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.     

4977‐bp mitochondrial DNA deletion in infertile patients with varicocele

Varicocele is the abnormal inflexion and distension of veins of the pampiniform plexus within spermatic cord and is one of the amendable causes of male infertility. It can increase reactive oxygen species (ROS) production in semen and cause oxidative stress. The purpose of this study was to analyse spermatozoa mtDNA 4977‐bp deletion in infertile men with varicocele. To detect 4977‐bp deletion in spermatozoa mtDNA, semen samples of 60 infertile patients with clinical varicocele and 90 normal men from northern Iran were prepared. After extraction of spermatozoa total DNA, Gap polymerase chain reaction (Gap PCR) was performed. 4977‐bp deletion was observed in 81.66% of patients with varicocele, while approximately 15.55% of controls had this deletion. As spermatozoa from patients with varicocele had a high frequency of occurrence of 4977‐bp deletion in mtDNA [OR = 24.18, 95% confidence interval (CI) = 10.15–57.57, P < 0.0001], varicocele may induce mtDNA deletion in spermatozoa and cause infertility in north Iranian men. However, to determine the relation between sperm mtDNA 4977‐bp deletion and varicocele‐induced infertility, larger population‐based studies are needed. It is concluded that there is an association between sperm mtDNA 4977‐bp deletion and varicocele‐induced infertility in the population studied.     

Sperm mitochondrial DNA deletion in Iranian infertiles with asthenozoospermia

Asthenozoospermia is an important cause of male infertility. The mutations in sperm mitochondrial DNA (mtDNA) result in either functionless or malfunctioning some proteins, subsequently affecting sperm motility leading to asthenozoospermia. The purpose of this study was to investigate sperm mtDNA 4,977‐bp deletion in infertile men with low sperm motility/immotile spermatozoa compared to healthy subjects with high sperm motility. Semen samples of 256 asthenozoospermic infertiles and 200 controls from northern Iran were collected. After extraction of spermatozoa total DNA, Gap‐polymerase chain reaction (Gap‐PCR) was performed. The deletion was observed in 85.93% of patients with asthenozoospermia compared with 14% in controls [OR = 37.5397, 95% confidence interval = 12.937–108.9276, p < .0001]. It is concluded that there is a strong association between sperm mtDNA 4,977‐bp deletion and asthenozoospermia‐induced infertility in the population examined. Large‐scale mtDNA deletions in spermatozoa may induce bioenergetic disorders. Nevertheless, to validate our results broader research may be needed.     

Association between methylenetetrahydrofolate reductase (MTHFR) gene promoter hypermethylation and the risk of idiopathic male infertility

Epigenetics has become a major field of reproductive medicine after the epigenetic regulation of gene expression was discovered. The aim of this study was to find out whether or not methylenetetrahydrofolate reductase (MTHFR) gene promoter hypermethylation in the spermatozoa of men who were offered assisted reproduction is associated with idiopathic male infertility. Sperm DNAs from 40 idiopathic infertile men with normozoospermia and 40 controls consisting of healthy fertile men were isolated. Following the modification of DNAs by sodium bisulphite, the methylation status of the MTHFR gene promoter was quantified by pyrosequencing. No significant differences were observed between the clinical characteristics of patients and controls. The percentage of MTHFR promoter methylation in infertile men with normozoospermia (11%) was significantly higher than that in the healthy control (4.3%) group (p = .01). A 9.5% of methylation level was determined via receiver operator characteristic (ROC) analysis as the cut‐off value. There were 21 (53%) hypermethylated men among the infertile men and 2 (5%) in the control group (p = .0001). The intragroup analysis of the infertile group did not reveal any statistically significant differences in terms of overall clinical characteristics between hyper‐ and normo‐methylated infertile men. Our results suggest that epigenetic silencing (hypermethylation) of MTHFR could result in an elevated risk of male infertility.     

