DNA methylation defects in spermatozoa of male partners from couples experiencing recurrent pregnancy loss

Abstract STUDY QUESTION What is the sperm DNA methylation status of imprinted genes in male partners from couples experiencing recurrent pregnancy loss (RPL)? SUMMARY ANSWER Aberrations in sperm DNA methylation status of several imprinted genes, such as insulin like growth factor-2-H19 differentially methylated region (IGF2-H19 DMR), intergenic differentially methylated region (IG-DMR), mesoderm specific transcript (MEST), zinc finger protein which regulates apoptosis and cell cycle arrest (ZAC), DMR in intron 10 of KCNQ1 gene (KvDMR), paternally expressed gene 3 (PEG3) and paternally expressed gene 10 (PEG10), as well as decreased sperm global 5-methylcytosine (5mC) levels, are associated with RPL. WHAT IS KNOWN ALREADY RPL is defined as loss of two or more pregnancies, affecting 1–2% of couples of reproductive age. Although there are several maternal and paternal aetiological factors contributing to RPL, nearly 50% of the cases remain idiopathic. Thus, there is a need to identify putative paternal factors that could be contributing towards pregnancy loss in cases of idiopathic RPL. STUDY DESIGN, SIZE, DURATION In this case–control study, 112 couples undergoing RPL with no identifiable cause were recruited from September 2015 to May 2018. The control group comprised of 106 healthy proven fertile couples with no history of infertility or miscarriage. PARTICIPANTS/MATERIALS, SETTING, METHODS In this study, we investigated the paternal genetic and epigenetic factors that could be associated with RPL. We studied DNA methylation, by pyrosequencing, of selected imprinted genes implicated in embryo development, such as IGF2-H19 DMR, IG-DMR, MEST, ZAC, KvDMR, PEG3, PEG10 and small nuclear ribonucleoprotein polypeptide N (SNRPN) in sperm of men whose partners present RPL. Global DNA methylation in sperm was evaluated by studying 5mC content and long interspersed nuclear element 1 (LINE1) promoter methylation. We also studied polymorphisms by pyrosequencing in the IGF2-H19 DMR as well in the IGF2 promoter in both groups. MAIN RESULTS AND THE ROLE OF CHANCE In the RPL group, we found a significant decrease in the global sperm 5mC levels and significant decrease in DNA methylation at three CpG sites in LINE1 promoter. For IGF2-H19 DMR and IG-DMR, a significant decrease in sperm DNA methylation at specific CpG sites was observed in RPL group. For maternally imprinted genes like MEST, ZAC, KvDMR, PEG3 and PEG10 hypermethylation was noted. Polymorphism studies for IGF2-H19 DMR and IGF2 revealed significant differences in the genotypic frequencies in males. LIMITATIONS, REASONS FOR CAUTION In this study, we analysed the methylation levels of selected candidate imprinted genes implicated in embryo development. Detection of methylation changes occurring at the genome-wide level may reveal further candidate genes having a better distinction between the control and study groups. WIDER IMPLICATIONS OF THE FINDINGS Our study demonstrates that certain polymorphisms and aberrant sperm methylation status in imprinted genes are associated with RPL and could contribute to the aetiology of RPL. This study suggests that investigation of paternal genetic and epigenetic factors could be useful in identification of possible causes of idiopathic RPL. STUDY FUNDING/COMPETING INTEREST(S) This study was funded by Department of Science and Technology-Science and Engineering Research Board (EMR/2014/000145) and National Institute for Research in Reproductive Health intramural funds (RA/872/01-2020). All authors declare no conflict of interest. TRIAL REGISTRATION NUMBER N/A.

