Search

EHMT1 (also known as GLP) is a multifunctional protein, best known for its role as an H3K9me1 and H3K9me2 methyltransferase through its reportedly obligatory dimerization with EHMT2 (also known as G9A). Here, we investigated the role of EHMT1 in the oocyte in comparison to EHMT2 using oocyte-specific conditional knockout mouse models (Ehmt2cKO,Ehmt1cKO,Ehmt1/2cDKO), with ablation from the early phase of oocyte growth. Loss of EHMT1 inEhmt1cKO andEhmt1/2cDKO oocytes recapitulated meiotic defects observed in theEhmt2cKO; however, there was a significant impairment in oocyte maturation and developmental competence inEhmt1cKO andEhmt1/2cDKO oocytes beyond that observed in theEhmt2cKO. Consequently, loss of EHMT1 in oogenesis results, upon fertilization, in mid-gestation embryonic lethality. To identify H3K9 methylation and other meaningful biological changes in each mutant to explore the molecular functions of EHMT1 and EHMT2, we performed immunofluorescence imaging, multi-omics sequencing, and mass spectrometry (MS)–based proteome analyses in cKO oocytes. Although H3K9me1 was depleted only upon loss of EHMT1, H3K9me2 was decreased, and H3K9me2-enriched domains were eliminated equally upon loss of EHMT1 or EHMT2. Furthermore, there were more significant changes in the transcriptome, DNA methylome, and proteome inEhmt1/2cDKO thanEhmt2cKO oocytes, with transcriptional derepression leading to increased protein abundance and local changes in genic DNA methylation inEhmt1/2cDKO oocytes. Together, our findings suggest that EHMT1 contributes to local transcriptional repression in the oocyte, partially independent of EHMT2, and is critical for oogenesis and oocyte developmental competence.

Funder: FP7 People: Marie-Curie Actions; Grant(s): 290123, Histone 3 lysine 4 trimethylation (H3K4me3) is an epigenetic mark found at gene promoters and CpG islands. H3K4me3 is essential for mammalian development, yet mechanisms underlying its genomic targeting are poorly understood. H3K4me3 methyltransferases SETD1B and MLL2 (KMT2B) are essential for oogenesis. We investigated changes in H3K4me3 in Setd1b conditional knockout (cKO) oocytes using ultra-low input ChIP-seq, with comparisons to DNA methylation and gene expression analyses. H3K4me3 was redistributed in Setd1b cKO oocytes showing losses at active gene promoters associated with downregulated gene expression. Remarkably, many regions also gained H3K4me3, in particular those that were DNA hypomethylated, transcriptionally inactive and CpG-rich, which are hallmarks of MLL2 targets. Consequently, loss of SETD1B disrupts the balance between MLL2 and de novo DNA methyltransferases in determining the epigenetic landscape during oogenesis. Our work reveals two distinct, complementary mechanisms of genomic targeting of H3K4me3 in oogenesis, with SETD1B linked to gene expression and MLL2 to CpG content.

GLP (EHMT1) is a multifunctional protein, best known for its role as an H3K9me1 and H3K9me2 methyltransferase through its reportedly obligatory dimerization with G9A (EHMT2). Here, we investigate the role of GLP in the oocyte in comparison to G9A using oocyte-specific conditional knockout mouse models (G9a cKO, Glp cKO, G9a-Glp cDKO). Loss of GLP in Glp cKO and G9a-Glp cDKO oocytes re-capitulated meiotic defects observed in the G9a cKO; however, there was a significant impairment in oocyte maturation and developmental competence in Glp cKO and G9a-Glp cDKO oocytes beyond that observed in the G9a cKO. Consequently, loss of GLP in oogenesis results upon fertilisation in mid-gestation embryonic lethality. To assess the molecular functions of GLP and G9A, we applied a multi-omics approach, supported by immunofluorescence, to identify changes in epigenomic, transcriptomic and proteomic signatures in cKO oocytes. H3K9me2 was equally depleted in all cKO oocytes, whereas H3K9me1 was decreased only upon loss of GLP. The transcriptome, DNA methylome and proteome were markedly more affected in G9a-Glp cDKO than G9a cKO oocytes, with transcriptional de-repression associated with increased protein abundance and gains in genic DNA methylation in G9a-Glp cDKO oocytes. Together, our findings suggest that GLP contributes to transcriptional repression in the oocyte, independent of G9A, and is critical for oogenesis and oocyte developmental competence.

