Association between alterations in DNA methylation level of spermatozoa at CpGs dinucleotide and male subfertility problems

The purpose of this study was to assess the relationship between alterations in sperm DNA methylation levels and sperm count and sperm motility. Five CpG sites underwent deep bisulphite sequencing to validate the observed methylation difference in 78 samples (28 proven fertile males “controls,” and 50 subfertile males “cases”). The results showed that variation in methylation levels was found in more than one CpG: the DNA methylation levels in CpG1, CpG2 and CpG3 of the PRRC2A gene‐related amplicon showed high significant differences in the case group compared to the control group (p ≤ .0001, p ≤ .003, and p ≤ .0001 respectively). Moreover, three CpGs of the four CpGs tested within the ANXA2 gene‐related amplicon (CpG1, CpG3 and CpG4) were significantly different (p ≤ .002, p ≤ .001, and p ≤ .0001, respectively) in the case group compared to the control group. In addition, a significant difference was found in seven CpGs of the twenty‐two CpGs tested within the MAPK8Ip3 gene‐related amplicon, besides six CpGs of the ten CpGs tested within the GAA gene‐related amplicon between case and control groups. In conclusion, this study identifies that CpGs have a significantly different in methylation levels of sperm DNA for subfertile males.     

Alterations in DNA methylation patterns and gene expression in spermatozoa of subfertile males

This study was designed to evaluate the DNA methylation patterns and gene expression in spermatozoa from subfertile males. Thirty samples were subjected to 450K arrays as a screening study to evaluate the variation in sperm DNA methylation levels between cases and controls groups, and then three CpG sites (cg07227024, cg05813498 and cg23081194) have the highest difference in methylation levels and located within ALS2CR12, GAA and UBE2G2 genes respectively; these were selected for further analysis using deep bisulphite sequencing and qPCR in 136 samples (64 proven fertile males “controls” and 72 subfertile males “cases”). A significant difference in the methylation level was found between cases and controls at two CpGs, six CpGs and three CpGs in ALS2CR12, GAA and UBE2G2 gene‐related amplicon respectively. Besides, the qPCR results showed a significant change in the expression levels of GAA, UBE2G2 and ALS2CR12 gene in cases compared to the controls (p ≤ .00001). In conclusion, the methylation levels at CpGs in GAA, UBE2G2 and ALS2CR12 gene amplicons were significantly different in subfertile compared to proven fertile males. In addition, a significantly different was showed in the expression levels of GAA, UBE2G2 and ALS2CR12 genes in subfertile males compared to proven fertile males.     

Impact of cigarette‐smoking on sperm DNA methylation and its effect on sperm parameters

DNA methylation is an epigenetic modification of the genome. The purpose of this study was to determine the influence of cigarette‐smoking on sperm DNA methylation from a genomewide survey of sperm samples and to ascertain its effect on sperm parameters. Twenty‐eight sperm DNA samples (from 14 fertile smokers as a case study and 14 proven fertile nonsmokers as controls) were subjected to Infinium 450K BeadChip arrays to identify the changes in the DNA methylation level between the two groups. Then, deep bisulphite sequencing was used to validate five CpGs on 78 samples. The results from the Infinium 450K found that only 11 CpGs showed a significant difference in DNA methylation between the case and the control groups. Five CpGs of the eleven (cg00648582, cg0932376, cg19169023, cg23841288 and cg27391564) underwent deep bisulphite sequencing where cg00648582, related to the PGAM5 gene, and the cg23841288 CpGs, related to the PTPRN2 gene amplicons, showed a significant increase in their DNA methylation level in more than one CpG in the case group. In contrast, a significant decrease was found at cg19169023 and at its various neighbouring CpGs in the TYRO3 gene‐related amplicons. Furthermore, this study demonstrated a significant correlation between the variation in sperm DNA methylation level and standard sperm parameters in the case group.     