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Epigenetics of Male Infertility: The Role of DNA Methylation

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.689624 ◽

2021 ◽

Vol 9 ◽

Author(s):

John Charles Rotondo ◽

Carmen Lanzillotti ◽

Chiara Mazziotta ◽

Mauro Tognon ◽

Fernanda Martini

Keyword(s):

Dna Methylation ◽

Male Infertility ◽

Methylation Status ◽

Reproductive Potential ◽

Diagnostic Tools ◽

Imprinted Genes ◽

Genome Methylation ◽

Sperm Dna ◽

Sperm Dna Methylation

In recent years, a number of studies focused on the role of epigenetics, including DNA methylation, in spermatogenesis and male infertility. We aimed to provide an overview of the knowledge concerning the gene and genome methylation and its regulation during spermatogenesis, specifically in the context of male infertility etiopathogenesis. Overall, the findings support the hypothesis that sperm DNA methylation is associated with sperm alterations and infertility. Several genes have been found to be differentially methylated in relation to impaired spermatogenesis and/or reproductive dysfunction. Particularly, DNA methylation defects of MEST and H19 within imprinted genes and MTHFR within non-imprinted genes have been repeatedly linked with male infertility. A deep knowledge of sperm DNA methylation status in association with reduced reproductive potential could improve the development of novel diagnostic tools for this disease. Further studies are needed to better elucidate the mechanisms affecting methylation in sperm and their impact on male infertility.

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Correlation of methylation status in MTHFR promoter region with recurrent pregnancy loss

Journal of Genetic Engineering and Biotechnology ◽

10.1186/s43141-021-00147-w ◽

2021 ◽

Vol 19 (1) ◽

Author(s):

Mai Mahmoud Shaker ◽

Taghreed Abdelmoniem shalabi ◽

Khalda said Amr

Keyword(s):

Dna Methylation ◽

Dna Sequences ◽

Promoter Region ◽

Pregnancy Loss ◽

Recurrent Pregnancy Loss ◽

Methylation Status ◽

Control Group ◽

Case Group ◽

Methyl Radical ◽

Fertile Women

Abstract Background DNA methylation is an epigenetic process for modifying transcription factors in various genes. Methylenetetrahydrofolate reductase (MTHFR) stimulates synthesis of methyl radical in the homocysteine cycle and delivers methyl groups needed in DNA methylation. Furthermore, numerous studies have linked gene polymorphisms of this enzyme with a larger risk of recurrent pregnancy loss (RPL), yet scarce information is available concerning the association between epigenetic deviations in this gene and RPL. Hypermethylation at precise DNA sequences can function as biomarkers for a diversity of diseases. We aimed by this study to evaluate the methylation status of the promoter region of MTHFR gene in women with RPL compared to healthy fertile women. It is a case–control study. Hundred RPL patients and hundred healthy fertile women with no history of RPL as controls were recruited. MTHFR C677T was assessed by polymerase chain reaction-restriction fragment length polymorphism (RFLP). Quantitative evaluation of DNA methylation was performed by high-resolution melt analysis by real-time PCR. Results The median of percentage of MTHFR promoter methylation in RPL cases was 6.45 [0.74–100] vs. controls was 4.50 [0.60–91.7], P value < 0.001. In the case group, 57 hypermethylated and 43 normo-methylated among RPL patients vs. 40 hypermethylated and 60 normo-methylated among controls, P< 0.005. Frequency of T allele in C677T MTHFR gene among RPL patients was 29% vs. 23% among the control group; C allele vs. T allele: odds ratio (OR) = 1.367 (95% confidence interval (CI) 0.725–2.581). Conclusion Findings suggested a significant association between hypermethylation of the MTHFR promoter region in RPL patients compared to healthy fertile women.

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High-resolution analyses of human sperm dynamic methylome reveal thousands of novel age-related epigenetic alterations

Clinical Epigenetics ◽

10.1186/s13148-020-00988-1 ◽

2020 ◽

Vol 12 (1) ◽

Author(s):

Mingju Cao ◽

Xiaojian Shao ◽

Peter Chan ◽

Warren Cheung ◽

Tony Kwan ◽

...

