sample heterogeneity in DNA methylation patterns. Furthermore, additional studies have underlined potential epigenetic heterogeneity among, and within, sperm cells, spermatogonia, and gonocytes.

18The impairment of both sperm epigenome and gene expression in relation to male reproductive dysfunction was recently explored using high-throughput techniques. Hypermethylation-induced silencing at SRY-Box Transcription Factor 30 gene has been detected in association with non-obstructive azoospermia. Two array-wide studies have described inverse correlations between DNA methylation and the mRNA expression of genes involved in different pathways, such as the response to hormone stimulus, activation of protein kinase activity, apoptosis, and reproduction, in sperm from both severe oligozoospermic and azoospermic patients.

19As high-throughput approaches are able to detect extensive variations in epigenetic marks, DNA methylation changes could potentially be used as a diagnostic tool to predict the risk of both male infertility and male infertility-related diseases. For instance, an array-based DNA methylation profile conducted on peripheral blood has revealed variations in DNA methylation patterns in infertile males compared to normozoospermic fertile controls, suggesting that epigenome-based blood markers can be used for diagnostic purposes. This has also been suggested by the results of another high-throughput study focused on congenital hypopituitarism (CH), which is a pituitary gland hormone deficiency that leads to metabolic disorders and male reproductive dysfunction. This study identified methylation at CpG sites on two spermatogenesis/testicular development-related genes in whole blood DNA isolated from a cohort of CH patients. In this context, the clinical application of these highly sensitive techniques cannot be ruled out. As high-throughput approaches can potentially support work on defining the forms of male infertility, these methods will deserve attention in andrological clinical practice.

20DNA methyltransferases and TET are two key regulative gene families involved in DNA methylation and demethylation pathways, respectively, during both embryogenesis and spermatogenesis. Despite their critical role in sperm methylation, the DNMT and TET genes have been poorly investigated in the context of male infertility. Decreased expression of DNMT1, -3A, and -3B enzymes in association with global DNA methylation changes has been detected in the testes of non-obstructive azoospermic patients, suggesting that the methylation-induced DNMT inactivation may be involved in male reproductive dysfunction. The presence of DNMT1 SNP variant rs4804490 was found to be associated with an increased risk of idiopathic infertility in males with abnormal semen parameters. In the same study, no associations were afterward determined between additional four DNMT variants, i.e., DNMT1 (rs4804490), DNMT3A (rs1550117), DNMT3B (rs2424909), and DNMT3L (rs7354779), and aberrant DNA methylation at several imprinted genes, including H19, GNAS complex locus (GNAS), and GTP-binding protein Di-Ras3 (DIRAS3). Conversely, two SNP variants, rs2656927/rs8103849, of ubiquitin-like, containing PHD and RING finger domains 1 (UHRF1) gene, which is tightly related to the DNMT1 pathway, have been identified in the blood of oligozoospermic infertile males. Other recent evidence indicated that UHRF1 inactivation may have a negative impact on male fertility potential, as the conditional loss of this gene in germ cells leads to DNA hypomethylation, upregulation of retrotransposons, and DNA damage response activation. UHRF1 also cooperates with the PIWI/piRNA pathway during spermatogenesis, suggesting a molecular link between DNA methylation and the PIWI/piRNA pathway in the germline.

21Ten-eleven translocation 1, -2, and -3 genes have been found to be consecutively expressed at different stages of spermatogenesis, and their expression levels have been positively correlated to male reproductive potential and pregnancy. Indeed sperms from oligozoospermic and/or asthenozoospermia males have significantly reduced TET1, -2 and -3 mRNAs compared to fertile donors, but not to normozoospermic males. TET1 protein deficiency in spermatogonia decreases the 5hmC levels and downregulates genes that are involved in several pathways, such as cell cycle, germ cell differentiation, meiosis, and reproduction, resulting in the reduction of spermatogonia stem cells and germ cell differentiation.

22When taken together, these studies suggest that DNMT and TET impairment in sperm cells may represent a potential risk factor for male infertility. Therefore, further investigations are needed to assess the role of DNMT and TET enzymes in the male infertility phenotype.

23Imbalances in the methylation patterns of imprinted genes could potentially impair spermatogenesis and/or favor the male infertility phenotype. The methylation status of a number of imprinted genes has been investigated in relation to impaired spermatogenesis and/or reproductive dysfunction (Table 2). DNA methylation defects of three maternally imprinted genes, i.e., PLAGL1, MEST, and DIRAS3, have been identified in sperm DNAs from infertile males with a low semen concentration. One of these genes, MEST, has been repeatedly found as hypermethylated in association with the male infertility phenotype. Specifically, the improper DNA methylation at MEST has been (i) linked to low sperm concentration and motility as well as poor sperm morphology in idiopathic infertile males, (ii) detected in primary spermatocytes from azoospermic patients presenting complete or incomplete maturation arrest, (iii) associated with decreased bi-testicular volume and increased follicle-stimulating hormone levels, and (iv) associated with abnormal protamine ratio in oligozoospermic patients. These data have been further confirmed by a meta-analytic study. Recently, MEST hypermethylation has been found in the spermatozoa of male partners from couples experiencing recurrent pregnancy loss.

