Aberrations in sperm DNA methylation patterns of males suffering from reduced fecundity

The purpose of this study was to evaluate the aberrations in sperm DNA methylation patterns of males suffering from reduced fecundity. A total of 108 males (65 males suffering from reduced fecundity as cases and 43 proven fertile males as a control) were included in the study. Thirty samples were subjected to 450K arrays as a screening phase, and then, three CpG sites located in the following genes: TYRO3, CGβ and FAM189A1 were selected to validate on 78 samples using deep bisulphite sequencing. A significant difference in the methylation level was found between cases and controls at all CpGs in TYRO3 gene‐related amplicon (CpG1, p ≤ .003, CpG2, p ≤ .0001, CpG3, p ≤ .003 and CpG4, p ≤ .030) and CpG1 in CGβ gene‐related amplicon (p ≤ .0001). Besides, a significant difference was found at two CpGs (CpG1, p ≤ .004 and CpG2, p ≤ .002) tested in the FAM189A1 gene‐related amplicon. A significant correlation was found between the methylation level at CpG1 in the FAM189A1 gene and the different types of sperm motility. In conclusion, an alteration in the methylation levels of sperm DNA from males with reduced fecundity was showed. In addition, a relationship between variations in the methylation level of these CpGs and sperm motility has been observed.     

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.     

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.     

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.     

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.     

DNA integrity and methylation changes of mouse spermatozoa following prolonged incubation

Sperm quality can be affected by different factors including the length of incubation time between sperm preparation and intracytoplasmic sperm injection. Here, we have evaluated the level of DNA methylation and expressions of related genes in mice spermatozoa. The spermatozoa were divided into three groups: fresh, spermatozoa incubated at room temperature (RT) and 37°C for 24 hr. The sperm chromatin structure assay was used to determine the DNA fragmentation index (DFI), and DNA methylation was analysed by flow cytometry. The expression levels of DNA methylation‐related genes were determined by quantitative real‐time PCR (qRT‐PCR). According to the results, we observed significantly higher sperm progressive motility and viability in the group incubated at RT compared to the spermatozoa incubated at 37°C (p < 0.05). Spermatozoa incubated at 37°C had a higher DFI compared to the other groups (p < 0.05), but the DNA methylation level significantly decreased (p < 0.05). qRT‐PCR analysis showed increased Dnmt‐1 expression in spermatozoa after 24‐hr incubation at 37°C. However, there were significantly higher expression levels of Dnmt‐3l, Dnmt‐3a and Dnmt‐3b after incubation at both RT and 37°C compared to the fresh group (p < 0.05). The 24‐hr incubation period affected both sperm DNA methylation and integrity. This study indicated that incubation at RT resulted in better sperm quality.     

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.     

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.     

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.     

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.     

Efficacy of antioxidant therapy on sperm quality measurements after varicocelectomy: A systematic review and meta‐analysis

Antioxidants were proved to be efficient to improve the quality of spermatozoa after varicocelectomy. We carried out a systematic review and performed a meta‐analysis to evaluate the efficacy of antioxidant therapy in sperm parameters' quality after varicocelectomy during 3 or 6 months' treatment cycle. During research, randomised controlled trials were searched by MEDLINE, EMBASE and the Cochrane Controlled Trials Register, and necessary parameters were compared between two groups after varicocelectomy. Finally, six studies including 576 patients were included in our meta‐analysis. As for sperm parameters, significant improvements of sperm concentration (p < .0001), sperm motility (p = .03), progressive sperm motility (p < .00001) and sperm morphology (p < .00001) were existed in antioxidant group 3 months after varicocelectomy. With regard to the 6 months' outcomes, sperm parameters were improved as well except sperm motility (p = .72) and progressive sperm motility (p = .57). Referring to pregnancy rate, no significant difference was existed between two groups (p = .36), and the FSH level of antioxidant group was lower than placebo group 3 or 6 months after varicocelectomy (3 months, p = .02; 6 months, p = .03). In conclusion, compared with the placebo, the antioxidant therapy after varicocelectomy can improve the quality of sperm parameters and construct a favourable living condition for spermatozoa by reducing FSH level.     

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.     

