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In recent decades, we have witnessed a progressive decline in male fertility. This is partly related to the increased prevalence of chronic diseases (e.g., obesity and diabetes mellitus) and risky lifestyle behaviors. These conditions alter male fertility through various non-genetic mechanisms. However, there is increasing evidence that they are also capable of causing sperm epigenetic alterations, which, in turn, can cause infertility. Furthermore, these modifications could be transmitted to offspring, altering their general and reproductive health. Therefore, these epigenetic modifications could represent one of the causes of the progressive decline in sperm count recorded in recent decades. This review focuses on highlighting epigenetic modifications at the sperm level induced by non-genetic causes of infertility. In detail, the effects on DNA methylation, histone modifications, and the expression profiles of non-coding RNAs are evaluated. Finally, a focus on the risk of transgenerational inheritance is presented. Our narrative review aims to demonstrate how certain conditions can alter gene expression, potentially leading to the transmission of anomalies to future generations. It emphasizes the importance of the early detection and treatment of reversible conditions (such as obesity and varicocele) and the modification of risky lifestyle behaviors. Addressing these issues is crucial for individual health, in preserving fertility, and in ensuring the well-being of future generations. [ABSTRACT FROM AUTHOR]

During respiration, particulate matter with a diameter of 2.5 µm or less (PM2.5) suspended in the atmosphere enters the terminal alveoli and blood. PM2.5 particles can attach to toxic substances, resulting in health problems. Limited information is available regarding the effects of prenatal exposure to water-soluble PM2.5 (WS-PM2.5) and water-insoluble PM2.5 (WI-PM2.5) on male reproduction. In addition, whether exposure to these particles has transgenerational effects remains unknown. We investigated whether prenatal exposure to WS-PM2.5 and WI-PM2.5 disrupts sperm function in generations F1, F2, and F3 of male mice. Pregnant BALB/c mice were treated using intratracheal instillation on gestation days 7, 11, and 15 with 10 mg of a water extract or insoluble PM2.5. On postnatal day 105, epididymal sperm count, motility, morphology, mitochondrial membrane potential (MMP), reactive oxygen species (ROS) production, the sperm chromatin DNA fragmentation index (DFI), and testicular DNA methyltransferase (Dnmt) levels were evaluated in all generations. Whole-genome bisulfite sequencing was used to analyze the DNA methylation status of generation F3. According to the results, exposure to WS-PM2.5 affected sperm morphology, ROS production, and mean DFI in generation F1; ROS production and mean DFI in generation F2; and sperm morphology and MMP in generation F3. Similarly, exposure to WI-PM2.5 affected sperm morphology, ROS production, mean DFI, %DFI, and Dnmt1 expression in generation F1; sperm morphology, MMP, and ROS production in generation F2; and sperm morphology, ROS, and %DFI in generation F3. Two hypermethylated genes, PRR16 and TJP2, were observed in the WS-PM2.5 and WI-PM2.5 groups, two hypomethylated genes, NFATC1 and APOA5, were observed in the WS-PM2.5 group, and two hypomethylated genes, ZFP945 and GSE1, were observed in the WI-PM2.5 group. Hence, prenatal exposure to PM2.5 resulted in transgenerational epigenetic effects, which may explain certain phenotypic changes in male reproduction.

To study the genome wide alterations in sperm DNA methylation in male partners of idiopathic recurrent pregnancy loss (iRPL) cases and note regions as potential diagnostic markers. Case-control study and methylome analysis of human sperm. Obstetrics and Gynaecology clinics. Control group consists of apparently healthy fertile men having fathered a child within the last 2 years (n = 39); and case group consists of male partners of iRPL cases having ≥2 consecutive 1st trimester pregnancy losses (n = 47). None. Sperm DNA samples of controls and cases were selected for whole genome bisulfite sequencing analysis based on the previously set thresholds of global methylation levels and methylation levels of imprinted genes (KvDMR and ZAC). Whole genome bisulfite sequencing of selected sperm genomic DNA was performed to identify differentially methylated CpG sites of iRPL cases compared with fertile controls. Pathway analysis of all the differentially methylated genes was done by Database for Annotation, Visualization, and Integrated Discovery annotation tool and Kyoto Encyclopedia of Genes and Genomes tool. Differentially methylated CpGs within genes relevant to embryo and placenta development were selected to further validate their methylation levels in study population by pyrosequencing. A total of 9497 differentially methylated CpGs with highest enrichment in intronic regions were obtained. In addition, 5352 differentially methylated regions and 2087 differentially methylated genes were noted. Signaling pathways involved in development were enriched on pathway analysis. Select CpGs within genes PPARG , KCNQ1 , SETD2, and MAP3K4 showed distinct hypomethylated subpopulations within iRPL study population. Our study highlights the altered methylation landscape of iRPL sperm, and their possible implications in pathways of embryo and placental development. The CpG sites that are hypomethylated specifically in sperm of iRPL subpopulation can be further assessed as predictive biomarkers. [ABSTRACT FROM AUTHOR]

Recently, more evidences of epigenetic impact on the male fertility, particularly on sperm DNA methylation have been reported. Data related to this issue in livestock males is still limited. The present study analyzed the DNA methylation status of the important gene for spermatogenesis, SIRT1, in ram sperm and its correspondence with semen quality and fertilizing ability. The ejaculates of 10 rams (5 rams - 1.5 years old, and 5 rams - 4 years old) from Synthetic Population Bulgarian Milk breed were evaluated and used for the artificial insemination of 174 ewes in breeding season. Two semen samples from each animal were used for DNA extraction followed by bisulfite conversion. The DNA methylation status of SIRT1 was detected through quantitative methylation-specific PCR using two sets of primers designed specifically for bisulfite-converted DNA sequences to attach methylated and unmethylated sites. On the base of age and conception rate the rams were divided in different groups. Data of semen quality, DNA methylation status of SIRT1 and reproductive performances of each group were statistically processed. Results showed a high average value of DNA methylation of SIRT1 in ram sperm (78.5±23.9%) and wide individual variability among investigated animals, with a coefficient of variation of 34.4%. The 1.5 years old animals tended to have a higher level of SIRT1 methylation than 4 years old animals. The rams in group with high fertilizing ability had significantly higher DNA methylation of SIRT1 in sperm than those with low fertilizing ability. In conclusion, results of this study provided evidence that the alteration of sperm SIRT1 methylation is associated with fertility performances of the rams and, probably, with their age.