24Altered methylation in H19 has been broadly documented to be associated with infertile males affected by oligozoospermia, asthenozoospermia, teratozoospermia, OA, AT, and OAT. Idiopathic infertile males with normal semen parameters have also been found to carry methylation defects at H19 in their sperm. Imprinting errors at H19 gene were detected in primary spermatocytes and elongated spermatids/spermatozoa with incomplete maturation arrest from two azoospermic patients, although the differences in H19 methylation levels between patients and controls were not statistically significant. In addition, abnormal methylation at H19 has been shown at the regulatory region CTCF-binding site 6 (CTCF6), located within the DMR of IGF2-H19, in sperm DNA from infertile males. Therefore, sperm cells carrying methylation defects at the IGF2-H19 CTCF6 region are at a high risk of causing biallelic inactivation of the IGF2 gene, which, in turn, could negatively impact embryo development and/or pregnancy outcome. Similarly, methylation defects at IGF2-H19 have been detected in placenta tissues from offspring conceived by assisted reproductive technologies (ART). Thus, these studies have pointed out that ART procedures may account for the epigenetic defects in the embryo.

25Altered DNA methylation patterns at overlapping transcript 1 (LIT1) and SNRPN have been found in infertile males tested with abnormal protamine ratio in sperm cells. Aberrant methylation at the SNRPN gene has also been associated with altered sperm motility and morphology as well as with OA and AT. Interestingly, imprinting errors at the SNRPN gene and IGF2-H19 have also been found in spermatozoa from asthenozoospermia patients and in ART-conceived human fetuses. Hypermethylation at SNRPN, alongside other maternally imprinted genes involved in embryonic germ cell development, such as MEG3, PEG1 (also known as MEST), PEG3, LIT1, and PLAG1 like zinc finger 1 (ZAC), as well as loss of methylation at the paternal imprinted gene GTL2 has been detected in sperm DNA derived from patients affected by moderate/severe oligozoospermia. Hypermethylation of PEG1, PEG3, PEG10, and ZAC genes has been shown in spermatozoa from male partners from couples experiencing recurrent pregnancy loss. Contrariwise, the lack of altered methylation has been reported for PEG1, ZAC, and GTL2 in the sperm of male partners from recurrent spontaneous miscarriages. Lastly, the maternally imprinted gene SNRPN upstream reading frame protein, which is related to the SNRPN pathway, has been detected as hypermethylated in association with oligozoospermia.

26An additional imprinted gene possibly involved in male reproductive dysfunction is zinc-finger CCHC-type containing 13 (ZCCHC13). ZCCHC13 is a novel imprinted gene involved in an epigenetic mechanism known as X-chromosome inactivation and has been detected both hypermethylated and down-regulated in testis biopsies isolated from non-obstructive azoospermia patients. Low methylation at GNAS and DIRAS3 imprinted genes has also been found to be more prevalent in idiopathic infertile males, especially in oligozoospermic males, than in fertile males. The evidence that improper DNA methylation at the GNAS gene is linked to male infertility is also supported by animal models.

27Imprinting errors could also be related to DNA fragmentation in sperm cells. Indeed, the improper methylation of the paternally imprinted gene Potassium Voltage-Gated Channel Subfamily Q Member 1 (KCNQ1), along with that of IGF2, has been found to be associated with a high level of DNA fragmentation in semen samples. In addition, the centromeric (domain 2) differentially methylated region (KvDMR) located in exon 10 of KCNQ1OT1 has been found to be hypermethylated in the spermatozoa of male partners from couples experiencing recurrent pregnancy loss.

28A recent array-wide methylation analysis conducted on sperm DNA from a cohort of males identified a number of genes associated with infertility, including the maternally imprinted genes DLG associated protein 2 (DLGAP2) and GATA binding protein 3 as well as the paternally imprinted genes catenin alpha 3, membrane-associated guanylate kinase, and tumor protein 73. An additional gene, brevican, which is a non-imprinted gene involved in brain extracellular matrix formation, has been identified in this study. All these genes might be considered as novel potential candidates accounting for male infertility phenotype, and their roles in spermatogenesis need to be further investigated.