Sperm DNA methylation of H19 imprinted gene and male infertility

Infertility affects up to 15% of reproductive‐aged couples worldwide, with male factor being detected in 40%–50% of the cases. Proper sperm production is associated with the establishment of appropriate epigenetic marks in developing germ cells. Several studies have demonstrated the association between abnormal spermatogenesis and epigenetic disturbances with the major focus on DNA methylation. Imprinted genes are expressed in a parent‐of‐origin‐specific manner, and the role of their DNA methylation in proper spermatogenesis has been documented recently. The existing evidence along with the absence of relevant data in south of Iran prompted us to study the methylation of H19 imprinted gene in spermatozoa of idiopathic infertile patients (males with abnormalities in sperm parameters) and healthy controls by Combined Bisulfite Restriction Analysis. According to our results, the lowest methylation percentage of H19 imprinted gene belongs to three cases with sperm characteristics under normal range (two cases Oligoasthenoteratozoospermia and one case Oligoteratozoospermia). However, our results show that the median of methylation percentage for H19 is not statistically significant between case and control groups. Our results and those of others introduce DNA methylation as a potential marker of fertility and should be investigated with more patients in future studies.     

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.     

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.     

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.     

Sperm chromatin quality and DNA integrity in partial versus total globozoospermia

Globozoospermia is a severe form of teratozoospermia with low incidence in infertile patients, considered as one of the important causes of male infertility. The objective was to investigate the chromatin/DNA integrity as well as apoptosis in ejaculated spermatozoa of cases with partial or total globozoospermia. Fifty‐seven semen samples were divided into three groups of partial globozoospermia (n = 17), total globozoospermia (n = 10) and normozoospermia (control; n = 30). Sperm chromatin condensation, DNA integrity and apoptosis were assessed using cytochemical assays. The results showed significant differences in sperm parameters of count and motility between two case groups versus controls. The percentages of spermatozoa with abnormal chromatin packaging and protamine deficiency were significantly higher in total and partial globozoospermic men compared to normozoospermic samples. Also, the rates of TUNEL‐positive spermatozoa were significantly increased in both globozoospermic cases with respect to the control (18.3 ± 10.1 and 12.3 ± 9.2 versus 5.9 ± 3 respectively). However, no significant differences were noticed between two subgroups of patients with regard to sperm DNA denaturation, DNA fragmentation and apoptosis. Abnormal chromatin packaging, DNA damage and apoptosis were significantly higher in cases than controls. The sperm chromatin/DNA anomalies may be considered as one of the main aetiology of ART failure in globozoospermic patients.     

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.     

Gsα activity is reduced in erythrocyte membranes of patients with psedohypoparathyroidism due to epigenetic alterations at the GNAS locus

In pseudohypoparathyroidism (PHP), PTH resistance results from impairment of signal transduction of G protein–coupled receptors caused by a deficiency of the Gsα‐cAMP signaling cascade due to diminished Gsα activity in maternally imprinted tissues. In PHP‐Ia, inactivating mutations of the GNAS gene lead to haploinsufficiency in some tissues with biallelic expression, so in addition to PHP, Albright's hereditary osteodystrophy (AHO) is also present. In PHP‐Ib, caused by methylation defects at the GNAS locus, diminished Gsα activity was thought to be limited to maternally imprinted tissues, such as the renal proximal tubule and the thyroid, leading to a lack of AHO. Recently, we demonstrated methylation defects in patients with AHO signs, indicating a connection between epigenetic changes and AHO. Our objective was to determine Gsα activity in erythrocyte membranes in patients with epigenetic defects at the GNAS locus compared to normal controls and patients with inactivating GNAS mutations. Gsα activity and expression, mutation of the GNAS locus, and methylation status were studied in patients with PHP and mild signs of AHO (PHP‐Ia: 12; PHP‐Ib: 17, of which 8 had some features of AHO). Then, we statistically compared the Gsα activity of the different PHP subtypes. Patients with methylation defects at the GNAS locus show a significant decrease in erythrocyte Gsα activity compared to normal controls (PHP‐Ib versus controls, p < .001). This was significantly lower in patients with AHO signs (PHP‐Ib + mild‐AHO versus PHP‐Ib, p < .05). Our research shows that PHP‐Ia and PHP‐Ib classification is not only overlapped genetically, as reported, but also in terms of Gsα activity. Reduced expression of GNAS due to methylation defects could downregulate Gsα activity in other tissues beyond those described and could also be causative of AHO. © 2011 American Society for Bone and Mineral Research     