Keyword(s):

Dna Methylation ◽

Human Sperm ◽

Distal Region ◽

Epigenetic Alterations ◽

Transcription Start Sites ◽

Neurodevelopmental Disease ◽

Cpg Sites ◽

Age Related ◽

Sperm Dna ◽

Sperm Dna Methylation

Abstract Background Children of aged fathers are at a higher risk of developing mental disorders. Alterations in sperm DNA methylation have been implicated as a potential cause. However, age-dependent modifications of the germ cells’ epigenome remain poorly understood. Our objective was to assess the DNA methylation profile of human spermatozoa during aging. Results We used a high throughput, customized methylC-capture sequencing (MCC-seq) approach to characterize the dynamic DNA methylation in spermatozoa from 94 fertile and infertile men, who were categorized as young, 48 men between 18–38 years or old 46 men between 46–71 years. We identified more than 150,000 age-related CpG sites that are significantly differentially methylated among 2.65 million CpG sites covered. We conducted machine learning using our dataset to predict the methylation age of subjects; the age prediction accuracy based on our assay provided a more accurate prediction than that using the 450 K chip approach. In addition, we found that there are more hypermethylated (62%) than hypomethylated (38%) CpG sites in sperm of aged men, corresponding to 798 of total differential methylated regions (DMRs), of which 483 are hypermethylated regions (HyperDMR), and 315 hypomethylated regions (HypoDMR). Moreover, the distribution of age-related hyper- and hypomethylated CpGs in sperm is not random; the CpG sites that were hypermethylated with advanced age were frequently located in the distal region to genes, whereas hypomethylated sites were near to gene transcription start sites (TSS). We identified a high density of age-associated CpG changes in chromosomes 4 and 16, particularly HyperDMRs with localized clusters, the chr4 DMR cluster overlaps PGC1α locus, a protein involved in metabolic aging and the chr16 DMR cluster overlaps RBFOX1 locus, a gene implicated in neurodevelopmental disease. Gene ontology analysis revealed that the most affected genes by age were associated with development, neuron projection, differentiation and recognition, and behaviour, suggesting a potential link to the higher risk of neurodevelopmental disorders in children of aged fathers. Conclusion We identified thousands of age-related and sperm-specific epigenetic alterations. These findings provide novel insight in understanding human sperm DNA methylation dynamics during paternal aging, and the subsequently affected genes potentially related to diseases in offspring.

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Comparison of DNA methylation patterns of parentally imprinted genes in placenta derived from IVF conceptions in two different culture media

Human Reproduction ◽

10.1093/humrep/deaa004 ◽

2020 ◽

Vol 35 (3) ◽

pp. 516-528

Author(s):

Callista L Mulder ◽

Tess M Wattimury ◽

Aldo Jongejan ◽

Cindy M de Winter-Korver ◽

Saskia K M van Daalen ◽

...

Keyword(s):