29In conclusion, many imprinted genes have been investigated for methylation in aberrant semen samples, such as oligozoospermia, asthenozoospermia, and teratozoospermia. H19 and MEST were the most investigated genes in aberrant sperm samples, showing repetitively aberrant loss or gain of methylation, respectively, which, in turn, may be causal factors in the development of male infertility. Of note is the fact that aberrant methylations have also been found in infertile males with normal sperm parameters. This aspect may have important implications in the diagnosis of male infertility, which, to date, lacks explanation in 30% of infertile males. It is also hoped that further studies will shed light into the etiology of these aberrant imprinting patterns in paternally or maternally imprinted genes in idiopathic infertility cases. Indeed the loss of methylation of paternally imprinted genes may be due to a deficiency of DNMTs, whereas hypermethylation of maternally imprinted genes might result from erroneous de novo methylation or a failure to erase maternal imprint in the male germ cell genomes.

30Habits and lifestyle factors, such as smoking, alcohol consumption, and diet, have been investigated in an effort to gain insight into the causes of aberrant sperm DNA methylation in relation to male infertility. A recent pilot study conducted on sperm DNA from a cohort of infertile males who underwent a supervised yoga practice regimen reported DNA methylation changes at nearly 400 genes, suggesting a link between positive lifestyle practices and male reproductive health.

31Tobacco smoke has a strong negative impact on sperm DNA methylation in a number of genes, also demonstrated in animal models. The imprinted gene SNRPN has been found to be aberrantly methylated in asthenozoospermia and teratozoospermic smokers. Other studies on humans and/or animal models have reported modifications to sperm DNA methylation following cannabis use. Improper DLGAP2 methylation has recently been reported in sperm from cannabis users, thereby suggesting the potentially negative effect of cannabis use prior to conception on imprinting marks. In this context, the impact of parental drug addiction on sperm DNA methylation in drug-sired offspring needs to be more deeply explored. Moreover, both alcohol and nicotine consumption seem to impair DNA methylation in several genes and genomic regions, leading to male infertility. Indeed data on DNA methylation changes at cyclin-dependent kinase inhibitor 2A and LINE1 and an increased risk of male reproductive dysfunction have been reported. More recently, evidence has also indicated that DNA methylation defects at MEST and GNAS genes, along with altered sperm motility, morphology, and concentration, could be linked to alcohol and nicotine exposure.

32Paternal diet is able to alter sperm epigenome and has been associated with negative pregnancy outcomes in mice. In addition, sperm from mice under a folate-deficient diet showed differential DNA methylation marks at genes implicated in development, diabetes, autism, and schizophrenia. In humans, several studies have reported on the relationship between paternal and/or maternal diet and sperm DNA methylation in male infertility. A recent study conducted on infertile males who underwent oral supplementation with micronutrients in support of folate, B vitamins, zinc, and cysteines reported an increase in DNA methylation levels, resulting in improved sperm nuclear maturation and antioxidant defenses, with a possible positive effect on reproductive function. Furthermore, evidences on sperm DNA from infertile males indicate a high methylation of vitamin D receptor promoter in vitamin D-deficient conditions, thus suggesting the role of diet in epigenetic modifications and male reproductive dysfunction. Similarly, one investigation indicated that parental diet can modulate the methylation levels of several genes, including liver-specific genes involved in cholesterol/lipid metabolism, whereas no effect of paternal diet on sperm DNA methylation was found in other studies. Similarly, parental pre-conceptional obesity could potentially impact on the establishment of imprinting marks during embryogenesis. Changes in methylation in two imprinted genes, PLAGL1 and MEG3, have been detected in umbilical cord blood leukocytes in a group of newborns from obese mothers, making a link between maternal overnutrition and the impairment of imprinting marks in the offspring. In males, imprinted genes involved in growth and development, including PEG3 and neuronatin alongside MEST, have been found to be hypomethylated in DNA derived from the blood of children of obese fathers. The relationship between parental obesity and the methylation status of the offspring clearly indicates that spermatogenesis may be susceptible to environmental factors from an epigenetic point of view. A recent animal model study supported and extended this conclusion by reporting data about environmental toxicant exposure and transgenerational inheritance of epigenetic marks. Additional animal models have shown similar results, reporting that epigenetic alterations at PEG3 and H19 can occur in sperm cells in offspring from diabetic and/or obese mice. These studies, when taken together, highlight that both defective DNA methylation and imprinting marks may be transferred through generations, potentially affecting the health of adult offspring. However, this assumption has been questioned in a recent meta-analysis of coordinated epigenome-wide association studies of paternal prenatal body mass index (BMI) in relation to DNA methylation. The study conclusions do not support the hypothesis that paternal BMI around the time of pregnancy is linked with offspring-blood DNA methylation, even at the imprinted regions. Considering the data available, more research is needed to address the role of paternal diet on sperm DNA methylation in infertile males.

33This review provides an overview of gene and genome methylation, its regulation during the spermatogenesis process, and the current knowledge on those DNA methylation defects which are known to be involved in male reproductive dysfunctions.