DNA methylation levels of imprinted and nonimprinted genes DMRs associated with defective human spermatozoa

Asthenozoospermia (AS) is a common cause of human male infertility. Recent studies have shown significant associations of aberrant DNA methylation in spermatozoa with male infertility. The aims of the this investigation were to assess the changes in DNA methylation of known imprinted genes (MEST, GNAS and H19), novel imprinted gene (FAM50B) and nonimprinted genes (LINE‐1 and P16) DMRs in the spermatozoa of infertile men with single‐factor AS. Semen samples were obtained from 46 AS patients and 49 age‐matched normal controls. DNA methylation levels of detected genes DMR were determined by MassARRAY quantitative methylation analysis. The average methylation level at the P16 and MEST DMRs was significantly lower in AS patients than in controls (patients 6.51 ± 0.32%, controls 7.66 ± 0.40%, P < 0.01). The methylation level of 6 CpG sites of P16 DMR, and 1 CpG site of MEST, GNAS, FAM50B and LINE‐1 DMRs, was lower in AS group than in control group. For the first time, the data presented here suggest that increased methylation defects of P16 DMR may be associated with low sperm motility. This study provides the potential association between low sperm motility infertile men and the aberrant DNA methylation of MEST, LINE‐1, GNAS and FAM50B DMRs.     

Methylation levels of IGF2 and KCNQ1 in spermatozoa from infertile men are associated with sperm DNA damage

Abnormal imprinted genes methylation in spermatozoa has been shown to be associated with subfertility. However, the relationship between sperm DNA damage and specific imprinted genes methylation remains unclear. In this study, DNA methylation levels were determined at seven imprinted genes loci (H19, INS‐IGF2, KCNQ1, MEG3, MEST, PEG3 and SNRPN) in 66 semen samples using the MSRE‐qPCR method. The semen samples were divided into two groups according to the threshold value (25%) of DNA fragmentation index (DFI). We found that the mean methylation level at IGF2 (cg17037101) in the group with DFI ≥ 25% was lower than that in the group with DFI < 25% (13.7 ± 3% vs. 31.5 ± 5.3%, p = 0.0053). However, the methylation levels of other CpGs did not differ from the imprinted genes. Correlation analysis of DFI with the methylation levels of imprinted genes demonstrated that the IGF2 (cg17037101) methylation level was negatively correlated with sperm DFI (r = ?0.448, p = 0.0038), and the KCNQ1 (cg24932449) methylation level was positively correlated with sperm DFI (r = 0.354, p = 0.0273). Our results suggest that the aberrant methylation of IGF2 and KCNQ1 genes may be associated with sperm DNA damage.     

Cigarette smoking induces only marginal changes in sperm DNA methylation levels of patients undergoing intracytoplasmic sperm injection treatment

DNA methylation plays important roles in genome stability and regulation of gene expression. This study was designed to determine the influence of cigarette smoking on sperm DNA methylation. From a genome‐wide survey on sperm samples, differentially methylated target CpGs should be selected and subjected to local deep bisulphite sequencing. Obtained methylation data are compared to sperm parameters and (ICSI) outcome. Similar to pilot study, samples were subjected to Infinium 450K BeadChip arrays to identify alterations in sperm DNA methylation between smokers and nonsmokers males. Routine testing on a significantly altered CpG site was performed on more samples using local deep bisulphite sequencing. Of approximately 485,000 CpG sites analysed, only seven CpGs were found to show a significant DNA methylation difference of >20% with the top six CpGs overlapping common SNP sites. The remaining CpG site (cg19455396) is located in intron 12 of the TAP2 gene. The results of deep bisulphite sequencing showed only a tendency towards hypomethylation in the smoking group. This study could not detect biologically relevant CpG positions that are altered in sperm DNA methylation on the influence of cigarette smoking beyond individual‐specific effects that may be caused by other environmental factors.     

Methylation patterns of methylenetetrahydrofolate reductase gene promoter in infertile males

Errors of folate/homocysteine pathways which are critical for transferring methyl groups have been suggested to affect male fertility. We aimed to evaluate the methylation patterns of the promoter of methylenetetrahydrofolate reductase (MTHFR) gene in infertile males and to investigate the association between MTHFR promoter methylation and success of sperm retrieval. Thirty-five nonobstructive azoospermic and 46 severe oligozoospermic patients constituted the study group and were compared with 49 fertile and/or normozoospermic men. The methylation status was analysed by methylation-specific polymerase chain reaction. MTHFR promoter methylation was detected in infertile men with NOA and SO in the ratio of 48.6% and 58.7%, respectively. Methylation was also observed in 51% of controls. MTHFR promoter was methylated in 65% of men with viable spermatozoon during TESE. No association was found regarding to the profile of MTHFR promoter methylation between both NOA and SO patients and controls (p = .621). There was no relation between the methylation status of MTHFR promoter and low motility and poor morphology (p = .682 and p = .413, respectively). No association was found between MTHFR promoter methylation and presence of viable spermatozoa (p = .382). Our data indicate that the promoter methylation of MTHFR gene may not be associated with male infertility.     