Objective: To identify differentially methylated regions and assess epigenetic age and instability scores within each cohort of differing income levels and educational attainment., Design: Retrospective analysis of epigenetic data., Setting: University., Patient(s): A total of 1,429 semen samples from participants in the Folic Acid and Zinc Supplementation Trial study., Intervention(s): Socioeconomic status, approximated by self-reported income, and education level., Main Outcome Measure(s): Differential sperm DNA methylation, epigenetic age, epigenetic stability., Result(s): There were four significant differentially methylated regions associated with the income cohorts and seven associated with the education cohorts, suggesting epigenetic patterns vary with socioeconomic status. Adjusted epigenetic age (germ-line age differential values) did not differ significantly in either cohort; however, a higher number of unstable promoters were observed in the lowest education cohort, indicating dysregulation in the epigenome., Conclusion(s): These findings suggest a biologic link between income and education level in sperm epigenetic patterns, which may influence reproductive health. The increase in unstable promoters in the lower education cohorts may contribute to health disparities associated with educational attainment; however, it is important to consider lifestyle factors and environmental exposures as potential underlying causes. Further research is needed to fully elucidate the implications of these epigenetic changes and their relationship to health outcomes., Clinical Trial Registration Number: Clinicaltrials.gov, NCT#01857310., Competing Interests: Declaration of Interests K.S. has nothing to disclose. C.P. has nothing to disclose. N.J.P. has nothing to disclose. E.A.D. has nothing to disclose. T.J. has nothing to disclose., (Copyright © 2024. Published by Elsevier Inc.)

Abstract Background DNA methylation has been shown to be involved in many biological processes, including X chromosome inactivation in females, paternal genomic imprinting, and others. Results Based on the correlation patterns of methylation levels of neighboring CpG sites among 28 sperm whole genome bisulfite sequencing (WGBS) data (486 × coverage), we obtained 31,272 methylation haplotype blocks (MHBs). Among them, we defined conserved methylated regions (CMRs), variably methylated regions (VMRs) and highly variably methylated regions (HVMRs) among individuals, and showed that HVMRs might play roles in transcriptional regulation and function in complex traits variation and adaptive evolution by integrating evidence from traditional and molecular quantitative trait loci (QTL), and selection signatures. Using a weighted correlation network analysis (WGCNA), we also detected a co-regulated module of HVMRs that was significantly associated with reproduction traits, and enriched for glycosyltransferase genes, which play critical roles in spermatogenesis and fertilization. Additionally, we identified 46 VMRs significantly associated with reproduction traits, nine of which were regulated by cis-SNPs, implying the possible intrinsic relationships among genomic variations, DNA methylation, and phenotypes. These significant VMRs were co-localized (± 10 kb) with genes related to sperm motility and reproduction, including ZFP36L1, CRISP2 and HGF. We provided further evidence that rs109326022 within a predominant QTL on BTA18 might influence the reproduction traits through regulating the methylation level of nearby genes JOSD2 and ASPDH in sperm. Conclusion In summary, our results demonstrated associations of sperm DNA methylation with reproduction traits, highlighting the potential of epigenomic information in genomic improvement programs for cattle.

Sperm DNA methylation is crucial for fertility and viability of offspring but epigenome evolution in mammals is largely understudied. By comparing sperm DNA methylomes and large-scale genome-wide association study (GWAS) signals between human and cattle, we aimed to examine the DNA methylome evolution and its associations with complex phenotypes in mammals. Our analysis revealed that genes with conserved non-methylated promoters (e.g., ANKS1A and WNT7A) among human and cattle were involved in common system and embryo development, and enriched for GWAS signals of body conformation traits in both species, while genes with conserved hypermethylated promoters (e.g., TCAP and CD80) were engaged in immune responses and highlighted by immune-related traits. On the other hand, genes with human-specific hypomethylated promoters (e.g., FOXP2 and HYDIN) were engaged in neuron system development and enriched for GWAS signals of brain-related traits, while genes with cattle-specific hypomethylated promoters (e.g., LDHB and DGAT2) mainly participated in lipid storage and metabolism. We validated our findings using sperm-retained nucleosome, preimplantation transcriptome, and adult tissue transcriptome data, as well as sequence evolutionary features, including motif binding sites, mutation rates, recombination rates and evolution signatures. In conclusion, our results demonstrate important roles of epigenome evolution in shaping the genetic architecture underlying complex phenotypes, hence enhance signal prioritization in GWAS and provide valuable information for human neurological disorders and livestock genetic improvement.

A growing number of sperm methylome analyses have identified genomic loci that are susceptible to paternal age effects in a variety of mammalian species, including human, bovine, and mouse. However, there is little overlap between different data sets. Here, we studied whether or not paternal age effects on the sperm epigenome have been conserved in mammalian evolution and compared methylation patterns of orthologous regulatory regions (mainly gene promoters) containing both conserved and non-conserved CpG sites in 94 human, 36 bovine, and 94 mouse sperm samples, using bisulfite pyrosequencing. We discovered three (NFKB2, RASGEF1C, and RPL6) age-related differentially methylated regions (ageDMRs) in humans, four (CHD7, HDAC11, PAK1, and PTK2B) in bovines, and three (Def6, Nrxn2, and Tbx19) in mice. Remarkably, the identified sperm ageDMRs were all species-specific. Most ageDMRs were in genomic regions with medium methylation levels and large methylation variation. Orthologous regions in species not showing this age effect were either hypermethylated (>80%) or hypomethylated (

