Does advanced paternal age affect global DNA methylation of human spermatozoa and intracytoplasmic sperm injection outcome?

Objective: This study was performed to (I) evaluate the potential effect of advanced paternal age on global DNA methylation in spermatozoa, and (II) to investigate the association between the outcome of intracytoplasmic sperm injection (ICSI), semen parameters, and advanced paternal age. Material and Methods: This study comprised 230 semen samples collected from males with a mean age of 38.2±8.5 years. Medical records were used to gather clinical information related to the female partner. The participants were divided into three groups depending on age: age <30 years; age 30-40 years; and age >40 years. The DNA was extracted from purified spermatozoa. Then the sperm global DNA methylation, sperm DNA fragmentation, and chromatin decondensation were evaluated by an ELISA, TUNEL, and Chromomycin A3 staining, respectively. Results: The sample counts were n=50 (21.8%), n=90 (39.1%) and n=90 (39.1%) for the <30, 30-40 and >40 year age-groups, respectively. A significant variation was found in the age of males included in this study (p<0.001). There was a significant reduction in sperm count, total motility, and non-progressive motility in the older group compared to the younger group (p<0.001). There was also a significant elevation in chromatin decondensation, DNA fragmentation, and global DNA methylation of spermatozoa in the older age group (p<0.001). Finally, there was a significant positive correlation between the percentage of non-motile sperm, sperm chromatin decondensation, DNA fragmentation, global DNA methylation status, and paternal age (p<0.001). Conclusion: These results suggest that advanced paternal age increased the DNA fragmentation, chromatin decondensation, and global DNA methylation level in human spermatozoa, which negatively affects the ICSI outcomes in couples undergoing ICSI cycles.

Relationship between nuclear DNA fragmentation, mitochondrial DNA damage and standard sperm parameters in spermatozoa of fertile and sub-fertile men before and after freeze-thawing procedure.

The aim of this study was to assess the stability of nuclear and mitochondrial DNA (n-DNA and mt-DNA) of spermatozoa under freeze-thawing and to find out the correlation between them and their association with standard sperm parameters. Forty-three semen samples were collected from fertile (G.1; n = 29) and sub-fertile (G.2; n = 14). N-DNA fragmentation was determined by TUNEL assay and mt-DNA using caspase 3 staining. Each semen sample was frozen at -196°C by the programmed freezer. Freeze-thawing decrease vitality, total motility and membrane integrity from (43.02 ± 22.74%; 31.63 ± 18.15%; 51.5 ± 24.82%) to (22.71 ± 17.3%; 9.21 ± 6.61%; 34.64 ± 19.92% respectively [p < .001]). G.1 native spermatozoa stained positive with TUNEL and caspase 3 were (14.85 ± 17.6% and 5.8 ± 11.59%) and increased after freeze-thawing to 27.54 ± 19.74% (p = .004) and 7.3 ± 6.13% (p = .01) respectively. In G.2, TUNEL and caspase 3 were (19.84 ± 17.52% and 7.53 ± 8.56%) and increased to (29.48 ± 16.97% [p = .03] and 10.21 ± 11.73%). In conclusion, freeze-thawing process affects not only semen parameters but also n-DNA and mt-DNA. Therefore, n-DNA and mt-DNA could be used as sensitive parameters for assessment of the cryodamage of human spermatozoa.

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.

Influence of extended incubation time on Human sperm chromatin condensation, sperm DNA strand breaks and their effect on fertilisation rate.