Association of XRCC1 and ERCC2 promoters’ methylation with chromatin condensation and sperm DNA fragmentation in idiopathic oligoasthenoteratozoospermic men

The aim of the study was to investigate whether the promoter methylation of XRCC1 and ERCC2 genes is associated with sperm DNA fragmentation and chromatin condensation in idiopathic oligoasthenoteratozoospermic men. This study involved 77 infertile men with idiopathic oligoasthenoteratozoospermia and 51 normozoospermic controls. The methylight method, TUNEL assay and aniline blue staining were used for the evaluation of XRCC1 and ERCC2 genes’ methylation, SDF and sperm chromatin condensation, respectively. SDF (p = .004) and XRCC1 methylation (p = .0056) were found to be significantly higher in men with idiopathic OAT than in the controls, while mature spermatozoa frequency was higher in controls as compared to infertile men (p < .0001). No significant association was found between SDF and methylation of XRCC1 and ERCC2 genes (p = .9277 and p = .8257, respectively). However, compared to the cut-off point obtained by receiver operating characteristic analysis, a significant association was found between SDF and XRCC1 methylation, positive and negative methylation groups, generated according to the cut-off value for XRCC1. XRCC1 methylation was found to have a significant effect on chromatin condensation (p = .0017). No significant difference was detected among ERCC2 methylation, male infertility and SDF. In conclusion, XRCC1 methylation may have a role in sperm chromatin condensation and idiopathic OAT.     

Normal sperm parameters per se do not reliably account for fertility: A case–control study in the real-life setting

A proportion of men are infertile despite having normal medical history/physical examination and normal semen analysis. We aimed to assess whether normal sperm parameters per se account for male factor fertility. 1,957 infertile men were compared with 103 age-comparable fertile controls. Semen analysis was based on 2010 World Health Organization reference criteria. Of all, 12.1% of infertile men and 40.8% of fertile men presented with normal sperm parameters. Among fertile men, 36.9% had isolated sperm abnormalities and 22.3% men showed two or more concomitant sperm abnormalities. Serum total testosterone was higher in infertile men with normal sperm parameters compared to those with ≥2 sperm abnormalities or azoospermia, but similar to those with isolated sperm abnormalities (p ≤ .001). Circulating hormones were similar among sperm parameters groups in fertile men. At multivariable analyses, testicular volume (OR 1.12, p ≤ .001) and FSH (OR 0.8, p ≤ .001) were associated with normal sperm parameters. Overall, the longer the infertility period, the greater the number of sperm parameters abnormalities (p < .01). In conclusion, we found that 12% of infertile men and only 41% of fertile men present with normal sperm parameters. Normal sperm parameters per se do not reliably account for fertility in the real-life setting.     

The effect of cigarette smoking on human seminal parameters,sperm chromatin structure and condensation

Considerable debate still exists regarding the effects of cigarette smoking on male fertility. This work aimed to explore effects of cigarette smoking on semen parameters and DNA fragmentation on 95 infertile patients who were divided into infertile male nonsmokers (45) and infertile male smokers (50). Smokers were subdivided according to a number of cigarettes smoked per day into mild (≤10), moderate (11‐20) and heavy smokers (≥21). Semen analysis, sperm chromatin condensation integrity with aniline blue staining and sperm viability were compared between the study groups. A significant decrease has been shown in sperm count (p = .006), progressive motility (p = <.001), percentage of normal forms (p = <.001) and viability (p = .002) between infertile nonsmoker and infertile smokers. The percentage of abnormal sperm chromatin condensation was significantly higher in smokers compared to nonsmokers (p = <.001). A linear correlation was detected between the extent of cigarette smoking and the degree of worsening in progressive motility (p = .001), total motility (p < .001), viability (p < .001) and normal morphology (p < .001). These results indicate that cigarette smoking has detrimental effects on semen parameters. It negatively affected all conventional semen parameters in addition to sperm chromatin condensation and sperm viability. These abnormalities were also proportional to the number of cigarettes smoked per day and to the duration of smoking.     