Abstract Background Testicular germ cell tumor such as seminoma is strongly associated with male reproductive problems commonly associated with the alteration of sperm parameters as described in testicular dysgenesis syndrome. Interestingly, numerous studies have reported that the precursor of germ cell cancer, germ cell neoplasia in situ (GCNIS), present similarities to fetal gonocytes, specifically characterized by global DNA hypomethylation particularly on imprinting sequences. These disorders may have a common origin derived from perturbations of embryonal programming during fetal development. Presently, there is no available information concerning the sperm DNA methylation patterns of testicular cancer patients. For the first time, we evaluated the sperm imprinting of seminoma patients. A total of 92 cryopreserved sperm samples were included, 31 before seminoma treatment (S): 23 normozoospermic (SN) and 8 oligozoospermic (SO) and 61 sperm controls samples: 31 normozoospermic (N) and 30 oligozoospermic (O). DNA methylation levels of seven differentially methylated regions (DMRs) of imprinted genes [H19/IGF2: IG-DMR (CTCF3 and CTCF6 of H19 gene); IGF2-DMRs (DMR0 and DMR2); MEG3/DLK1:IG-DMR; SNURF:TSS-DMR; KCNQ1OT1:TSS-DMR] were assessed by pyrosequencing. All comparative analyses were adjusted for age. Results Comparisons of sperm DNA methylation levels between seminoma (S) and normozoospermic (N) samples showed a significant difference for the SNURF sequence (p = 0.017), but after taking into account the sperm parameters, no difference was observed. However, we confirmed a significant association between oligozoospermia (O) and imprinting defects for H19/IGF2-CTCF6 (p = 0.001), MEG3/DLK1 (p = 0.017), IGF2-DMR2 (p = 0.022), and SNURF (p = 0.032) in comparison with control groups (N). Conclusions This study highlights the high risk of sperm imprinting defects in cases of oligozoospermia and shows for the first time that seminoma patients with normal spermatogenesis present sperm imprinting integrity. These data suggest a low probability of the involvement of a common imprinting defect in fetal cells leading to both TGCT and subfertility.

Background: Many environmental and lifestyle factors have been implicated in the decline of sperm quality, with diet being one of the most plausible factors identified in recent years. Moreover, several studies have reported a close association between the alteration of specific sperm DNA methylation signatures and semen quality. Objectives: To evaluate the effect of tree nut consumption on sperm DNA methylation patterns in healthy individuals reporting eating a Western‐style diet. Material and methods: This is a post hoc analysis conducted in a subset of participants (healthy, non‐smoking, and young) from the FERTINUTS 14‐wk randomized‐controlled, parallel trial, recruited between December 2015 and February 2017. The participants included in the current study (n = 72) were randomly selected in a proportion 2:1 from the original FERTINUTS trial between the 98 participants that completed the entire dietary intervention (nut group, n = 48; control group, n = 24). Sperm DNA methylation patterns were examined at baseline and after 14 weeks in 48 individuals consuming 60 g/d of mixed nuts (nut group) and in 24 individuals following the usual Western‐style diet avoiding consumption of nuts (control group). Results: Over the course of the trial, no significant changes in global methylation were observed between groups. However, in the nut group, we identified 36 genomic regions that were significantly differentially methylated between the baseline and the end of the trial and 97.2% of the regions displayed hypermethylation. We identified no such change in the control group over the same period of time. We also utilized the recently developed germ line age calculator to determine if nut consumption resulted in alterations to the epigenetic age of cells and no significant differences were found. Discussion and conclusion: Adding nuts to a regular Western‐style diet subtly impacts sperm DNA methylation in specific regions, demonstrating that there are some sperm epigenome regions that could respond to diet. [ABSTRACT FROM AUTHOR]

In somatic cells/tissues, methylation of ribosomal DNA (rDNA) increases with age and age‐related pathologies, which has a direct impact on the regulation of nucleolar activity and cellular metabolism. Here, we used bisulfite pyrosequencing and show that methylation of the rDNA transcription unit including upstream control element (UCE), core promoter, 18S rDNA, and 28S rDNA in human sperm also significantly increases with donor's age. This positive correlation between sperm rDNA methylation and biological age is evolutionarily conserved among mammals with widely different life spans such as humans, marmoset, bovine, and mouse. Similar to the tandemly repeated rDNA, methylation of human α‐satellite and interspersed LINE1 repeats, marmoset α‐satellite, bovine alpha‐ and testis satellite I, mouse minor and major satellite, and LINE1‐T repeats increases in the aging male germline, probably related to their sperm histone packaging. Deep bisulfite sequencing of single rDNA molecules in human sperm revealed that methylation does not only depend on donor's age, but also depend on the region and sequence context (A vs. G alleles). Both average rDNA methylation of all analyzed DNA molecules and the number of fully (>50%) methylated alleles, which are thought to be epigenetically silenced, increase with donor's age. All analyzed CpGs in the sperm rDNA transcription unit show comparable age‐related methylation changes. Unlike other epigenetic aging markers, the rDNA clock appears to operate in similar ways in germline and soma in different mammalian species. We propose that sperm rDNA methylation, directly or indirectly, influences nucleolar formation and developmental potential in the early embryo. [ABSTRACT FROM AUTHOR]

Sperm DNA methylation is crucial for fertility and viability of offspring but epigenome evolution in mammals is largely understudied. By comparing sperm DNA methylomes and large-scale genome-wide association study (GWAS) signals between human and cattle, we aimed to examine the DNA methylome evolution and its associations with complex phenotypes in mammals. Our analysis revealed that genes with conserved non-methylated promoters (e.g., ANKS1A and WNT7A) among human and cattle were involved in common system and embryo development, and enriched for GWAS signals of body conformation traits in both species, while genes with conserved hypermethylated promoters (e.g., TCAP and CD80) were engaged in immune responses and highlighted by immune-related traits. On the other hand, genes with human-specific hypomethylated promoters (e.g., FOXP2 and HYDIN) were engaged in neuron system development and enriched for GWAS signals of brain-related traits, while genes with cattle-specific hypomethylated promoters (e.g., LDHB and DGAT2) mainly participated in lipid storage and metabolism. We validated our findings using sperm-retained nucleosome, preimplantation transcriptome, and adult tissue transcriptome data, as well as sequence evolutionary features, including motif binding sites, mutation rates, recombination rates and evolution signatures. In conclusion, our results demonstrate important roles of epigenome evolution in shaping the genetic architecture underlying complex phenotypes, hence enhance signal prioritization in GWAS and provide valuable information for human neurological disorders and livestock genetic improvement. [ABSTRACT FROM AUTHOR]

