Idiopathic male infertility is strongly associated with aberrant promoter methylation of methylenetetrahydrofolate reductase (MTHFR)

Background

Abnormal germline DNA methylation in males has been proposed as a possible mechanism compromising spermatogenesis of some men currently diagnosed with idiopathic infertility. Previous studies have been focused on imprinted genes with DNA methylation in poor quality human sperms. However, recent but limited data have revealed that sperm methylation abnormalities may involve large numbers of genes or shown that genes that are not imprinted are also affected.

Methodology/Principal Findings

Using the methylation-specific polymerase chain reaction and bisulfite sequencing method, we examined methylation patterns of the promoter of methylenetetrahydrofolate reductase (MTHFR) gene ({"type":"entrez-nucleotide","attrs":{"text":"NG_013351","term_id":"262331545","term_text":"NG_013351"}}NG_013351: 1538–1719) in sperm DNA obtained from 94 idiopathic infertile men and 54 normal fertile controls. Subjects with idiopathic infertility were further divided into groups of normozoospermia and oligozoospermia. Overall, 45% (41/94) of idiopathic infertile males had MTHFR hypermethylation (both hemimethylation and full methylation), compared with 15% of fertile controls (P<0.05). Subjects with higher methylation level of MTHFR were more likely to have idiopathic male infertility (P-value for trend  = 0.0007). Comparing the two groups of idiopathic infertile subjects with different sperm concentrations, a higher methylation pattern was found in the group with oligozoospermia.

Conclusions

Hypermethylation of the promoter of MTHFR gene in sperms is associated with idiopathic male infertility. The functional relevance of hypermathylation of MTHFR to male fertility warrants further investigation.     

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

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.     

Epimedium protects against dyszoospermia in mice with Pex3 knockout by exerting antioxidant effects and regulating the expression level of P16

Oxidative stress (OS) is one of the primary factors leading to male infertility. Oral administration of antioxidants has thus far been found to significantly improve the quality of human sperm. Therefore, antioxidant treatment has become the consensus among international experts on male infertility. In this study, peroxisomal biogenesis factor 3 (Pex3)-knockout (KO, −/−) mice were used as a model to compare the efficacy of three types of traditional Chinese medicine (TCM) granules (Epimedium [YYH], Cuscuta [TSZ], and Rhodiola [HJT]) for male reproductive function rescue. YYH was revealed to be the best and exerted a rescue effect on Pex3−/− mice with spermatogenesis defects. In addition, YYH prominently reduced ROS levels in the testes, inhibited DNA oxidative damage in spermatogenic cells, promoted the proliferation of spermatogenic cells, and inhibited apoptosis in Pex3−/− male mice. Furthermore, the mechanism by which YYH ameliorated dyszoospermia was confirmed via the establishment of cyclin-dependent kinase inhibitor 2 A (P16Ink4a)-KO mice. Specifically, Pex3−/− mice produced elevated amounts of ROS, which damaged germ cell DNA and further activated the signaling pathway of the cell senescence regulatory protein P16-CDK6, resulting in cell cycle arrest and eventually contributing to spermatogenesis dysfunction. YYH supplementation partially corrected the associated phenotype in gene KO mice by affecting P16 expression levels, thus improving the reproductive outcome to a certain extent.Subject terms: Senescence, Embryology, Infertility, Experimental models of disease, Translational research     

Loss of TDP-43 in male germ cells causes meiotic failure and impairs fertility in mice