Variations of SOST mRNA expression in human bone are associated with DNA polymorphism and DNA methylation in the SOST gene

SOST encodes sclerostin, an inhibitor of bone formation that antagonizes canonical Wnt signaling. Variations of SOST expression have an impact on bone mineral density (BMD) and bone strength. We hypothesized that genetic and epigenetic DNA modifications have an impact on SOST gene expression. By analyzing 43 bone samples from women, we found that rs851054 (G/A) is associated with SOST mRNA expression, donors with one or two G allele(s) displaying higher SOST expression (p < 0.05). Beside this polymorphism, we also investigated the role of CpG methylation in SOST mRNA expression, and analyzed methylation variation at 13 CpG sites on the 1st exon of SOST in 14 human bone samples. Principal component analysis identified three groups of CpG sites that explained most of the methylation variation. We calculated the percentage of methylation in the main group of CpGs, and showed that higher rates of methylated CpGs are associated with higher SOST mRNA expression. To demonstrate that change in SOST expression might be related to human bone disease, we analyzed 131 patients with osteogenesis imperfecta (OI), a rare disease characterized by low BMD, bone fragility, and marked intra-familial variability of bone phenotypes. We found an association between rs851054 of the SOST promoter and the fracture rate only during childhood (p < 0.01). In conclusion, genetic and epigenetic changes contribute to variation in SOST expression in human bone. Our data also indicate that these variations may be related to the severity of OI.     

Association between promoter methylation of MLH1 and MSH2 and reactive oxygen species in oligozoospermic men—A pilot study

MLH1 and MSH2 are important genes for DNA mismatch repair and crossing over during meiosis and are implicated in male infertility. Therefore, the methylation patterns of the DNA mismatch repair genes MLH1 and MSH2 in oligozoospermic males were investigated. Ten oligozoospermic patients and 29 normozoospermic donors were analysed. Methylation profiles of the MLH1 and MSH2 promotors were analysed. In addition, sperm motility and seminal reactive oxygen species (ROS) were recorded. Receiver operating characteristic (ROC) analysis was conducted to determine the accuracy of the DNA methylation status of MLH1 and MSH2 to distinguish between oligozoospermic and normozoospermic men. In oligozoospermic men, MLH1 was significantly (p = .0013) more methylated compared to normozoospermic men. Additionally, there was a significant positive association (r = .384; p = .0159) between seminal ROS levels and MLH1 methylation. Contrary, no association between MSH2 methylation and oligozoospermia was found. ROC curve analysis for methylation status of MLH1 was significant (p = .0275) with an area under the curve of 61.1%, a sensitivity of 22.2% and a specificity of 100.0%. This pilot study indicates oligozoospermic patients have more methylation of MLH1 than normozoospermic patients. Whether hypermethylation of the MLH1 promoter plays a role in repairing relevant mismatches of sperm DNA strands in idiopathic oligozoospermia warrants further investigation.     

Protective effect of vitamin E on sperm parameters in rats infected with Candida albicans

Candida albicans is one of the most frequent pathogens present in the reproductive system. The negative in vitro effects of C. albicans on sperm functions have previously been studied. The current study was undertaken to investigate the effects of C. albicans infection in vivo on sperm quality and to evaluate the efficacy of vitamin E administration in rats infected with C. albicans. In this study, 5 days after infection induction, animals were treated with vitamin E for 5 weeks. Thereafter, sperm parameters, lipid peroxidation (LPO), total antioxidant capacity (TAC), hormonal analysis and testis histology were evaluated. Based on the results, sperm parameters and TAC significantly reduced, while LPO and tissue damage increased (p ≤ .05) following the infection. Hormone analysis showed low LH and testosterone levels in serum of the infected rats. Treatment with vitamin E significantly (p ≤ .05) improved sperm quality and testis histology, increased TAC and reduced LPO. In addition, vitamin E administration significantly increased (p ≤ .05) serum LH and testosterone levels. These results clearly indicate that vitamin E is effective in attenuating the adverse effects of C. albicans infection on male fertility and could be used as a complementary treatment for patients who suffer from fertility disorders following C. albicans infection.     