Purpose: This study was conducted in order to investigate the effects of reactive oxygen species (ROS) levels on the seminal plasma (SP) metabolite milieu and sperm dysfunction.Methods: Semen specimens of 151 normozoospermic men were analyzed for ROS by chemiluminescence and classified according to seminal ROS levels [in relative light units (RLU)/s/106 sperm]: group 1 (n = 39): low (ROS < 20), group 2 (n = 38): mild (20 ≤ ROS < 40), group 3 (n = 31): moderate (40 ≤ ROS < 60), and group 4 (n = 43): high (ROS ≥ 60). A comprehensive analysis of SP and semen parameters, including conventional semen characteristics, measurement of total antioxidant capacity (TAC), sperm DNA fragmentation index (DFI), chromatin maturation index (CMI), H19-Igf2 methylation status, and untargeted seminal metabolic profiling using nuclear magnetic resonance spectroscopy (1H-NMR), was carried out.Result(s): The methylation status of H19 and Igf2 was significantly different in specimens with high ROS (P < 0.005). Metabolic fingerprinting of these SP samples showed upregulation of trimethylamine N-oxide (P < 0.001) and downregulations of tryptophan (P < 0.05) and tyrosine/tyrosol (P < 0.01). High ROS significantly reduced total sperm motility (P < 0.05), sperm concentration (P < 0.001), and seminal TAC (P < 0.001) but increased CMI and DFI (P < 0.005). ROS levels have a positive correlation with Igf2 methylation (r = 0.19, P < 0.05), DFI (r = 0.40, P < 0.001), CMI (r = 0.39, P < 0.001), and trimethylamine N-oxide (r = 0.45, P < 0.05) and a negative correlation with H19 methylation (r = − 0.20, P < 0.05), tryptophan (r = − 0.45, P < 0.05), sperm motility (r = − 0.20, P < 0.05), sperm viability (r = − 0.23, P < 0.01), and sperm concentration (r = − 0.30, P < 0.001).Conclusion(s): Results showed significant correlation between ROS levels and H19-Igf2 gene methylation as well as semen parameters. These findings are critical to identify idiopathic male infertility and its management through assisted reproduction technology (ART). [ABSTRACT FROM AUTHOR]

During respiration, particulate matter with a diameter of 2.5 µm or less (PM 2.5) suspended in the atmosphere enters the terminal alveoli and blood. PM 2.5 particles can attach to toxic substances, resulting in health problems. Limited information is available regarding the effects of prenatal exposure to water-soluble PM 2.5 (WS-PM 2.5) and water-insoluble PM 2.5 (WI-PM 2.5) on male reproduction. In addition, whether exposure to these particles has transgenerational effects remains unknown. We investigated whether prenatal exposure to WS-PM 2.5 and WI-PM 2.5 disrupts sperm function in generations F1, F2, and F3 of male mice. Pregnant BALB/c mice were treated using intratracheal instillation on gestation days 7, 11, and 15 with 10 mg of a water extract or insoluble PM 2.5. On postnatal day 105, epididymal sperm count, motility, morphology, mitochondrial membrane potential (MMP), reactive oxygen species (ROS) production, the sperm chromatin DNA fragmentation index (DFI), and testicular DNA methyltransferase (Dnmt) levels were evaluated in all generations. Whole-genome bisulfite sequencing was used to analyze the DNA methylation status of generation F3. According to the results, exposure to WS-PM 2.5 affected sperm morphology, ROS production, and mean DFI in generation F1; ROS production and mean DFI in generation F2; and sperm morphology and MMP in generation F3. Similarly, exposure to WI-PM 2.5 affected sperm morphology, ROS production, mean DFI, %DFI, and Dnmt1 expression in generation F1; sperm morphology, MMP, and ROS production in generation F2; and sperm morphology, ROS, and %DFI in generation F3. Two hypermethylated genes, PRR16 and TJP2 , were observed in the WS-PM 2.5 and WI-PM 2.5 groups, two hypomethylated genes, NFATC1 and APOA5 , were observed in the WS-PM 2.5 group, and two hypomethylated genes, ZFP945 and GSE1 , were observed in the WI-PM 2.5 group. Hence, prenatal exposure to PM 2.5 resulted in transgenerational epigenetic effects, which may explain certain phenotypic changes in male reproduction. [Display omitted] • WI-PM 2.5 exposure induces sperm ROS and morphological abnormalities in the F1-F3. • WS-PM 2.5 and WI-PM 2.5 increases DFI in the F1 and F2 and F1 and F3, respectively. • Exposure to WS-PM 2.5 increases the testicular expression of Dnmt1 in generation F3. • PRR16 and TJP2 are hypermethylated genes in the WS-PM2.5 and WI-PM2.5 groups. • PM 2.5 exerts epigenetic transgenerational effects on male offspring. [ABSTRACT FROM AUTHOR]

Study Question: Do methylation changes in sperm DNA correlate with infertility?Study Answer: Loss of spermatogenesis and fertility was correlated with 1680 differentially-methylated CpGs (DMCs) across 1052 genes.What Is Known Already: Methylation changes in a number of genes have been correlated with reduced sperm count and motility.Study Design, Size, Duration: This case-control study used spermatozoal DNA from 38 oligo-/oligoastheno-zoospermic infertile patients and 26 normozoospermic fertile men.Participants/materials, Settings, Methods: Genome-wide methylation analysis was undertaken using 450 K BeadChip on spermatozoal DNA from six infertile and six fertile men to identify DMCs. This was followed by deep sequencing of spermatozoal DNA from 32 infertile patients and 20 fertile controls.Main Results and the Role Of Chance: A total of 1680 DMCs were identified, out of which 1436 were hypermethylated and 244 were hypomethylated. Classification of DMCs according to the genes identified BCAN, CTNNA3, DLGAP2, GATA3, MAGI2 and TP73 among imprinted genes, SPATA5, SPATA7, SPATA16 and SPATA22 among spermatogenesis-associated genes, KDM4C and JMJD1C, EZH2 and HDAC4 among genes which regulate methylation and gene expression, HLA-C, HLA-DRB6 and HLA-DQA1 among complementation and immune response genes, and CRISPLD1, LPHN3 and CPEB2 among other genes. Genes showing significant differential methylation in deep sequencing, i.e. HOXB1, GATA3, EBF3, BCAN and TCERG1L, are strong candidates for further investigations. The role of chance was ruled out by deep sequencing of select genes.Large-scale Data: N/A.Limitations, Reason For Caution: Genome-wide analyses are fairly accurate, but may not be exactly validated in replication studies across all DMCs. We used the 't' test in the genome-wide methylation analysis, whereas other tests could provide a more robust and powerful analysis.Wider Implications Of the Findings: DMCs can serve as markers for inclusion in infertility screening panels, particularly those in the genes showing differential methylation consistent with previous studies. The genes validated by deep sequencing are strong candidates for investigations of their roles in spermatogenesis.Study Funding/competing Interest(s): The study was funded by the Council of Scientific and Industrial Research (CSIR), Govt. of India with grant number BSC0101 awarded to Rajender Singh. None of the authors has any competing interest to declare. [ABSTRACT FROM AUTHOR]

Summary: 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. [ABSTRACT FROM AUTHOR]