Oxidative stress‐induced alterations in seminal plasma antioxidants: Is there any association with keap1 gene methylation in human spermatozoa?

Kelch‐like ECH‐associated protein 1 (keap1)‐nuclear factor‐erythroid 2‐related factor 2 (Nrf2) pathway is one of the master regulators of cellular defence against oxidative stress. Epigenetic alterations like hypermethylation of keap1 gene impair keap1‐Nrf2 system in several oxidative stress–associated diseases. The objective of this study was to evaluate the epigenetic status of keap1 in sperm DNA of normozoospermic subjects, having different levels of reactive oxygen species (ROS) in seminal plasma. Semen samples were obtained from 151 apparently healthy male partners of couples who attended the Avicenna infertility clinic. Samples were categorised into four groups according to their ROS levels: group A (n = 39, ROS < 20 RLU/s per 10⁶ spermatozoa), group B (n = 38, 20 ≤ ROS < 40 RLU/s per 10⁶ spermatozoa), group C (n = 31, 40 ≤ ROS < 60 RLU/s per 10⁶ spermatozoa) and group D; (n = 43, ROS ≥ 60 RLU/s per 10⁶ spermatozoa). Keap1 methylation status was assessed using methylation‐specific PCR along with seminal total antioxidant capacity. The results showed no significant alterations in keap1 methylation in any groups, whereas the total antioxidant capacity enhanced with increasing levels of ROS exposure. These results indicate that keap1 was not methylated during ROS elevation and oxidative stress, suggesting that the cells have adopted other mechanisms to elevate antioxidant level.     

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.     

The role of N-acetyl-cysteine (NAC) orally daily on the sperm parameters and serum hormones in idiopathic infertile men: A systematic review and meta-analysis of randomised controlled trials

The meta-analysis was performed to access the role of N-acetyl-cysteine (NAC) orally daily on the sperm parameters and serum hormones in idiopathic infertile men. Randomised controlled trials (RCTs) were retrieved using PubMed, EMBASE and Cochrane register databases. The references of included studies were also searched. Finally, three articles including 431 infertile men were analysed. The results indicated that the NAC group had a considerable improvement in sperm concentration (mean difference [MD], 3.80; p < .00001), ejaculate volume (MD, 0.69; p = .002), sperm motility (MD, 4.69; p < .0001) and normal morphology (MD, 1.68; p = .0002) compared with the placebo group. However, in terms of serum hormones, the NAC group did not show significant difference in increasing the serum levels of testosterone (MD, 1.35; p = .21), luteinising hormone (MD, 0.82; p = .40), follicle-stimulating hormone (MD, −7.48; p = .29) and prolactin (MD, −0.34; p = .32) compared with the placebo group. In conclusion, NAC orally daily produced a greater improvement in sperm concentration, ejaculate volume, sperm motility and normal morphology for idiopathic infertile men, whereas no significant influence in serum hormones, which required more high-quality RCTs with sufficient sample sizes and statistics to prove.     

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.     

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.     

Do seasonal variations in ambient temperature,humidity and daylight duration affect semen parameters? A retrospective analysis over eight years

We aimed to evaluate the possible effects of seasonal variation on semen parameters. We retrospectively analysed the data of 6,116 semen samples collected at a university hospital for eight years. The past ambient temperature, relative humidity and daylight duration records, and birth registry of the province were obtained to examine the relationship of seasonal changes in semen parameters with annual birth rates and environmental factors. The mean age was 33.03 ± 6.86 years. We found a significant difference between months for sperm concentration (p < .0001), total sperm count (p < .0001), progressively motile sperm count (p < .0001) and normal sperm morphology (p = .028). The sperm concentration and total count were significantly lower in July and August compared with December, May and June. The progressively motile sperm count in October was 23.6% less than the value of May. The temperature and temperature–humidity index were negatively correlated with semen parameters. The highest number of births was in the summer. However, no correlation was present between deliveries and the semen concentration regarding months (r_s = 0.199, p = .083). In conclusion, we observed significant seasonal and monthly differences in sperm concentration, sperm count and progressively motile sperm count. Increased ambient temperature due to seasonal changes may be a detrimental factor for semen parameters.