Dna Methylation ◽

Embryo Culture ◽

Culture Medium ◽

Culture Media ◽

Methylation Status ◽

Culture Conditions ◽

Imprinted Genes ◽

Cpg Sites ◽

Embryo Culture Medium ◽

The Mean

Abstract Study question Is there a difference in DNA methylation status of imprinted genes in placentas derived from IVF conceptions where embryo culture was performed in human tubal fluid (HTF) versus G5 culture medium? Summary answer We found no statistically significant differences in the mean DNA methylation status of differentially methylated regions (DMRs) associated with parentally imprinted genes in placentas derived from IVF conceptions cultured in HTF versus G5 culture medium. What is known already Animal studies indicate that the embryo culture environment affects the DNA methylation status of the embryo. In humans, birthweight is known to be affected by the type of embryo culture medium used. The effect of embryo culture media on pregnancy, birth and child development may thus be mediated by differential methylation of parentally imprinted genes in the placenta. Study design, size, duration To identify differential DNA methylation of imprinted genes in human placenta derived from IVF conceptions exposed to HTF or G5 embryo culture medium, placenta samples (n = 43 for HTF, n = 54 for G5) were collected between 2010 and 2012 s as part of a multi-center randomized controlled trial in the Netherlands comparing these embryo culture media. Placenta samples from 69 naturally conceived (NC) live births were collected during 2008–2013 in the Netherlands as reference material. Participants/materials, setting, methods To identify differential DNA methylation of imprinted genes, we opted for an amplicon-based sequencing strategy on an Illumina MiSeq sequencing platform. DNA was isolated and 34 DMRs associated with well-defined parentally imprinted genes were amplified in a two-step PCR before sequencing using MiSeq technology. Sequencing data were analyzed in a multivariate fashion to eliminate possible confounding effects. Main results and the role of chance We found no statistically significant differences in the mean DNA methylation status of any of the imprinted DMRs in placentas derived from IVF conceptions cultured in HTF or G5 culture medium. We also did not observe any differences in the mean methylation status per amplicon nor in the variance in methylation per amplicon between the two culture medium groups. A separate surrogate variable analysis also demonstrated that the IVF culture medium was not associated with the DNA methylation status of these DMRs. The mean methylation level and variance per CpG was equal between HTF and G5 placenta. Additional comparison of DNA methylation status of NC placenta samples revealed no statistically significant differences in mean amplicon and CpG methylation between G5, HTF and NC placenta; however, the number of placenta samples exhibiting outlier methylation levels was higher in IVF placenta compared to NC (P < 0.00001). Also, we were able to identify 37 CpG sites that uniquely displayed outlier methylation in G5 placentas and 32 CpG sites that uniquely displayed outlier methylation in HTF. In 8/37 (G5) and 4/32 (HTF) unique outliers CpGs, a medium-specific unique outlier could be directly correlated to outlier methylation of the entire amplicon. Limitations, reasons for caution Due to practical reasons, not all placentas were collected during the trial, and we collected the placentas from natural conceptions from a different cohort, potentially creating bias. We limited ourselves to the DNA methylation status of 34 imprinted DMRs, and we studied only the placenta and no other embryo-derived tissues. Wider implications of the findings It has often been postulated, but has yet to be rigorously tested, that imprinting mediates the effects of embryo culture conditions on pregnancy, birth and child development in humans. Since we did not detect any statistically significant effects of embryo culture conditions on methylation status of imprinted genes in the placenta, this suggests that other unexplored mechanisms may underlie these effects. The biological and clinical relevance of detected outliers with respect to methylation levels of CpGs and DMR require additional analysis in a larger sample size as well. Given the importance and the growing number of children born through IVF, research into these molecular mechanisms is urgently needed. Study funding/competing interest(s) This study was funded by the March of Dimes grant number #6-FY13-153. The authors have no conflicts of interest. Trial registration number Placental biopsies were obtained under Netherlands Trial Registry number 1979 and 1298.

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Refraining from use diminishes cannabis-associated epigenetic changes in human sperm

Environmental Epigenetics ◽

10.1093/eep/dvab009 ◽

2021 ◽

Vol 7 (1) ◽

Author(s):

Rose Schrott ◽

Susan K Murphy ◽

Jennifer L Modliszewski ◽

Dillon E King ◽

Bendu Hill ◽

...

Keyword(s):

Dna Methylation ◽

Early Development ◽

Bisulfite Sequencing ◽

Human Sperm ◽

Whole Genome ◽

Epigenetic Changes ◽

Cpg Sites ◽

Sperm Dna ◽

Genome Bisulfite Sequencing ◽

Sperm Dna Methylation

Abstract Cannabis use alters sperm DNA methylation, but the potential reversibility of these changes is unknown. Semen samples from cannabis users and non-user controls were collected at baseline and again following a 77-day period of cannabis abstinence (one spermatogenic cycle). Users and controls did not significantly differ by demographics or semen analyses. Whole-genome bisulfite sequencing identified 163 CpG sites with significantly different DNA methylation in sperm between groups (P < 2.94 × 10−9). Genes associated with altered CpG sites were enriched with those involved in development, including cardiogenesis and neurodevelopment. Many of the differences in sperm DNA methylation between groups were diminished after cannabis abstinence. These results indicate that sustained cannabis abstinence significantly reduces the number of sperm showing cannabis-associated alterations at genes important for early development.