Testicular toxicity is a frequent adverse effect of cancer chemotherapy that has no effective clinical biomarker. To find new biomarkers, we focused on epigenetic mechanisms in the male germline. We investigated the DNA methylation status of the male germline during testicular toxicity induced by doxorubicin (DXR), a widely used anticancer agent. We established mouse models of early stage testicular toxicity and testicular pre-toxicity by the administration of 0.2 mg/kg and 0.02 mg/kg DXR, respectively, twice weekly for 5 weeks. Histological analysis showed sparse abnormalities in testicular tissue; however, western blotting analysis revealed reduced testicular expression levels of DNA methyltransferases DNMT3a and DNMT3b in both DXR-treated groups. Interestingly, comprehensive sperm DNA methylation analysis using Methyl-CpG binding domain protein-enriched genome sequencing revealed that hypomethylation was the most frequent change induced by DXR. These findings suggest that sperm DNA methylation status may be used as an early diagnostic marker for testicular changes not detected by conventional toxicity analysis. [ABSTRACT FROM AUTHOR]

Decreased male fertility is a big concern in both human society and the livestock industry. Sperm DNA methylation is commonly believed to be associated with male fertility. However, due to the lack of accurate male fertility records (i.e., limited mating times), few studies have investigated the comprehensive impacts of sperm DNA methylation on male fertility in mammals. In this study, we generated 10 sperm DNA methylomes and performed a preliminary correlation analysis between signals from sperm DNA methylation and signals from large-scale (n = 27,214) genome-wide association studies (GWAS) of 35 complex traits (including 12 male fertility-related traits). We detected genomic regions, which experienced DNA methylation alterations in sperm and were associated with aging and extreme fertility phenotypes (e.g., sire-conception rate or SCR). In dynamic hypomethylated regions (HMRs) and partially methylated domains (PMDs), we found genes (e.g., HOX gene clusters and microRNAs) that were involved in the embryonic development. We demonstrated that genomic regions, which gained rather than lost methylations during aging, and in animals with low SCR were significantly and selectively enriched for GWAS signals of male fertility traits. Our study discovered 16 genes as the potential candidate markers for male fertility, including SAMD5 and PDE5A. Collectively, this initial effort supported a hypothesis that sperm DNA methylation may contribute to male fertility in cattle and revealed the usefulness of functional annotations in enhancing biological interpretation and genomic prediction for complex traits and diseases.

Numerous health consequences of tobacco smoke exposure have been characterized, and the effects of smoking on traditional measures of male fertility are well described. However, a growing body of data indicates that pre-conception paternal smoking also confers increased risk for a number of morbidities on offspring. The mechanism for this increased risk has not been elucidated, but it is likely mediated, at least in part, through epigenetic modifications transmitted through spermatozoa. In this study, we investigated the impact of cigarette smoke exposure on sperm DNA methylation patterns in 78 men who smoke and 78 never-smokers using the Infinium Human Methylation 450 beadchip. We investigated two models of DNA methylation alterations: (i) consistently altered methylation at specific CpGs or within specific genomic regions and (ii) stochastic DNA methylation alterations manifest as increased variability in genome-wide methylation patterns in men who smoke. We identified 141 significantly differentially methylated CpGs associated with smoking. In addition, we identified a trend toward increased variance in methylation patterns genome-wide in sperm DNA from men who smoke compared with never-smokers. These findings of widespread DNA methylation alterations are consistent with the broad range of offspring heath disparities associated with pre-conception paternal smoke exposure and warrant further investigation to identify the specific mechanism by which sperm DNA methylation perturbation confers risk to offspring health and whether these changes can be transmitted to offspring and transgenerationally. [ABSTRACT FROM AUTHOR]

Considering the widespread use of assisted reproductive techniques ( ART), DNA methylation of specific genes involved in spermatogenesis achieves increasingly clinical relevance, representing a possible explanation of increased incidence of syndromes related to genomic imprinting in medically assisted pregnancies. Several trials suggested a relationship between male sub-fertility and sperm DNA methylation, although its weight on seminal parameters alteration is still a matter of debate. To evaluate whether aberrant sperm DNA methylation of imprinted genes is associated with impaired sperm parameters. Meta-analysis of controlled clinical trials evaluating imprinted genes sperm DNA methylation comparing men with idiopathic infertility to fertile controls. Twenty-four studies were included, allowing a meta-analytic evaluation for H19, MEST, SNRPN, and LINE-1. When a high heterogeneity of the results was demonstrated, the random effect model was used. H19 methylation levels resulted significantly lower in 879 infertile compared with 562 fertile men (7.53%, 95% CI: 5.14-9.93%, p < 0.001), suggesting a 9.91-fold higher risk ratio to show aberrant sperm DNA methylation (95% CI: 5.55-17.70, p < 0.001, I2 = 19%) in infertile men. The mean MEST methylation level was significantly higher in 846 infertile compared with 353 fertile men (3.35%, 95% CI: 1.41-5.29%, p < 0.001), as well as for SNRPN comparing 301 infertile men with 124 controls (3.23%, 95% CI: 0.75-5.72%, p < 0.001). LINE-1 methylation levels did not differ between 291 infertile men and 198 controls (0.44%, 95% CI: −2.04-1.16%, p = 0.63). The meta-analytic approach demonstrated that male infertility is associated with altered sperm methylation at H19, MEST, and SNRPN. Although its role in infertility remains unclear, sperm DNA methylation could be associated with the epigenetic risk in ART. In this setting, before proposing this analysis in clinical practice, an accurate identification of the most representative genes and a cost-effectiveness evaluation should be assessed in ad hoc prospective studies. [ABSTRACT FROM AUTHOR]