The purpose of this study was to determine influence of extended incubation time on sperm chromatin condensation and DNA strand breaks and their effect on fertilisation rate. Forty couples undergoing ICSI therapy were included. Semen was prepared by PureSperm gradient centrifugation and divided into two parts. The first part (G1) was used immediately for ICSI, whereas the second part (G2) was kept in the incubator at 37°C, 5% and 90% Humidity for 5 hr, and thereafter, the capacitated spermatozoa were used for ICSI. The TUNEL test and chromomycin CMA3 were used to evaluate the DNA strand breaks and chromatin condensation respectively. The percentage of condensed chromatin was 73.92 ± 12.70 in the group 1 and 81.13 ± 10.31% in group 2 (p = .001). However, the double-strand breaks were 11.15 ± 8.67% in G.1 and 16.30 ± 11.12% in G.2. (p = .001). Fertilisation rate in the (Group 1) was 62.45% and 69.17% in (Group 2). There was a positive correlation between condensed chromatin and fertilisation rate (r = 0.846, p = .001) and a negative correlation with DNA double-strand breaks (r = -0.802; p = .001). In conclusion, the prolonged sperm incubation (5 hr) leads to a higher chromatin condensation and to a significantly increased number of DNA strands double breaks with no influence on fertilisation rates.

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.

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.

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.

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, CGß 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 CGß 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.

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.

Aberrant DNA methylation patterns of human spermatozoa in current smoker males.

The purpose of this study was to investigate the impact of current cigarette smoking on sperm DNA methylation patterns. A total of 108 males (51 current smokers and 57 never smoked males) were included in the study. Using 450 BeadChip Arrays, the differentially methylated CpGs between current smokers (n=15) and never smoked males (n=15) were identified. Out of significantly 11 CpGs identified, 2 CpGs namely cg07869343 and cg19169023, which are located in the MAPK8IP3 and TKR genes were selected for further analysis. Using deep bisulfite sequencing in an independent cohort of current smokers (n=36) and never smoked males (n=42), 6 and 1 CpGs showed a significant difference in the MAPK8IP (CpG3, CpG5, CpG6, CpG7, CpG8, and CpG21) and in the TKR (CpG4) were identified, respectively (P≤0.05). Our results indicate that cigarette smoking causes biochemical changes in the sperm DNA methylation in many regions and could adversely affect semen parameters.

Spermatozoa from males with reduced fecundity exhibit differential DNA methylation patterns.

Infertility affects 10-15% of couples, and approximately 50% of cases are linked to male factor infertility. The purpose of this study was to evaluate the DNA methylation patterns in spermatozoa from males who are suffering from a reduction in fecundity. 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 four CpG sites (cg05799088, cg07227024, cg16338278, and cg08408433) underwent to deep bisulfite sequencing to validate the observed methylation differences in 111 samples (56 proven fertile males as 'controls' and 55 males suffering from a reduction in fecundity as 'cases'). A significant difference in the mean methylation level was found between cases and controls in the CpGs of PRICKLE2 gene-related amplicon (CpG1, p ≤ 0.002, and CpG2, p ≤ 0.004) and CpG of ALS2CR12 gene-related amplicon (CpG1, p ≤ 0.015, and CpG2, p ≤ 0.009). Besides, a significant difference was found at seven from thirteen CpGs tested in the ALDH3B2 gene amplicon CpG2, CpG6, CpG9, CpG10, CpG11, CpG12, and CpG13 (p ≤ 0.005, p ≤ 0.004, p ≤ 0.012, p ≤ 0.028, p ≤ 0.012, p ≤ 0.009, and p ≤ 0.001, respectively). In addition, the results showed that nine CpGs out of the twenty-six within the PTGIR gene-related amplicon (CpG4, CpG6, CpG8, CpG9, CpG11, CpG15, CpG19, CpG23, and CpG26) had a significant difference in their mean methylation level (p ≤ 0.006, p ≤ 0.009, p ≤ 0.003, p ≤ 0.003, p ≤ 0.007, p ≤ 0.002, p ≤ 0.018, p ≤ 0.018, and p ≤ 0.040, respectively) in the case vs. CONTROL GROUP: In conclusion, an alteration in the methylation levels of sperm DNA from males with reduced fecundity was observed. In addition, an association between changes in the methylation level for these CpGs and different semen parameters has been found.