The importance of the placenta in supporting mammalian development has long been recognized, but our knowledge of the molecular, genetic and epigenetic requirements that underpin normal placentation has remained remarkably under-appreciated. Both the in vivo mouse model and in vitro-derived murine trophoblast stem cells have been invaluable research tools for gaining insights into these aspects of placental development and function, with recent studies starting to reshape our view of how a unique epigenetic environment contributes to trophoblast differentiation and placenta formation. These advances, together with recent successes in deriving human trophoblast stem cells, open up new and exciting prospects in basic and clinical settings that will help deepen our understanding of placental development and associated disorders of pregnancy.

Genomic imprinting is the monoallelic expression of a gene based on parent of origin and is a consequence of differential epigenetic marking between the male and female germlines. Canonically, genomic imprinting is mediated by allelic DNA methylation. However, recently it has been shown that maternal H3K27me3 can result in DNA methylation-independent imprinting, termed “noncanonical imprinting.” In this review, we compare and contrast what is currently known about the underlying mechanisms, the role of endogenous retroviral elements, and the conservation of canonical and noncanonical genomic imprinting.

Histone 3 lysine 4 trimethylation (H3K4me3) is an epigenetic mark found at gene promoters and CpG islands. H3K4me3 is essential for mammalian development, yet mechanisms underlying its genomic targeting are poorly understood. H3K4me3 methyltransferases SETD1B and MLL2 are essential for oogenesis. We investigated changes in H3K4me3 in Setd1b conditional knockout (cKO) oocytes using ultra-low input ChIP-seq, with comparisons to DNA methylation and gene expression analyses. H3K4me3 was redistributed in Setd1b cKO oocytes showing losses at active gene promoters associated with downregulated gene expression. Remarkably, many regions also gained H3K4me3, in particular those that were DNA hypomethylated, transcriptionally inactive and CpG-rich, which are hallmarks of MLL2 targets. Consequently, loss of SETD1B disrupts the balance between MLL2 and de novo DNA methyltransferases in determining the epigenetic landscape during oogenesis. Our work reveals two distinct, complementary mechanisms of genomic targeting of H3K4me3 in oogenesis, with SETD1B linked to gene expression and MLL2 to CpG content.Graphical AbstractIn oogenesis, SETD1B and CXXC1 target H3K4me3 to actively transcribed gene promoters, while MLL2 targets transcriptionally inactive regions based on underlying CpG composition (upper panel). When SETD1B is ablated, H3K4me3 is lost at a subset of active promoters, resulting in downregulation of transcription (lower panel). Loss of SETD1B alters the activity of MLL2, permitting MLL2 to deposit H3K4me3 at CpG-rich regions, many of which should otherwise be DNA methylated. Thus, it is evident that MLL2 and de novo DNMTs compete for genomic occupancy late in oogenesis, and loss of SETD1B disrupts the balance of these mechanisms.