STUDY QUESTION Which are the main determinants, if any, of sperm DNA methylation levels? SUMMARY ANSWER Geographical region resulted associated with the sperm methylation status assessed on genome-wide repetitive sequences. WHAT IS KNOWN ALREADY DNA methylation level, assessed on repetitive sequences from peripheral blood lymphocyte, can vary with age, gender, alcohol consumption and white blood cell counts. STUDY DESIGN, SIZE, DURATION A cross-sectional study. Individual data were collected from 269 young healthy men of proven fertility living in three geographical regions: Inuits from Greenland, Caucasians from Warsaw (Poland) and Kharkiv (Ukraine). Semen samples were collected between May 2002 and February 2004 and aliquots were immediately frozen. PARTICIPANTS/MATERIALS, SETTING, METHODS We estimated sperm DNA global methylation level (DGML) in two ways. First DNA methylation in repetitive DNA sequences (LINE-1, Satα and Alu) was quantified by PCR pyrosequencing after bisulfite conversion and second by flow cytometry (FCM) using fluorescently labeled monoclonal antibodies anti-5-methylcytosine. We analyzed whether personal characteristics and habits, body mass index, semen quality parameters, sperm chromatin integrity, biomarkers of accessory gland function and the plasma concentration of reproductive hormones were associated with sperm DNA methylation levels in men. Associations were evaluated by analysis of variance and linear regression analyses. MAIN RESULTS AND THE ROLE OF CHANCE The geographical location emerged as the main determinant when using the methylation level in repetitive sequences. FCM DGML results were not associated with those from repetitive sequence analysis. No other consistent associations between methylation markers and the assessed variables were identified across countries. LIMITATIONS, REASONS FOR CAUTION The methods used are only surrogates of the actual sperm methylome and the methylation levels at individual specific loci were not explored. WIDER IMPLICATIONS OF THE FINDINGS Sperm DGML is relatively independent from semen quality parameters and is a new candidate biomarker for epidemiological studies of the impact of environmental contaminants on male fertility. STUDY FUNDING/COMPETING INTEREST(S) The study is part of the project CLEAR (Climate change, Environmental contaminants and Reproductive health) supported by the European Commission 7th framework program, contract no: FP7-ENV-2008-1-226217. No competing interest is declared. [ABSTRACT FROM PUBLISHER]

Aberrant sperm DNA methylation patterns, mainly in imprinted genes, have been associated with male subfertility and oligospermia. Here, we performed a genome-wide methylation analysis in sperm samples representing a wide range of semen parameters. Sperm DNA samples of 38 males attending a fertility centre were analysed with Illumina HumanMethylation27 BeadChips, which quantify methylation of >27 000 CpG sites in cis-regulatory regions of almost 15 000 genes. In an unsupervised analysis of methylation of all analysed sites, the patient samples clustered into a major and a minor group. The major group clustered with samples from normozoospermic healthy volunteers and, thus, may more closely resemble the normal situation. When correlating the clusters with semen and clinical parameters, the sperm counts were significantly different between groups with the minor group exhibiting sperm counts in the low normal range. A linear model identified almost 3000 CpGs with significant methylation differences between groups. Functional analysis revealed a broad gain of methylation in spermatogenesis-related genes and a loss of methylation in inflammation- and immune response-related genes. Quantitative bisulfite pyrosequencing validated differential methylation in three of five significant candidate genes on the array. Collectively, we identified a subgroup of sperm samples for assisted reproduction with sperm counts in the low normal range and broad methylation changes (affecting approximately 10% of analysed CpG sites) in specific pathways, most importantly spermatogenesis-related genes. We propose that epigenetic analysis can supplement traditional semen parameters and has the potential to provide new insights into the aetiology of male subfertility. [ABSTRACT FROM AUTHOR]

A growing number of sperm methylome analyses have identified genomic loci that are susceptible to paternal age effects in a variety of mammalian species, including human, bovine, and mouse. However, there is little overlap between different data sets. Here, we studied whether or not paternal age effects on the sperm epigenome have been conserved in mammalian evolution and compared methylation patterns of orthologous regulatory regions (mainly gene promoters) containing both conserved and non-conserved CpG sites in 94 human, 36 bovine, and 94 mouse sperm samples, using bisulfite pyrosequencing. We discovered three (NFKB2, RASGEF1C, and RPL6) age-related differentially methylated regions (ageDMRs) in humans, four (CHD7, HDAC11, PAK1, and PTK2B) in bovines, and three (Def6, Nrxn2, and Tbx19) in mice. Remarkably, the identified sperm ageDMRs were all species-specific. Most ageDMRs were in genomic regions with medium methylation levels and large methylation variation. Orthologous regions in species not showing this age effect were either hypermethylated (>80%) or hypomethylated (<20%). In humans and mice, ageDMRs lost methylation, whereas bovine ageDMRs gained methylation with age. Our results are in line with the hypothesis that sperm ageDMRs are in regions under epigenomic evolution and may be part of an epigenetic mechanism(s) for lineage-specific environmental adaptations and provide a solid basis for studies on downstream effects in the genes analyzed here. [ABSTRACT FROM AUTHOR]

B10.BR-Y del male mice with large deletion in the male-specific region of the Y chromosome long arm (MSYq) are very useful experimental model which requires, however, more detailed characterization. In the present study, the influence of the deletion on transcript levels of MSYq genes (Ssty1, Ssty2, Sly, Srsy, Asty, Orly) and homologous to them X-linked genes (Sstx, Slx, Slxl1, Srsx) was assessed. Quantitative PCR analysis showed that in testes of B10.BR-Y del males activity of Ssty1 is unchanged, but transcription from all other MSYq genes is highly reduced and reaches from 59 % to only 5 % of the control levels. The decrease in expression of MSYq genes is accompanied by the two-fold increase in expression of Slx and Slxl1 genes. This is the first functional characterization of the deletion in B10.BR-Y del strain. Another aim of the study was to reveal the mechanism through which deleted Y chromosome of B10.BR-Y del males could alter phenotype of their female progeny, what was documented in our previous works. Epigenetic inheritance hypothesis was tested by microarray analysis of DNA methylation in B10.BR-Y del and control B10.BR sperm. The assessment revealed moderate differences and allowed concluding that the mutated Y chromosome can influence traits of females from the next generation partially through altering sperm DNA methylation, but probably some additional mechanisms are engaged here. Breeding data indicate that feminization of pre- and neonatal environment in which next generation females develop is one of such additional mechanisms., (Copyright © 2022 Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn. Published by Elsevier B.V. All rights reserved.)