Meiotic arrest is a common cause of human male infertility, but the causes of this arrest are poorly understood. Transactive response DNA-binding protein of 43 kDa (TDP-43) is highly expressed in spermatocytes in the preleptotene and pachytene stages of meiosis. TDP-43 is linked to several human neurodegenerative disorders wherein its nuclear clearance accompanied by cytoplasmic aggregates underlies neurodegeneration. Exploring the functional requirement for TDP-43 for spermatogenesis for the first time, we show here that conditional KO (cKO) of the Tardbp gene (encoding TDP-43) in male germ cells of mice leads to reduced testis size, depletion of germ cells, vacuole formation within the seminiferous epithelium, and reduced sperm production. Fertility trials also indicated severe subfertility. Spermatocytes of cKO mice showed failure to complete prophase I of meiosis with arrest at the midpachytene stage. Staining of synaptonemal complex protein 3 and γH2AX, markers of the meiotic synaptonemal complex and DNA damage, respectively, and super illumination microscopy revealed nonhomologous pairing and synapsis defects. Quantitative RT–PCR showed reduction in the expression of genes critical for prophase I of meiosis, including Spo11 (initiator of meiotic double-stranded breaks), Rec8 (meiotic recombination protein), and Rad21L (RAD21-like, cohesin complex component), as well as those involved in the retinoic acid pathway critical for entry into meiosis. RNA-Seq showed 1036 upregulated and 1638 downregulated genes (false discovery rate <0.05) in the Tardbp cKO testis, impacting meiosis pathways. Our work reveals a crucial role for TDP-43 in male meiosis and suggests that some forms of meiotic arrest seen in infertile men may result from the loss of function of TDP-43.     

Human Spermatogenic Failure Purges Deleterious Mutation Load from the Autosomes and Both Sex Chromosomes,including the Gene DMRT1

Gonadal failure, along with early pregnancy loss and perinatal death, may be an important filter that limits the propagation of harmful mutations in the human population. We hypothesized that men with spermatogenic impairment, a disease with unknown genetic architecture and a common cause of male infertility, are enriched for rare deleterious mutations compared to men with normal spermatogenesis. After assaying genomewide SNPs and CNVs in 323 Caucasian men with idiopathic spermatogenic impairment and more than 1,100 controls, we estimate that each rare autosomal deletion detected in our study multiplicatively changes a man''s risk of disease by 10% (OR 1.10 [1.04–1.16], p<2×10⁻³), rare X-linked CNVs by 29%, (OR 1.29 [1.11–1.50], p<1×10⁻³), and rare Y-linked duplications by 88% (OR 1.88 [1.13–3.13], p<0.03). By contrasting the properties of our case-specific CNVs with those of CNV callsets from cases of autism, schizophrenia, bipolar disorder, and intellectual disability, we propose that the CNV burden in spermatogenic impairment is distinct from the burden of large, dominant mutations described for neurodevelopmental disorders. We identified two patients with deletions of DMRT1, a gene on chromosome 9p24.3 orthologous to the putative sex determination locus of the avian ZW chromosome system. In an independent sample of Han Chinese men, we identified 3 more DMRT1 deletions in 979 cases of idiopathic azoospermia and none in 1,734 controls, and found none in an additional 4,519 controls from public databases. The combined results indicate that DMRT1 loss-of-function mutations are a risk factor and potential genetic cause of human spermatogenic failure (frequency of 0.38% in 1306 cases and 0% in 7,754 controls, p = 6.2×10⁻⁵). Our study identifies other recurrent CNVs as potential causes of idiopathic azoospermia and generates hypotheses for directing future studies on the genetic basis of male infertility and IVF outcomes.     

FertilityOnline: A Straightforward Pipeline for Functional Gene Annotation and Disease Mutation Discovery

Exploring the genetic basis of human infertility is currently under intensive investigation.However, only a handful of genes have been validated in animal models as disease-causing genes in infertile men. Thus, to better understand the genetic basis of human spermatogenesis and bridge the knowledge gap between humans and other animal species, we construct the Fertility Online, a database integrating the literature-curated functional genes during spermatogenesis into an existing spermatogenic dat...     

Analysis of tissue-specific differentially methylated regions (TDMs) in humans

Alterations in DNA methylation have been implicated in mammalian development. Hence, the identification of tissue-specific differentially methylated regions (TDMs) is indispensable for understanding its role. Using restriction landmark genomic scanning of six mouse tissues, 150 putative TDMs were identified and 14 were further analyzed. The DNA sequences of the 14 mouse TDMs are analyzed in this study. Six of the human homologous regions show TDMs to both mouse and human and genes in five of these regions have conserved tissue-specific expression: preferential expression in testis. A TDM, DDX4, is further analyzed in nine testis tissues. An increase in methylation of the promoter region is significantly associated with a marked reduction of the gene expression and defects in spermatogenesis, suggesting that hypomethylation of the DDX4 promoter region regulates DDX4 gene expression in spermatogenic cells. Our results indicate that some genomic regions with tissue-specific methylation and expression are conserved between mouse and human and suggest that DNA methylation may have an important role in regulating differentiation and tissue-/cell-specific gene expression of some genes.     