Stability of the epigenetic landscape underpins maintenance of the cell-type-specific transcriptional profile. As one of the main repressive epigenetic systems, DNA methylation has been shown to be important for long-term gene silencing; its loss leads to ectopic and aberrant transcription in differentiated cells and cancer1. The developing mouse germ line endures global changes in DNA methylation in the absence of widespread transcriptional activation. Here, using an ultra-low-input native chromatin immunoprecipitation approach, we show that following DNA demethylation the gonadal primordial germ cells undergo remodelling of repressive histone modifications, resulting in a sex-specific signature in mice. We further demonstrate that Polycomb has a central role in transcriptional control in the newly hypomethylated germline genome as the genetic loss of Ezh2 leads to aberrant transcriptional activation, retrotransposon derepression and dramatic loss of developing female germ cells. This sex-specific effect of Ezh2 deletion is explained by the distinct landscape of repressive modifications observed in male and female germ cells. Overall, our study provides insight into the dynamic interplay between repressive chromatin modifications in the context of a developmental reprogramming system.

Advancing maternal age causes a progressive reduction in fertility. The decline in developmental competence of the oocyte with age is likely to be a consequence of multiple contributory factors. Loss of epigenetic quality of the oocyte could impair early developmental events or programme adverse outcomes in offspring that manifest only later in life. Here, we undertake joint profiling of the transcriptome and DNA methylome of individual oocytes from reproductively young and old mice undergoing natural ovulation. We find reduced complexity as well as increased variance in the transcriptome of oocytes from aged females. This transcriptome heterogeneity is reflected in the identification of discrete sub‐populations. Oocytes with a transcriptome characteristic of immature chromatin configuration (NSN) clustered into two groups: one with reduced developmental competence, as indicated by lower expression of maternal effect genes, and one with a young‐like transcriptome. Oocytes from older females had on average reduced CpG methylation, but the characteristic bimodal methylation landscape of the oocyte was preserved. Germline differentially methylated regions of imprinted genes were appropriately methylated irrespective of age. For the majority of differentially expressed transcripts, the absence of correlated methylation changes suggests a post‐transcriptional basis for most age‐related effects on the transcriptome. However, we did find differences in gene body methylation at which there were corresponding changes in gene expression, indicating age‐related effects on transcription that translate into methylation differences. Interestingly, oocytes varied in expression and methylation of these genes, which could contribute to variable competence of oocytes or penetrance of maternal age‐related phenotypes in offspring., Early embryo development is heavily supported by maternal transcripts and epigenetic states produced in the oocyte. To investigate the effects of age, we compare the transcriptome and methylome of germinal vesicle oocytes from young and old mice using parallel single‐cell RNA sequencing and whole‐genome bisulphite sequencing. We find increased variance in the transcriptome of oocytes from aged females, altered expression of maternal effect genes and differences in gene body methylation at which there were corresponding changes in gene expression.

As the maternal–foetal interface, the placenta is essential for the establishment and progression of healthy pregnancy, regulating both foetal growth and maternal adaptation to pregnancy. The evolution and functional importance of genomic imprinting are inextricably linked to mammalian placentation. Recent technological advances in mapping and manipulating the epigenome in embryogenesis in mouse models have revealed novel mechanisms regulating genomic imprinting in placental trophoblast, the physiological implications of which are only just beginning to be explored. This review will highlight important recent discoveries and exciting new directions in the study of placental imprinting., Author summary The placenta is essential for healthy pregnancy because it supports the growth of the baby, helps the mother’s body adapt, and provides a connection between mother and the developing baby. Studying gene regulation and the early steps in placental development is challenging in human pregnancy, so mouse models have been key in building our understanding of these processes. In particular, these studies have identified a subset of genes that are essential for placentation, termed imprinted genes. Imprinted genes are those that are expressed from only one copy, depending on whether they were inherited from mom or dad. In this review, I describe recent novel approaches used to study the mechanisms regulating these imprinted genes in mouse models, and I highlight several new discoveries. It has become apparent that the regulation of imprinted genes in placenta is often unique from other tissues and that there are species-specific mechanisms allowing the evolution of new imprinted genes specifically in the placenta.