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Simulated Wildfire Smoke Significantly Alters Sperm DNA Methylation Patterns in a Murine Model

Toxics ◽

10.3390/toxics9090199 ◽

2021 ◽

Vol 9 (9) ◽

pp. 199

Author(s):

Adam Schuller ◽

Chiara Bellini ◽

Timothy G. Jenkins ◽

Matthew Eden ◽

Jacqueline Matz ◽

...

Keyword(s):

Dna Methylation ◽

Prolonged Exposure ◽

Smoke Exposure ◽

Differentially Methylated Region ◽

Respiratory Morbidity ◽

Differentially Methylated Regions ◽

Wildfire Smoke ◽

Adult Mice ◽

Sperm Dna ◽

Sperm Dna Methylation

Wildfires are now a common feature of the western US, increasing in both intensity and number of acres burned over the last three decades. The effects of this changing wildfire and smoke landscape are a critical public and occupational health issue. While respiratory morbidity due to smoke exposure is a priority, evaluating the molecular underpinnings that explain recent extrapulmonary observations is necessary. Here, we use an Apoe−/− mouse model to investigate the epigenetic impact of paternal exposure to simulated wildfire smoke. We demonstrate that 40 days of exposure to smoke from Douglas fir needles induces sperm DNA methylation changes in adult mice. DNA methylation was measured by reduced representation bisulfite sequencing and varied significantly in 3353 differentially methylated regions, which were subsequently annotated to 2117 genes. The differentially methylated regions were broadly distributed across the mouse genome, but the vast majority (nearly 80%) were hypermethylated. Pathway analyses, using gene-derived and differentially methylated region-derived gene ontology terms, point to a number of developmental processes that may warrant future investigation. Overall, this study of simulated wildfire smoke exposure suggests paternal reproductive risks are possible with prolonged exposure.

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Male obesity impacts DNA methylation reprogramming in sperm

Clinical Epigenetics ◽

10.1186/s13148-020-00997-0 ◽

2021 ◽

Vol 13 (1) ◽

Author(s):

Sanaz Keyhan ◽

Emily Burke ◽

Rose Schrott ◽

Zhiqing Huang ◽

Carole Grenier ◽

...

Keyword(s):

Dna Methylation ◽

System Development ◽

Human Sperm ◽

Nervous System Development ◽

Cpg Sites ◽

Epididymal Maturation ◽

Sperm Dna ◽

Stem Cell Pluripotency ◽

The Impact ◽

Sperm Dna Methylation

Abstract Background Male obesity has profound effects on morbidity and mortality, but relatively little is known about the impact of obesity on gametes and the potential for adverse effects of male obesity to be passed to the next generation. DNA methylation contributes to gene regulation and is erased and re-established during gametogenesis. Throughout post-pubertal spermatogenesis, there are continual needs to both maintain established methylation and complete DNA methylation programming, even during epididymal maturation. This dynamic epigenetic landscape may confer increased vulnerability to environmental influences, including the obesogenic environment, that could disrupt reprogramming fidelity. Here we conducted an exploratory analysis that showed that overweight/obesity (n = 20) is associated with differences in mature spermatozoa DNA methylation profiles relative to controls with normal BMI (n = 47). Results We identified 3264 CpG sites in human sperm that are significantly associated with BMI (p < 0.05) using Infinium HumanMethylation450 BeadChips. These CpG sites were significantly overrepresented among genes involved in transcriptional regulation and misregulation in cancer, nervous system development, and stem cell pluripotency. Analysis of individual sperm using bisulfite sequencing of cloned alleles revealed that the methylation differences are present in a subset of sperm rather than being randomly distributed across all sperm. Conclusions Male obesity is associated with altered sperm DNA methylation profiles that appear to affect reprogramming fidelity in a subset of sperm, suggestive of an influence on the spermatogonia. Further work is required to determine the potential heritability of these DNA methylation alterations. If heritable, these changes have the potential to impede normal development.