High Resolution X Chromosome-Specific Array-CGH Detects New CNVs in Infertile Males

Context

The role of CNVs in male infertility is poorly defined, and only those linked to the Y chromosome have been the object of extensive research. Although it has been predicted that the X chromosome is also enriched in spermatogenesis genes, no clinically relevant gene mutations have been identified so far.

Objectives

In order to advance our understanding of the role of X-linked genetic factors in male infertility, we applied high resolution X chromosome specific array-CGH in 199 men with different sperm count followed by the analysis of selected, patient-specific deletions in large groups of cases and normozoospermic controls.

Results

We identified 73 CNVs, among which 55 are novel, providing the largest collection of X-linked CNVs in relation to spermatogenesis. We found 12 patient-specific deletions with potential clinical implication. Cancer Testis Antigen gene family members were the most frequently affected genes, and represent new genetic targets in relationship with altered spermatogenesis. One of the most relevant findings of our study is the significantly higher global burden of deletions in patients compared to controls due to an excessive rate of deletions/person (0.57 versus 0.21, respectively; p = 8.785×10⁻⁶) and to a higher mean sequence loss/person (11.79 Kb and 8.13 Kb, respectively; p = 3.435×10⁻⁴).

Conclusions

By the analysis of the X chromosome at the highest resolution available to date, in a large group of subjects with known sperm count we observed a deletion burden in relation to spermatogenic impairment and the lack of highly recurrent deletions on the X chromosome. We identified a number of potentially important patient-specific CNVs and candidate spermatogenesis genes, which represent novel targets for future investigations.     

The histone methyltransferase ESET is required for the survival of spermatogonial stem/progenitor cells in mice

Self-renewal and differentiation of spermatogonial stem cells (SSCs) are the foundation of spermatogenesis throughout a male''s life. SSC transplantation will be a valuable solution for young male patients to preserve their fertility. As SSCs in the collected testis tissue from the patients are very limited, it is necessary to expansion the SSCs in vitro. Previous studies suggested that histone methyltransferase ERG-associated protein with SET domain (ESET) represses gene expression and is essential for the maintenance of the pool of embryonic stem cells and neurons. The objective of this study was to determine the role of ESET in SSCs using in vitrocell culture and germ cell transplantation. Cell transplantation assay showed that knockdown of ESET reduced the number of seminiferous tubules with spermatogenesis when compared with that of the control. Knockdown of ESET also upregulated the expression of apoptosis-associated genes (such as P53, Caspase9, Apaf1), whereas inhibited the expression of apoptosis-suppressing genes (such as Bcl2l1, X-linked inhibitor of apoptosis protein). In addition, suppression of ESET led to increase in expression of Caspase9 and activation of Caspase3 (P17) as well as cleavage of poly (ADP-ribose) polymerase. Among the five ESET-targeting genes (Cox4i2, spermatogenesis and oogenesis Specific Basic Helix-Loop-Helix 2, Nobox, Foxn1 and Dazl) examined by ChIP assay, Cox4i2 was found to regulate SSC apoptosis by the rescue experiment. BSP analyses further showed that DNA methylation in the promoter loci of Cox4i2was influenced by ESET, indicating that ESET also regulated gene expression through DNA methylation in addition to histone methylation. In conclusion, we found that ESET regulated SSC apoptosis by suppressing of Cox4i2 expression through histone H3 lysine 9 tri-methylation and DNA methylation. The results obtained will provide unique insights that would broaden the research on SSC biology and contribute to the treatment of male infertility.     

DNA Methylation as a Biomarker for Cardiovascular Disease Risk

Background

Elevated serum homocysteine is associated with an increased risk of cardiovascular disease (CVD). This may reflect a reduced systemic remethylation capacity, which would be expected to cause decreased genomic DNA methylation in peripheral blood leukocytes (PBL).