Histone 3 K4 trimethylation (depositing H3K4me3 marks) is typically associated with active promoters yet paradoxically occurs at untranscribed domains. Research to delineate the mechanisms of targeting H3K4 methyltransferases is ongoing. The oocyte provides an attractive system to investigate these mechanisms, because extensive H3K4me3 acquisition occurs in nondividing cells. We developed low-input chromatin immunoprecipitation to interrogate H3K4me3, H3K27ac and H3K27me3 marks throughout oogenesis. In nongrowing oocytes, H3K4me3 was restricted to active promoters, but as oogenesis progressed, H3K4me3 accumulated in a transcription-independent manner and was targeted to intergenic regions, putative enhancers and silent H3K27me3-marked promoters. Ablation of the H3K4 methyltransferase gene Mll2 resulted in loss of transcription-independent H3K4 trimethylation but had limited effects on transcription-coupled H3K4 trimethylation or gene expression. Deletion of Dnmt3a and Dnmt3b showed that DNA methylation protects regions from acquiring H3K4me3. Our findings reveal two independent mechanisms of targeting H3K4me3 to genomic elements, with MLL2 recruited to unmethylated CpG-rich regions independently of transcription.

Formation of the three primary germ layers during gastrulation is an essential step in the establishment of the vertebrate body plan and is associated with major transcriptional changes(1-5). Global epigenetic reprogramming accompanies these changes(6-8), but the role of the epigenome in regulating early cell-fate choice remains unresolved, and the coordination between different molecular layers is unclear. Here we describe a single-cell multi-omics map of chromatin accessibility, DNA methylation and RNA expression during the onset of gastrulation in mouse embryos. The initial exit from pluripotency coincides with the establishment of a global repressive epigenetic landscape, followed by the emergence of lineage-specific epigenetic patterns during gastrulation. Notably, cells committed to mesoderm and endoderm undergo widespread coordinated epigenetic rearrangements at enhancer marks, driven by ten-eleven translocation (TET)-mediated demethylation and a concomitant increase of accessibility. By contrast, the methylation and accessibility landscape of ectodermal cells is already established in the early epiblast. Hence, regulatory elements associated with each germ layer are either epigenetically primed or remodelled before cell-fate decisions, providing the molecular framework for a hierarchical emergence of the primary germ layers.

The importance of the placenta in supporting mammalian development has long been recognized, but our knowledge of the molecular, genetic and epigenetic requirements that underpin normal placentation has remained remarkably under-appreciated. Both the in vivo mouse model and in vitro-derived murine trophoblast stem cells have been invaluable research tools for gaining insights into these aspects of placental development and function, with recent studies starting to reshape our view of how a unique epigenetic environment contributes to trophoblast differentiation and placenta formation. These advances, together with recent successes in deriving human trophoblast stem cells, open up new and exciting prospects in basic and clinical settings that will help deepen our understanding of placental development and associated disorders of pregnancy.

At the heart of genomic imprinting in mammals are imprinting control regions (ICRs), which are the discrete genetic elements that confer imprinted monoallelic expression to several genes in imprinted gene clusters. A characteristic of the known ICRs is that they acquire different epigenetic states, exemplified by differences in DNA methylation, in the sperm and egg, and these imprint marks remain on the sperm- and oocyte-derived alleles into the next generation as a lifelong memory of parental origin. Although there has been much focus on gametic marking of ICRs as the point of imprint specification, recent mechanistic studies and genome-wide DNA methylation profiling do not support the existence of a specific imprinting machinery in germ cells. Rather, ICRs are part of more widespread methylation events that occur during gametogenesis. Instead, a decisive component in the specification of imprints is the choice of which sites of gamete-derived methylation to maintain in the zygote and preimplantation embryo at a time when much of the remainder of the genome is being demethylated. Among the factors involved in this selection, the zinc-finger protein Zfp57 can be regarded as an imprint-specific, sequence-specific DNA binding factor responsible for maintaining methylation at most ICRs. The recent insights into the balance of gametic and zygotic contributions to imprint specification should help understand mechanistic opportunities and constraints on the evolution of imprinting in mammals.