34Most of the aforementioned studies have added sufficient strength to the hypothesis that sperm methylation is associated with sperm alterations and infertility. In spite of these reports, the causative role of improper DNA methylation marks in inducing male infertility remains poorly characterized, particularly due to the lack of studies on the mechanisms involving DNA methylation in sperm cells.

35It is convincible that aberrations in methylation at specific target genes may reflect whole methylome defects due to altered DNMT and TET activities during sperm cell development and spermatogenesis. On the other hand, several risk factors, such as lifestyle, drugs, hormones, and diet, may account for the gain or loss of methylation in key genes in sperm cells. New functional studies on this topic are needed to better elucidate the mechanisms affecting methylation in sperm. Nevertheless, extensive knowledge of sperm DNA methylation status in association with reduced reproductive capability is useful in developing novel diagnostic tools for male infertility. The current primary diagnostic protocol for identifying infertile males relies on the assessment of sperm number, motility, and morphology. This diagnostic approach has several limitations in differentiating fertile and infertile males, as abnormalities causing male reproductive dysfunction are still unknown in a fraction of males. In addition, this approach also provides limited information on male reproductive potential and cannot be employed as a prognostic tool in predicting pregnancy success and possible outcome. When taken together, these drawbacks in the current protocols make DNA methylation evaluation a putative useful tool in clinical practice. A large number of studies have already demonstrated that aberrant DNA methylation in spermatozoa is linked to defective sperm parameters and male infertility phenotype as well as a negative pregnancy outcome. In this context, sperm DNA methylation could become a novel diagnostic and prognostic parameter for assessing male infertility and pregnancy outcome, respectively. This approach has also been reported in monitoring other diseases. Thanks to high-throughput technology, which is rapidly increasing in epigenetic studies, methylome analyses may soon allow the methylation differences between infertile and fertile males to be characterized, thereby greatly improving the current knowledge on the relationship between sperm DNA methylation and male infertility. Hopefully, the translation of this technology into clinical andrological practice will improve the diagnostic and prognostic assessment of infertile males.

36The role of DNA methylation in spermatogenesis, sperm function, and male infertility is an important research area that deserves attention. Although recent evidence seems to indicate a possible overestimation of aberrant DNA methylation in male infertility, the aforementioned studies have provided significant associations between improper DNA methylation in sperm and infertile males.

37A number of genes have been found to be differentially methylated in relation to impaired spermatogenesis and/or reproductive dysfunction. DNA methylation defects in genes, including MEST and H19 within imprinted genes and MTHFR within non-imprinted genes, have been previously identified in a wide number of studies, and their defective marks could be potentially employed as useful tools in clinical practice for assessing male infertility.

38The lack of studies for several of the additional genes reported herein and/or the conflicting results for others make the use of defective DNA methylation marks as a diagnostic tool still unlikely. Further studies are necessary to identify novel genes that could potentially be employed as tools in clinical practice.

39As suggested in several works, the dysregulation of epigenetic mechanisms, including the aberrant methylation of DNA, may play an important role in the development of infertility with unknown etiology in a high fraction of males. However, further research is needed to epigenetically explore the etiology of this disease. Indeed the use of DNA methylation as a marker or cause of sperm abnormalities in the field of fertility is only beginning to be explored thoroughly and is currently not completely clarified. Moreover, the use of methylation changes in DNA as a marker to identify the male infertility factor is difficult, as these changes may have little or even no significant biological impact or multiple different changes may be necessary to establish infertile phenotypes. In this effort, methylation signatures in normal sperm from fertile individuals should provide useful knowledge for elucidating these topics. Moreover, as aging is correlated with changes in DNA methylation, investigating sperm DNA methylation in age-stratified normal fertile individuals should also be taken into account as well as to improve our knowledge in this field. Another point deserving attention in the context of methodological approaches is the potential contamination by somatic cells during epigenetic analyses, such as blood, white, and epithelial cells, as a result of incorrect sperm cell isolation/processing. Standardized and detailed protocols for processing sperm samples should be used in studying DNA methylation in male infertility, as even only a few contaminated somatic cells might alter the epigenetic signatures of sperm cells. Research into sperm DNA methylation in male infertility might be an important future area of study. We thus encourage further studies focused on the role of DNA methylation in spermatogenesis and male reproductive potential as well as embryo development and pregnancy outcome. Such newly acquired data could improve the diagnostic and prognostic setup of male infertility phenotypes and pregnancy outcomes, respectively, with high health benefits for humans.

40JR, MT, and FM contributed to conceptualization and writing—review and editing. JR contributed to writing—original draft preparation. CM and CL contributed to visualization. MT and FM contributed to supervision, contributed to funding acquisition, and project administration. All the authors have read and agreed to the published version of the manuscript.

41The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.