Methodology/Principal Findings

We examined the association between prevalence of CVD (myocardial infarction, stroke) and its predisposing conditions (hypertension, diabetes) and PBL global genomic DNA methylation as represented by ALU and Satellite 2 (AS) repetitive element DNA methylation in 286 participants of the Singapore Chinese Health Study, a population-based prospective investigation of 63,257 men and women aged 45–74 years recruited during 1993–1998. Men exhibited significantly higher global DNA methylation [geometric mean (95% confidence interval (CI)): 159 (143, 178)] than women [133 (121, 147)] (P = 0·01). Global DNA methylation was significantly elevated in men with a history of CVD or its predisposing conditions at baseline (P = 0·03) but not in women (P = 0·53). Fifty-two subjects (22 men, 30 women) who were negative for these CVD/predisposing conditions at baseline acquired one or more of these conditions by the time of their follow-up I interviews, which took place on average about 5·8 years post-enrollment. Global DNA methylation levels of the 22 incident cases in men were intermediate (AS, 177) relative to the 56 male subjects who remained free of CVD/predisposing conditions at follow-up (lowest AS, 132) and the 51 male subjects with a diagnosis of CVD or predisposing conditions reported at baseline (highest AS 184) (P for trend = 0.0008) No such association was observed in women (P = 0.91). Baseline body mass index was positively associated with AS in both men and women (P = 0·007).

Conclusions/Significance

Our findings indicate that elevated, not decreased, PBL DNA methylation is positively associated with prevalence of CVD/predisposing conditions and obesity in Singapore Chinese.     

Epigenetics, spermatogenesis and male infertility

Epigenetic modifications characterized by DNA methylation, histone modifications, and chromatin remodeling are important regulators in a number of biological processes, including spermatogenesis. Several genes in the testes are regulated through epigenetic mechanisms, indicating a direct influence of epigenetic mechanisms on the process of spermatogenesis. In the present article, we have provided a comprehensive review of the epigenetic processes in the testes, correlation of epigenetic aberrations with male infertility, impact of environmental factors on the epigenome and male fertility, and significance of epigenetic changes/aberrations in assisted reproduction. The literature review suggested a significant impact of epigenetic aberrations (epimutations) on spermatogenesis, and this could lead to male infertility. Epimutations (often hypermethylation) in several genes, namely MTHFR, PAX8, NTF3, SFN, HRAS, JHM2DA, IGF2, H19, RASGRF1, GTL2, PLAG1, D1RAS3, MEST, KCNQ1, LIT1, and SNRPN, have been reported in association with poor semen parameters or male infertility. Environmental toxins/drugs may affect fertility via epigenetic modifications. For example, 5-aza-2'-deoxycytidine, an anticancer agent, causes a decrease in global DNA methylation that leads to altered sperm morphology, decreased sperm motility, decreased fertilization capacity, and decreased embryo survival. Similarly, Endocrine disruptors, such as methoxychlor (an estrogenic pesticide) and vinclozolin (an anti-androgenic fungicide) have been found by experiments on animals to affect epigenetic modifications that may cause spermatogenic defects in subsequent generations. Assisted reproduction procedures that have been considered rather safe, are now being implicated in inducing epigenetic changes that could affect fertility in subsequent generations. Techniques such as intracytoplasmic sperm injection (ICSI) and round spermatid injection (ROSI) may increase the incidence of imprinting disorders and adversely affect embryonic development by using immature spermatozoa that may not have established proper imprints or global methylation. Epigenetic changes, in contrast to genetic aberrations, may be less deleterious because they are potentially reversible. Further research could identify certain drugs capable of reversing epigenetic changes.     

The Relationship of DNA Methylation with Age,Gender and Genotype in Twins and Healthy Controls

Cytosine-5 methylation within CpG dinucleotides is a potentially important mechanism of epigenetic influence on human traits and disease. In addition to influences of age and gender, genetic control of DNA methylation levels has recently been described. We used whole blood genomic DNA in a twin set (23 MZ twin-pairs and 23 DZ twin-pairs, N = 92) as well as healthy controls (N = 96) to investigate heritability and relationship with age and gender of selected DNA methylation profiles using readily commercially available GoldenGate bead array technology. Despite the inability to detect meaningful methylation differences in the majority of CpG loci due to tissue type and locus selection issues, we found replicable significant associations of DNA methylation with age and gender. We identified associations of genetically heritable single nucleotide polymorphisms with large differences in DNA methylation levels near the polymorphism (cis effects) as well as associations with much smaller differences in DNA methylation levels elsewhere in the human genome (trans effects). Our results demonstrate the feasibility of array-based approaches in studies of DNA methylation and highlight the vast differences between individual loci. The identification of CpG loci of which DNA methylation levels are under genetic control or are related to age or gender will facilitate further studies into the role of DNA methylation and disease.     