Workshops are an important part of the IFPA annual meeting as they allow for discussion of specialized topics. At IFPA meeting 2015 there were twelve themed workshops, three of which are summarized in this report. These workshops covered areas of placental regulation and nutrient handling: 1) placental epigenetics; 2) placental mitochondrial function; 3) placental transport systems.

Background Genomic imprinting is an epigenetic phenomenon that allows a subset of genes to be expressed mono-allelically based on the parent of origin and is typically regulated by differential DNA methylation inherited from gametes. Imprinting is pervasive in murine extra-embryonic lineages, and uniquely, the imprinting of several genes has been found to be conferred non-canonically through maternally inherited repressive histone modification H3K27me3. However, the underlying regulatory mechanisms of non-canonical imprinting in post-implantation development remain unexplored. Results We identify imprinted regions in post-implantation epiblast and extra-embryonic ectoderm (ExE) by assaying allelic histone modifications (H3K4me3, H3K36me3, H3K27me3), gene expression, and DNA methylation in reciprocal C57BL/6 and CAST hybrid embryos. We distinguish loci with DNA methylation-dependent (canonical) and independent (non-canonical) imprinting by assaying hybrid embryos with ablated maternally inherited DNA methylation. We find that non-canonical imprints are localized to endogenous retrovirus-K (ERVK) long terminal repeats (LTRs), which act as imprinted promoters specifically in extra-embryonic lineages. Transcribed ERVK LTRs are CpG-rich and located in close proximity to gene promoters, and imprinting status is determined by their epigenetic patterning in the oocyte. Finally, we show that oocyte-derived H3K27me3 associated with non-canonical imprints is not maintained beyond pre-implantation development at these elements and is replaced by secondary imprinted DNA methylation on the maternal allele in post-implantation ExE, while being completely silenced by bi-allelic DNA methylation in the epiblast. Conclusions This study reveals distinct epigenetic mechanisms regulating non-canonical imprinted gene expression between embryonic and extra-embryonic development and identifies an integral role for ERVK LTR repetitive elements.

Formation of the three primary germ layers during gastrulation is an essential step in the establishment of the vertebrate body plan. Recent studies employing single cell RNA-sequencing have identified major transcriptional changes associated with germ layer specification. Global epigenetic reprogramming accompanies these changes, but the role of the epigenome in regulating early cell fate choice remains unresolved, and the coordination between different epigenetic layers is unclear. Here we describe the first single cell triple-omics map of chromatin accessibility, DNA methylation and RNA expression during the exit from pluripotency and the onset of gastrulation in mouse embryos. We find dynamic dependencies between the different molecular layers, with evidence for distinct modes of epigenetic regulation. The initial exit from pluripotency coincides with the establishment of a global repressive epigenetic landscape, followed by the emergence of local lineage-specific epigenetic patterns during gastrulation. Notably, cells committed to mesoderm and endoderm undergo widespread coordinated epigenetic rearrangements, driven by loss of methylation in enhancer marks and a concomitant increase of chromatin accessibility. In striking contrast, the epigenetic landscape of ectodermal cells is already established in the early epiblast. Hence, regulatory elements associated with each germ layer are either epigenetically primed or epigenetically remodelled prior to overt cell fate decisions during gastrulation, providing the molecular logic for a hierarchical emergence of the primary germ layers.HighlightsFirst map of mouse gastrulation using comprehensive single cell triple-omic analysis.Exit from pluripotency is associated with a global repressive epigenetic landscape, driven by a sharp gain of DNA methylation and a gradual decrease of chromatin accessibility.DNA methylation and chromatin accessibility changes in enhancers, but not in promoters, are associated with germ layer formation.Mesoderm and endoderm enhancers become open and demethylated upon lineage commitment.Ectoderm enhancers are primed in the early epiblast and protected from the global repressive dynamics, supporting a default model of ectoderm commitment in vivo.