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Early-Life Exposure to Environmental Contaminants Perturbs the Sperm Epigenome and Induces Negative Pregnancy Outcomes for Three Generations via the Paternal Lineage

Epigenomes ◽

10.3390/epigenomes5020010 ◽

2021 ◽

Vol 5 (2) ◽

pp. 10

Author(s):

Clotilde Maurice ◽

Mathieu Dalvai ◽

Romain Lambrot ◽

Astrid Deschênes ◽

Marie-Pier Scott-Boyer ◽

...

Keyword(s):

Dna Methylation ◽

Pregnancy Outcomes ◽

Sperm Quality ◽

The Arctic ◽

Congenital Defects ◽

Developmental Defects ◽

Early Life Exposure ◽

Sperm Dna ◽

Inuit Population ◽

Sperm Dna Methylation

Due to the grasshopper effect, the Arctic food chain in Canada is contaminated with persistent organic pollutants (POPs) of industrial origin, including polychlorinated biphenyls and organochlorine pesticides. Exposure to POPs may be a contributor to the greater incidence of poor fetal growth, placental abnormalities, stillbirths, congenital defects and shortened lifespan in the Inuit population compared to non-Aboriginal Canadians. Although maternal exposure to POPs is well established to harm pregnancy outcomes, paternal transmission of the effects of POPs is a possibility that has not been well investigated. We used a rat model to test the hypothesis that exposure to POPs during gestation and suckling leads to developmental defects that are transmitted to subsequent generations via the male lineage. Indeed, developmental exposure to an environmentally relevant Arctic POPs mixture impaired sperm quality and pregnancy outcomes across two subsequent, unexposed generations and altered sperm DNA methylation, some of which are also observed for two additional generations. Genes corresponding to the altered sperm methylome correspond to health problems encountered in the Inuit population. These findings demonstrate that the paternal methylome is sensitive to the environment and that some perturbations persist for at least two subsequent generations. In conclusion, although many factors influence health, paternal exposure to contaminants plays a heretofore-underappreciated role with sperm DNA methylation contributing to the molecular underpinnings involved.

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Sperm DNA methylation mediates the association of male age on reproductive outcomes among couples undergoing infertility treatment

Scientific Reports ◽

10.1038/s41598-020-80857-2 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Oladele A. Oluwayiose ◽

Haotian Wu ◽

Hachem Saddiki ◽

Brian W. Whitcomb ◽

Laura B. Balzer ◽

...

Keyword(s):

Dna Methylation ◽

Developed Countries ◽

Infertility Treatment ◽

Reproductive Outcomes ◽

Neurodevelopmental Outcomes ◽

Male Age ◽

Age Related ◽

Sperm Dna ◽

Increased Risk ◽

Sperm Dna Methylation

AbstractParental age at time of offspring conception is increasing in developed countries. Advanced male age is associated with decreased reproductive success and increased risk of adverse neurodevelopmental outcomes in offspring. Mechanisms for these male age effects remain unclear, but changes in sperm DNA methylation over time is one potential explanation. We assessed genome-wide methylation of sperm DNA from 47 semen samples collected from male participants of couples seeking infertility treatment. We report that higher male age was associated with lower likelihood of fertilization and live birth, and poor embryo development (p < 0.05). Furthermore, our multivariable linear models showed male age was associated with alterations in sperm methylation at 1698 CpGs and 1146 regions (q < 0.05), which were associated with > 750 genes enriched in embryonic development, behavior and neurodevelopment among others. High dimensional mediation analyses identified four genes (DEFB126, TPI1P3, PLCH2 and DLGAP2) with age-related sperm differential methylation that accounted for 64% (95% CI 0.42–0.86%; p < 0.05) of the effect of male age on lower fertilization rate. Our findings from this modest IVF population provide evidence for sperm methylation as a mechanism of age-induced poor reproductive outcomes and identifies possible candidate genes for mediating these effects.

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