Effect of selenium and pentoxifylline on expression of CATSPER1 and 2 genes and FSH/LH levels in treated mice by dexamethasone

Dexamethasone has deleterious effects on male fertility and sperm parameters. In this study, the effect of dexamethasone on expression of CATSPER1 and 2 genes was investigated. These two genes play an important role in sperm motility. Selenium and pentoxifylline were subsequently used to protect testis tissue against the destructive effects of dexamethasone. Each group received one of the following treatments for 7 days: dexamethasone (7 mg/kg) , pentoxifylline (200 mg/kg), selenium (0.3 mg/kg), dexamethasone + pentoxifylline or selenium + dexamethasone. Animals in the control group received a normal saline injection. The expression of CATSPER1 and 2 genes was analysed by real‐time PCR and serum levels of FSH and LH were determined with the enzyme‐linked immunosorbent assay method. Based on the results, dexamethasone decreases not only CATSPER1 and 2 gene expression but also serum levels of LH (p ≤ 0.05); however, it has no effect on FSH (p > 0.05). Treating with selenium significantly increased the gene expression of both CATSPER1 and 2 (p ≤ 0.05), while pentoxifylline enhanced only CATSPER2 gene expression (p ≤ 0.05). These two antioxidants were shown to increase serum levels of LH (p ≤ 0.05). Our data suggest that selenium is more effective than pentoxifylline in overcoming adverse effects of dexamethasone on male fertility.     

Semiquantitative promoter methylation of MLH1 and MSH2 genes and their impact on sperm DNA fragmentation and chromatin condensation in infertile men

To investigate the semiquantitative methylation alterations of MLH1 and MSH2 and the possible association among methylation of MLH1 and MSH2, sperm DNA fragmentation and sperm chromatin condensation in idiopathic oligoasthenoteratozoospermic men. Seventy-five idiopathic infertile men and 52 fertile and/or normozoospermic men were included in the study. SDF was analysed using the TUNEL assay in semen samples of 100 men. Promoter methylation of MLH1 and MSH2 genes was assessed by semiquantitative methylight analysis in semen samples of 39 and 40 men respectively. Sperm chromatin condensation was evaluated using aniline blue staining in 114 men. MLH1 promoter methylation was positively correlated with the percentage of aniline blue positive spermatozoa (r = 0.401, p = 0.0188). On the other hand, MSH2 promoter methylation was negatively correlated with sperm concentration and total sperm count (r = −0.421, p = 0.0068 and r = 0.4408, p = 0.009 respectively). The percentage of aniline blue positive spermatozoa in the control group was significantly lower than in the OAT group (p < 0.0001) and negatively correlated with total sperm count (r = −0.683, p < 0.0001), progressive sperm motility (r = −0.628, p < 0.0001), total motility (r = −0.639, p < 0.0001) and normal morphology (r = −0.668, p < 0.0001). Promoter methylation profile of MLH1 and MSH2 genes may play role on sperm DNA packaging and conventional semen parameters respectively.     

The correlation between mammalian target of rapamycin (mTOR) gene expression and sperm DNA damage among infertile patients with and without varicocele

This study aimed to assess the possible correlation between mammalian target of rapamycin (mTOR) gene expression and sperm DNA damage among infertile patients with and without varicocele. The study included sixty infertile males and fifty fertile males as controls. The infertile group was subdivided into the following subgroups: thirty males with varicocele and thirty males without varicocele. All subjects underwent medical history collection, clinical examination, semen analysis, sperm DNA integrity assessment, mTOR gene expression assessment and scrotal colour Doppler ultrasound. The mean mTOR gene expression in infertile patients with varicocele (23.52 ± 14.65) was significantly higher than that in infertile patients without varicocele (12.24 ± 12.44) and fertile control subjects (3.92 ± 3.26; p = 0.003 and p < 0.001 respectively). In the infertile varicocele‐positive group, mTOR gene expression showed a significant negative correlation with sperm count (p = 0.028, r = ?0.400) and progressive sperm motility (p = 0.038, r = ?0.381), as well as a significant positive correlation with the sperm DNA fragmentation index (DFI; p = 0.001, r = 0.578). In the infertile varicocele‐negative group, mTOR gene expression showed a significant negative correlation with progressive sperm motility (p = 0.018, r = ?0.429) and a significant positive correlation with sperm DFI (p < 0.001, r = 0.673). In conclusion, according to these results, there is a significant positive correlation between mTOR gene expression and sperm DFI among infertile patients with and without varicocele.     