Early development in mammals is the culmination of periods of reading, writing and erasing epigenetic instructions that define and endow fully differentiated gametes with the potential to create new life. The pathways that support these processes are highly dynamic, yet they must also balance the strict enforcement of cell fate decision at critical junctures, to ensure the elaboration of fixed lineages at precise time points. The complex and multilayered information of the epigenome must instruct a single genome in the zygote and orchestrate the differentiation of all subsequent cell types giving rise to the adult organism replete with new germ cells to perpetuate the process in the next generation. The mechanisms and machinery of the epigenetic pathways required in early mammalian development have been studied extensively, especially in the mouse. Here we consider epigenetic regulation required to establish both totipotency and pluripotency, a prerequisite for preimplantation development, leading to embryonic and extraembryonic lineage commitment at the blastocyst stage setting the cellular trajectory for full-term gestation. The processes and mechanistic regulation in these early developmental decisions are critical to ensure the linear direction and high fidelity of cell fate determination. Applying the principles of reprogramming in development to experimental reprogramming in patient-specific regenerative medicine has opened the possibility to form new replacement tissues and organs in the treatment of chronic diseases and to rejuvenate aging pathology extending a longer healthy lifespan.

BACKGROUND Over the past few years, advances in molecular technologies have allowed unprecedented mapping of epigenetic modifications in gametes and during early embryonic development. This work is allowing a detailed genomic analysis, which for the first time can answer long-standing questions about epigenetic regulation and reprogramming, and highlights differences between mouse and human, the implications of which are only beginning to be explored. OBJECTIVE AND RATIONALE In this review, we summarise new low-cell molecular methods enabling the interrogation of epigenetic information in gametes and early embryos, the mechanistic insights these have provided, and contrast the findings in mouse and human. SEARCH METHODS Relevant studies were identified by PubMed search. OUTCOMES We discuss the levels of epigenetic regulation, from DNA modifications to chromatin organisation, during mouse gametogenesis, fertilisation and pre- and post-implantation development. The recently characterised features of the oocyte epigenome highlight its exceptionally unique regulatory landscape. The chromatin organisation and epigenetic landscape of both gametic genomes are rapidly reprogrammed after fertilisation. This extensive epigenetic remodelling is necessary for zygotic genome activation, but the mechanistic link remains unclear. While the vast majority of epigenetic information from the gametes is erased in pre-implantation development, new insights suggest that repressive histone modifications from the oocyte may mediate a novel mechanism of imprinting. To date, the characterisation of epigenetics in human development has been almost exclusively limited to DNA methylation profiling; these data reinforce that the global dynamics are conserved between mouse and human. However, as we look closer, it is becoming apparent that the mechanisms regulating these dynamics are distinct. These early findings emphasise the importance of investigations of fundamental epigenetic mechanisms in both mouse and humans. WIDER IMPLICATIONS Failures in epigenetic regulation have been implicated in human disease and infertility. With increasing maternal age and use of reproductive technologies in countries all over the world, it is becoming ever more important to understand the necessary processes required to establish a developmentally competent embryo. Furthermore, it is essential to evaluate the extent to which these epigenetic patterns are sensitive to such technologies and other adverse environmental exposures.

Background 5,10-Methylenetetrahydrofolate reductase (MTHFR) is a key enzyme in one-carbon metabolism that ensures the availability of methyl groups for methylation reactions. Two single-nucleotide polymorphisms (SNPs) in the MTHFR gene, 677C>T and 1298A>C, result in a thermolabile enzyme with reduced function. These variants, in both the maternal and/or fetal genes, have been associated with pregnancy complications including miscarriage, neural tube defects (NTDs), and preeclampsia (PE), perhaps due to altered capacity for DNA methylation (DNAm). In this study, we assessed the association between MTHFR 677TT and 1298CC genotypes and risk of NTDs, PE, or normotensive intrauterine growth restriction (nIUGR). Additionally, we assessed whether these high-risk genotypes are associated with altered DNAm in the placenta. Results In 303 placentas screened for this study, we observed no significant association between the occurrence of NTDs (N = 55), PE (early-onset: N = 28, late-onset: N = 20), or nIUGR (N = 21) and placental (fetal) MTHFR 677TT or 1298CC genotypes compared to healthy pregnancies (N = 179), though a trend of increased 677TT genotype in PE/IUGR together was observed (OR 2.53, p = 0.048). DNAm was profiled in 10 high-risk 677 (677TT + 1298AA), 10 high-risk 1298 (677CC + 1298CC), and 10 reference (677CC + 1298AA) genotype placentas. Linear modeling identified no significantly differentially methylated sites between high-risk 677 or 1298 and reference placentas at a false discovery rate