Relationship between DYNLT1 and Beclin1 expression and the fertilising potential of human spermatozoa

This study aimed to evaluate dynein light chain type 1 (DYNLT1) mRNA expression in mature spermatozoa and to investigate its association with Beclin1 expression to help in understanding of pathogenesis of male infertility. It included 60 infertile men divided into idiopathic (n = 20), accessory gland inflammation (n = 20), and varicocele (n = 20) groups, and 20 healthy fertile men as a control group. Semen parameters were evaluated according to the 2010 World Health Organization criteria. Mature spermatozoa were isolated by Sil‐select gradient. Relative quantification of DYNLT1 and Beclin1 mRNA expression in whole sperm pellet and mature spermatozoa was done using real‐time PCR. Beclin1 protein was assessed in whole sperm pellet and mature spermatozoa by ELISA. Beclin1 mRNA and protein were significantly increased in spermatozoa from infertile patients of different aetiologies in comparison to healthy controls (p < .05). However, DYNLT1 mRNA expression was significantly decreased in infertile groups than controls (p < .05). Mature spermatozoa extracted from all studied subjects showed increased DYNLT1 mRNA and decreased Beclin1 mRNA and protein expression compared with the whole sample. It is concluded that decreased Beclin1 and increased DYNLT1 mRNA expression in mature spermatozoa may provide an insight into the biological processes that are activated or suppressed during sperm maturation.     

Investigating the relationship between BRCA1 and BRCA2 genes methylation profile and sperm DNA fragmentation in infertile men

The purpose of the study was to investigate whether the promoter methylation status of BRCA1 and BRCA2 DNA repair genes is associated with sperm DNA fragmentation (sDF) in infertile men with oligoasthenoteratozoospermia (OAT) which emerges due to various reasons and is effective in male infertility. Seventy‐three infertile men with OAT and 20 normozoospermic volunteers participated in the study. To investigate sDF and methylation patterns of BRCA1 and BRCA2 gene promoters, TUNEL assay and methylation‐specific PCR (MS‐PCR) were used. The mean sDF ratio for the patients was calculated as 22.50%. The calculated cut‐off value for sDF ratio was 17.0% in ROC curve analysis. Regarding sDF, a significant difference between the normozoospermic group and the OAT group with abnormal semen parameters (p < 0.001) was found. sDF demonstrated a significant effect on the semen parameters and negative correlations on sDF ratios and sperm motility, concentration and morphology. There was no statistically significant association between sDF and the methylation status of the promoter of either BRCA1 or BRCA2 genes. In routine clinical practice, sperm DNA integrity should be investigated before applying assisted reproductive techniques. To understand better the relationship between epigenetic regulation of DNA repair genes and male infertility, additional studies are required.     

MMP2, MMP9 and TIMP2 polymorphisms affect sperm parameters but not fertility in Polish males

Proper function of the blood–testis barrier is pivotal to spermatogenesis. Synchronised action of matrix metalloproteinases (MMP) and their inhibitors (TIMP) is mandatory to maintain dynamic balance of the barrier. Therefore, the association of functional genetic variants of MMP‐2, MMP‐9 and TIMP‐2 and male infertility was studied. A total of 416 infertile males and 421 healthy subjects were genotyped for 7 SNPs within MMP2, MMP9 and TIMP2 genes, along with the assessment of semen parameters (concentration, motility and morphology of spermatozoa). No association was observed between the studied genotypes and male infertility. However, higher sperm concentration was associated with TIMP2 rs8080623 C and rs2277698 T variants among infertile men, and with MMP9 rs17576 A minor allele in controls (p < .05). TIMP2 rs9900972 T and rs2277698 T allele were associated with higher percentage of morphologically normal spermatozoa among controls. MMP2 rs2285053 TT homozygous infertile patients presented higher percentage of spermatozoa displaying nonprogressive motility. Haplotype analysis revealed strong linkage disequilibrium between the studied loci (5 of 8 possible TIMP2 haplotypes, and 3 of 4 possible MMP2 and MMP9 were found). None of the haplotypes showed association with infertility. This study results suggest an association between MMP9 and TIMP2 SNPs with sperm parameters, but not infertility.     