Identification of inter-individual variations of sperm DNA methylation is very essential for elucidating phenotypic variation of reproduction and production traits. We had generated 28 sperm whole genome sequencing (WGBS) data from healthy individuals. We detected 46 variably methylated regions (VMRs) significantly (P < 5.77 × 10-5) associated with reproduction traits. These significant VMRs were also co-localized (±10 kb) with genes related to sperm motility and reproduction, including ZFP36L1, CRISP2 and HGF. To further identify epigenomic markers associated with reproduction and production traits, we generated WGBS dada for a specific pair of monozygotic twin artificial insemination Holstein bulls with moderately different sperm qualities (Bull1 > Bull2). We found 528 differentially methylated regions between the twin bulls, which spanned or overlapped with 309 differentially methylated genes (DMG). These DMG were particularly associated with reproduction, embryo development and the nervous system. To further obtain DNA methylation markers involved in the diversity of porcine reproduction and production traits due to long-term artificial selection, we profiled sperm DNA methylation of three commercial pig breeds--Duroc, Landrace and Large White. we found the hypomethylated regions (HMRs) were highly conserved among pig breeds, while 1040-1666 breed-specific HMRs were identified and associated with the QTLs of embryonic developmental and phenotypic changes. By integrating reduced representation bisulfite sequencing public data of pig testis, we further defined 1743 conservative HMRs between sperm and testis, which may play a role in spermatogenesis process. Overall, our studies demonstrated associations of sperm DNA methylation with bovine and porcine production and reproduction traits, highlighting the potential of epigenomic information in genomic improvement programs for livestock. [ABSTRACT FROM AUTHOR]

Decabromodiphenyl ether (BDE-209), a congener of polybrominated diphenyl ethers, is a commonly used brominated flame retardant and a known endocrine disrupting chemical (EDC). Knowledge about the effects of prenatal BDE-209 exposure on male reproduction and whether transgenerational effects occur in subsequent generations are scant. Therefore, in this study, we tested the hypothesis that prenatal exposure to BDE-209 disrupted sperm function in the F1, F2, and F3 generations of male rats. Pregnant Sprague-Dawley rats were treated by gavage from gestation day 0 to birth with 5 mg BDE-209/kg/day. This treatment was based on the lowest-observed-adverse-effect level for DNA damage to sperm in male offspring. On postnatal day 84 for all generations, epididymal sperm counts, motility, morphology, reactive oxygen species generation, sperm chromatin DNA structure integrity, testicular DNA content in spermatogenesis, and serum testosterone levels were assessed. DNA methyltransferase (Dnmts) mRNA expression and methyl-CpG binding domain sequencing were also examined to analyze DNA methylation status in the F3 generation. In the F1 generation, prenatal exposure to BDE-209 disrupted body weight, decreased anogenital distance (AGD), sperm count, and motility; and increased bent tail rates of sperm. In the F2 generation, exposure to BDE-209 decreased AGD, sperm count, normal morphology rates, Dnmt1 expression, and increased Dnmt3a expression. In the F3 generation, BDE-209 exposure decreased AGD and normal sperm morphology, disrupted testicular elongated spermatid and round spermatid rates, reduced serum testosterone levels, and inhibited the mRNA expression of Dnmt1 and Dnmt3b. Compared with the control group, there existed 215 differentially hyper-methylated and 83 hypo-methylated genes in the BDE-209 group. BDE-209 is an EDC to disrupt the male reproduction from F1 to F3. BDE-209-induced changes in sperm function and hyper- or hypo-DNA methylation in the F3 generation might therefore explain the possible mechanism underlying BDE-209-mediated epigenetic transgenerational effects on the male reproductive system. [Display omitted] • BDE-209 exposure reduced AGD and normal sperm morphology rates in the F1–F3. • BDE-209 altered testicular cell populations and serum testosterone level in the F3. • BDE-209 affected the testicular Dnmt1 and Dnmt3b expression in the F3 generation. • BDE-209 changed sperm hyper- and hypo-DNA methylation in the F3 generation. • BDE-209-mediated epigenetic transgenerational effects on the male reproduction. [ABSTRACT FROM AUTHOR]

Di(2-ethylhexyl) phthalate (DEHP) is widely used as a plasticizer in the manufacture of polyvinylchloride plastics and has been associated with concerns regarding male reproductive toxicity. In this study, we hypothesized that maternal exposure to DEHP induces transgenerational inheritance of adult-onset adverse reproductive outcomes through the male germline in the F1, F2, and F3 generations of male offspring. Pregnant rats were treated with 5 or 500 mg of DEHP/kg/day through gavage from gestation day 0 to birth. The offspring body weight, anogenital distance (AGD), anogenital index (AGI), sperm count, motility, and DNA fragmentation index (DFI) were measured for all generations. Methyl-CpG binding domain sequencing was performed to analyze sperm DNA methylation status in the F3. DEHP exposure at 500 mg/kg affected AGD, AGI, sperm count, mean DFI, and %DFI in the F1; AGD, sperm count, and mean DFI in the F2; and AGD, AGI, mean DFI, and %DFI in the F3. DEHP exposure at 5 mg/kg affected AGD, AGI, sperm count, and %DFI in the F1; sperm count in the F2; and AGD and AGI in F3. Compared with the control group, 15 and 45 differentially hypermethylated genes were identified in the groups administered 5 mg/kg and 500 mg/kg DEHP, respectively. Moreover, 130 and 6 differentially hypomethylated genes were observed in the groups administered 5 mg/kg and 500 mg/kg DEHP. Overall, these results demonstrated that prenatal exposure to DEHP caused transgenerational epigenetic effects, which may explain the observed phenotypic changes in the male reproductive system. [ABSTRACT FROM AUTHOR]

In this study, the complexity of chromatin integrity was investigated in frozen-thawed semen samples from 37 sires with contrasting fertility, expressed as 56-day non-return rates (NR56). Protamine deficiency, thiols, and disulfide bonds were assessed and compared with previously published data for DNA fragmentation index (DFI) and high DNA stainability (HDS). In addition, in vitro embryo development and sperm DNA methylation were assessed using semen samples from 16 of these bulls. The percentages of DFI and HDS were negatively associated with NR56 and cleavage rate and positively associated with sperm protamine deficiency (p < 0.05). Significant differences in cleavage and blastocyst rates were observed between bulls of high and low NR56. However, once fertilization occurred, further development into blastocysts was not associated with NR56. The differential methylation analysis showed that spermatozoa from bulls of low NR56 were hypermethylated compared to bulls of high NR56. Pathway analysis showed that genes annotated to differentially methylated cytosines could participate in different biological pathways and have important biological roles related to bull fertility. In conclusion, sperm cells from Norwegian Red bulls of inferior fertility have less compact chromatin structure, higher levels of DNA damage, and are hypermethylated compared with bulls of superior fertility., (© 2021 The Authors. Molecular Reproduction and Development published by Wiley Periodicals LLC.)