The association of rs11457523 in HSP90AA1 with idiopathic male infertility in the Chinese population

The association of single nucleotide polymorphisms (SNPs) in heat shock protein 90 (HSP90) genes with idiopathic male infertility remains unclear. In this study, the five selected SNPs in HSP90AA1 namely rs10133307, rs10873531, rs11547523, rs11621560 and rs7145597 were genotyped in 116 idiopathic infertile males and 185 ethnically matched fertile males using the Sequenom MassARRAY assay. The role of these SNPs in male infertility was then studied using multiple genetic models. We observed that genotype distribution (p = .028) and allelic frequency (p = .032) of rs11547523 were significantly different between the infertile and fertile groups. In particular, A genotype of rs11547523 was associated with an increased risk of infertility in the allele (OR = 2.508, p = .048), dominant (OR = 2.733, p = .030) and additive models (OR = 0.366, p = .031). However, there were no significant differences in semen parameters including seminal volume (p = .452), sperm concentration (p = .727), total sperm number (p = .588), motility (p = .282) and morphology (p = .975) between A and A/G genotypes of rs11547523. These results indicate that rs11547523 in HSP90AA1 may be associated with idiopathic male infertility in the Chinese population. The outcome of this study contributes to the development of the diagnosis of male infertility.     

DNA methylation level of spermatozoa from subfertile and proven fertile and its relation to standard sperm parameters

The epigenetic mechanism plays an important role in spermatogenesis such as DNA methylation where this episode is represented by either switching genes on or off. Twenty‐eight samples (14 case and 14 controls) were subjected to Infinium 450K BeadChip arrays to identify genomic regions that differ in sperm DNA methylation patterns in the subfertile compared to the proven fertile group. Then two CpGs were validated by deep bisulphite sequencing on 82 sperm samples. The results screening study revealed eight CpGs were significantly different in their sperm DNA methylation levels between cases and control group. The results of the validation study for the two CpGs (cg19779893 and cg19406113) showed that a significant variation in the methylation level at 2 CpGs of 3 CpGs related to cg19779893 site amplicon in cases compared to the controls. Moreover, six CpGs related to the cg19406113 site amplicon showed significant differences in sperm DNA methylation between the cases and the control group. Furthermore, there was a significant decrease in the sperm parameters in the cases compared to the control group. This study found two CpGs altered in their sperm DNA methylation levels. In addition, a strong association was found between changes in the sperm DNA methylation levels in these CpGs sites and sperm parameters.     

Preparation and observation methods can produce misleading artefacts in human sperm ultrastructural morphology

The aim of this study was to evaluate the production of artefacts during preparation and observation of human sperm for ultrastructural morphology and analyse the possible reasons of causing these artefacts. Under the scanning electron microscopy, damaged sperm heads (crack or/and rupture), necks (head‐neck or head‐midpiece separation) and midpiece (disassembled and denuded axoneme ultrastructures, bent midpiece) were analysed to be the consequence of exogenous effects. Thirty infertile men with teratozoospermia revealed more spermatozoa with damage to head, neck and midpiece than did thirty fertile males (p < .01). After the samples from fertile males underwent five repeated observations, most sperm heads and necks in the samples were destroyed when compared with the single observation (p < .01). Destroyed sperm heads were full of cracks and peelings, even sperm tails were broken and fragmented, and separations of the sperm head‐neck or head‐midpiece became common. Spermatozoa from fertile males with centrifugation of 600 g for washing sperm exhibited more damage to the midpiece than those with the 300 g (p < .01). These results demonstrate that preparation and observation methods can damage sperm ultrastructures, leading to producing artefacts of ultrastructural morphology. The artefacts of sperm ultrastructural morphology may be associated with sperm structural fragility, preparation conditions and electron beam damage.