Loss of Imprinting and Allelic Switching at the DLK1-MEG3 Locus in Human Hepatocellular Carcinoma

Deregulation of imprinted genes is an important molecular mechanism contributing to the development of cancer in humans. However, knowledge about imprinting defects in human hepatocellular carcinoma (HCC), the third leading cause of cancer mortality worldwide, is still limited. Therefore, a systematic meta-analysis of the expression of 223 imprinted loci in human HCC was initiated. This screen revealed that the DLK1-MEG3 locus is frequently deregulated in HCC. Deregulation of DLK1 and MEG3 expression accompanied by extensive aberrations in DNA methylation could be confirmed experimentally in an independent series of human HCC (n = 40) in more than 80% of cases. Loss of methylation at the DLK1-MEG3 locus correlates linearly with global loss of DNA methylation in HCC (r² = 0.63, p<0.0001). Inhibition of DNMT1 in HCC cells using siRNA led to a reduction in MEG3-DMR methylation and concomitant increase in MEG3 RNA expression. Allele-specific expression analysis identified loss of imprinting in 10 out of 31 informative samples (32%), rendering it one of the most frequent molecular defects in human HCC. In 2 cases unequivocal gain of bi-allelic expression accompanied by substantial loss of methylation at the IG-DMR could be demonstrated. In 8 cases the tumour cells displayed allelic switching by mono-allelic expression of the normally imprinted allele. Allelic switching was accompanied by gains or losses of DNA methylation primarily at IG-DMR1. Analysis of 10 hepatocellular adenomas (HCA) and 5 cases of focal nodular hyperplasia (FNH) confirmed that this epigenetic instability is specifically associated with the process of malignant transformation and not linked to increased proliferation per se. This widespread imprint instability in human HCC has to be considered in order to minimize unwanted side-effects of therapeutic approaches targeting the DNA methylation machinery. It might also serve in the future as predictive biomarker and for monitoring response to epigenetic therapy.     

Identification of a DNA Methylome Profile of Esophageal Squamous Cell Carcinoma and Potential Plasma Epigenetic Biomarkers for Early Diagnosis

DNA methylation is a critical epigenetic mechanism involved in key cellular processes. Its deregulation has been linked to many human cancers including esophageal squamous cell carcinoma (ESCC). This study was designed to explore the whole methylation status of ESCC and to identify potential plasma biomarkers for early diagnosis. We used Infinium Methylation 450k array to analyze ESCC tissues (n = 4), paired normal surrounding tissues (n = 4) and normal mucosa from healthy individuals (n = 4), and combined these with gene expression data from the GEO database. One hundred and sixty eight genes had differentially methylated CpG sites in their promoter region and a gene expression pattern inverse to the direction of change in DNA methylation. These genes were involved in several cancer-related pathways. Three genes were validated in additional 42 ESCC tissues and paired normal surrounding tissues. The methylation frequency of EPB41L3, GPX3, and COL14A1 were higher in tumor tissues than in normal surrounding tissues (P<0.017). The higher methylation frequency of EPB41l3 was correlated with large tumor size (P = 0.044) and advanced pT tumor stage (P = 0.001). The higher methylation frequency of GPX3 and COL14A1 were correlated with advanced pN tumor stage (P = 0.001 and P<0.001). The methylation of EPB41L3, GPX3, and COL14A1 genes were only found in ESCC patients'' plasma, but not in normal individuals upon testing 42 ESCC patients and 50 healthy individuals. Diagnostic sensitivity was increased when methylation of any of the 3 genes were counted (64.3% sensitivity and 100% specificity). These differentially methylated genes in plasma may be used as biomarkers for early diagnosis of ESCC.