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.     

精子MTCYB基因缺失与男性不育的关系

目的 探讨精子ＭＴＣＹＢ基因缺失与男性不育的关系。方法 应用ＰＣＲ技术对１００例不育男性（精子活动力差５０例,精子活动力正常５０例）,和３０例已育男性的精子ＭＴＣＹＢ基因进行检测。结果 男性不育组精子ＭＴＣＹＢ基因缺失率为１６％,其中精子活力差的５０例标本检出９例,精子活动力正常的５０例标本中检出７例,经统计学检验二组差异无显著性。     

将表观遗传学引入男科实验室：挑战与前景

近期研究发现精子DNA甲基化的异常与自发性男性不育有显著关联，自然流产及胚胎发育紊乱频发。因此，对精子中某些特定基因DNA甲基化进行研究分析有可能成为临床男科中一种新的有价值的诊断标记。本文讨论了DNA甲基化分析在男性不育症诊断中的应用现状和价值，并且概述了面临的挑战和未来的应用前景。本文突出介绍了DNA甲基化作用在男科诊断学的应用前景，同时也对概述了其在自发性不育症的诊断价值。     

4,977-bp human mitochondrial DNA deletion is associated with asthenozoospermic infertility in Jordan

Male infertility is commonly associated with sperm abnormalities including asthenozoospermia. The molecular basis of asthenozoospermia was linked to mitochondrial DNA (mtDNA) mutations. The 4,977-bp human mtDNA deletion is one of the most common mutations of spermatozoa and results in loss of about 33% of the mitochondrial genome. In this preliminary study, we aimed to investigate the presence of 4,977-bp mtDNA deletion in asthenozoospermic infertile men in Jordan. Semen specimens of 120 asthenozoospermic infertile men and 80 normozoospermic individuals were collected at the in vitro fertilization unit. MtDNA was extracted after the enrichment of spermatozoa; then, polymerase chain reaction was performed using 4,977-bp mtDNA deletion-specific primers. The deletion of 4,977-bp mtDNA was detected in 79.2% of asthenozoospermic patients compared to 10% in normozoospermic controls. The results showed a significant association between the presence of 4,977-bp mtDNA deletion and the asthenozoospermia and infertility (OR = 34.2000, 95% CI = 14.57–80.26, p-value < .001). In conclusion, our findings underscored a strong association between 4,977-bp mtDNA deletion and asthenozoospermia in the Jordanian population.     

Analysis of the methylation pattern of six gene promoters in sperm of men with abnormal protamination

It has recently been shown that alteration of the methylation pattern of imprinted genes is associated with different types of male infertility. The objective of our study was to investigate the methylation pattern of selected gene promoters in sperm of patients with abnormal protamine replacement. The promoters of OCT4, SOX2, NANOG, HOXC11, miR-17 and CREM were analyzed using bisulfite sequencing and the percentage of DNA methylation was compared between patients with an abnormal protamine 1/protamine 2 (P1/P2) ratio and normozoospermic controls. No significant quantitative differences were found between groups of patients with either an abnormally high or low P1/P2 ratio compared to normal controls. However, two individual samples from infertile subjects (2/20, 10%) showed an altered methylation pattern for the CREM gene promoter that was not found in control samples. These two samples had a significantly higher (P<0.05) promoter methylation (5.58 and 4.23%, respectively) compared to the control group (0.46%). In conclusion, in our pilot study, extreme methylations defects were not seen broadly in severely infertile men. However, two patients exhibited altered methylation of the CREM gene, which may be either causative or a result of abnormal protmaine replacement.