Background: DNA methylation has been shown to be involved in many biological processes, including X chromosome inactivation in females, paternal genomic imprinting, and others. Results: Based on the correlation patterns of methylation levels of neighboring CpG sites among 28 sperm whole genome bisulfite sequencing (WGBS) data (486 × coverage), we obtained 31,272 methylation haplotype blocks (MHBs). Among them, we defined conserved methylated regions (CMRs), variably methylated regions (VMRs) and highly variably methylated regions (HVMRs) among individuals, and showed that HVMRs might play roles in transcriptional regulation and function in complex traits variation and adaptive evolution by integrating evidence from traditional and molecular quantitative trait loci (QTL), and selection signatures. Using a weighted correlation network analysis (WGCNA), we also detected a co-regulated module of HVMRs that was significantly associated with reproduction traits, and enriched for glycosyltransferase genes, which play critical roles in spermatogenesis and fertilization. Additionally, we identified 46 VMRs significantly associated with reproduction traits, nine of which were regulated by cis-SNPs, implying the possible intrinsic relationships among genomic variations, DNA methylation, and phenotypes. These significant VMRs were co-localized (± 10 kb) with genes related to sperm motility and reproduction, including ZFP36L1, CRISP2 and HGF. We provided further evidence that rs109326022 within a predominant QTL on BTA18 might influence the reproduction traits through regulating the methylation level of nearby genes JOSD2 and ASPDH in sperm. Conclusion: In summary, our results demonstrated associations of sperm DNA methylation with reproduction traits, highlighting the potential of epigenomic information in genomic improvement programs for cattle. [ABSTRACT FROM AUTHOR]

Background: Testicular germ cell tumor such as seminoma is strongly associated with male reproductive problems commonly associated with the alteration of sperm parameters as described in testicular dysgenesis syndrome. Interestingly, numerous studies have reported that the precursor of germ cell cancer, germ cell neoplasia in situ (GCNIS), present similarities to fetal gonocytes, specifically characterized by global DNA hypomethylation particularly on imprinting sequences. These disorders may have a common origin derived from perturbations of embryonal programming during fetal development. Presently, there is no available information concerning the sperm DNA methylation patterns of testicular cancer patients. For the first time, we evaluated the sperm imprinting of seminoma patients. A total of 92 cryopreserved sperm samples were included, 31 before seminoma treatment (S): 23 normozoospermic (SN) and 8 oligozoospermic (SO) and 61 sperm controls samples: 31 normozoospermic (N) and 30 oligozoospermic (O). DNA methylation levels of seven differentially methylated regions (DMRs) of imprinted genes [H19/IGF2: IG-DMR (CTCF3 and CTCF6 of H19 gene); IGF2-DMRs (DMR0 and DMR2); MEG3/DLK1:IG-DMR; SNURF:TSS-DMR; KCNQ1OT1:TSS-DMR] were assessed by pyrosequencing. All comparative analyses were adjusted for age. Results: Comparisons of sperm DNA methylation levels between seminoma (S) and normozoospermic (N) samples showed a significant difference for the SNURF sequence (p = 0.017), but after taking into account the sperm parameters, no difference was observed. However, we confirmed a significant association between oligozoospermia (O) and imprinting defects for H19/IGF2-CTCF6 (p = 0.001), MEG3/DLK1 (p = 0.017), IGF2-DMR2 (p = 0.022), and SNURF (p = 0.032) in comparison with control groups (N). Conclusions: This study highlights the high risk of sperm imprinting defects in cases of oligozoospermia and shows for the first time that seminoma patients with normal spermatogenesis present sperm imprinting integrity. These data suggest a low probability of the involvement of a common imprinting defect in fetal cells leading to both TGCT and subfertility. [ABSTRACT FROM AUTHOR]

Objective: To characterize a potential genetic cause for methylation errors described in oligozoospermia. Design: Analysis of PEG1/MEST-DMR and H19-DMR methylation level in sperm, in parallel with the study of several genes on the Y chromosome, DNMT3A, and DNMT3L. Clinical outcome was also looked at regarding PEG1/MEST-DMR and H19-DMR methylation level in sperm. Setting: Research and diagnostic laboratories. Patient(s): One hundred nineteen normospermic and 175 oligozoospermic men consulting for couple infertility. Intervention(s): We studied PEG1/MEST-DMR and H19-DMR methylation profiles in 294 men. We searched for Y chromosome gene aberrations and for mutations in both DNMT3A and DNMT3L genes in men showing epimutations. Assisted reproductive technology (ART) outcomes were also investigated. Main Outcome Measure(s): Sperm samples were collected from 294 volunteers for genomic DNA isolation that was used to study methylation profiles in imprinted loci and Y chromosome SMCY, DNMT3A, and DNMT3L genes. Pregnancy rate was also studied after ART treatment using sperm showing epimutations. Result(s): Epimutations in H19-DMR and PEG1/MEST-DMR were found in 20% and 3% of oligozoospermic men, respectively. We identified an amino acid change in DNMT3A in one case and in DNMT3L in eight men with altered methylation profiles. No mutations were detected in SMCY or in selected Y chromsome genes. No correlation between ART outcome and epimutations was found. Conclusion(s): We observed epimethylations in spermatozoa of oligozoospermic individuals, but no association was found with genetic variants or in the ART outcome. [ABSTRACT FROM AUTHOR]

Objective: To evaluate the relative intraindividual changes in sperm 5-methylcytosine (5-mC) and 5-hydroxymethylcytosine (5-hmC) levels associated with age and to compare the levels of 5-hmC in mature human sperm to blood DNA. Design: Prospective research study. Setting: University-based andrology and in vitro fertilization (IVF) laboratory. Patient(s): Fifteen known fertile sperm donors, 22 other known fertile controls, and 41 male blood donors from a general population tissue bank. Intervention(s): None. Main Outcome Measure(s): Measurements of global 5-mC and 5-hmC levels via an enzyme-linked immunosorbent assay (ELISA)-based assay. Result(s): Global sperm 5-mC levels exhibit a statistically significant increase with age, and a similar trend was seen in 5-hmC levels. On average, in the age ranges we analyzed, 5-mC increased by 1.76% per year, and 5-hmC, though more variable, increased by approximately 5% per year. Additionally, we found that 5-hmC levels in sperm are 32.59% of that found in blood DNA. Conclusion(s): Global sperm DNA methylation patterns are stable over short periods of time but increase with age, which raises important questions regarding the risks of advanced paternal age. Additionally, as we would predict for a transcriptionally quiescent cell type, 5-hmC levels are statistically significantly lower in human sperm than in blood DNA. [ABSTRACT FROM AUTHOR]