Psychological challenges, including traumatic events, have been hypothesized to increase the age-related pace of biological aging. Here we test the hypothesis that psychological challenges can affect the pace of telomere attrition, a marker of cellular aging, using data from an ongoing longitudinal-cohort study of Kaqchikel Mayan women living in a population with a high frequency of child mortality, a traumatic life event. Specifically, we evaluate the associations between child mortality, maternal telomere length and the mothers’ hypothalamic-pituitary-adrenal axis (HPAA), or stress axis, activity. Child mortality data were collected in 2000 and 2013. HPAA activity was assessed by quantifying cortisol levels in first morning urinary specimens collected every other day for seven weeks in 2013. Telomere length (TL) was quantified using qPCR in 55 women from buccal specimens collected in 2013. Results: Shorter TL with increasing age was only observed in women who experienced child mortality (p = 0.015). Women with higher average basal cortisol (p = 0.007) and greater within-individual variation (standard deviation) in basal cortisol (p = 0.053) presented shorter TL. Non-parametric bootstrapping to estimate mediation effects suggests that HPAA activity mediates the effect of child mortality on TL. Our results are, thus, consistent with the hypothesis that traumatic events can influence cellular aging and that HPAA activity may play a mediatory role. Future large-scale longitudinal studies are necessary to confirm our results and further explore the role of the HPAA in cellular aging, as well as to advance our understanding of the underlying mechanisms involved.

Miscarriage occurs in 15% of clinical pregnancies. Although chromosomal errors are observed in50%, causes of karyotypically normal losses are poorly understood. DNA methylation undergoes reprogramming during development and must be appropriately set to maintain a healthy pregnancy. We hypothesize that aberrant DNA methylation may cause karyotypically normal miscarriage, particularly among women experiencing recurrent miscarriage (RM). DNA methylation in first-trimester chorionic villi was assessed in chromosomally normal miscarriages from women with RM (N = 33) or isolated miscarriage (M; N = 21) and elective terminations (TA; N = 16). Differentially methylated candidate loci were identified using the Illumina Infinium HumanMethylation27 BeadChip array. Follow-up bisulfite pyrosequencing at promoter regions showed an increase in methylation in M compared with TA at cytochrome P450, subfamily 1A, polypeptide 2 (CYP1A2; P = 0.002) and RM compared with TA at AXL receptor tyrosine kinase (P = 0.02), and a decrease in RM and M compared with TA at defensin β 1 (DEFB1; P = 0.008). Gene ontology analysis showed an enrichment of imprinted genes (P = 9.53 × 10(-10)) and genes previously associated with RM (P = 9.51 × 10(-6)). An increase of outliers at seven imprinted loci was observed in RM (3.9%) compared with M (0%) and TA (0.9%) (P = 0.02), with increased average methylation at H19/IGF2 ICR1 in M samples (P0.0001). Altered DNA methylation in the placenta at specific loci, and global dysregulation in specific cases, may contribute to or be a consequence of poor placental function in karyotypically normal miscarriage.

Maternal effect genes control early events of embryogenesis. Maternal homozygous and compound mutations in two such genes, NLRP7 and c6orf221, have been detected in the majority of women experiencing recurrent biparental hydatidiform moles. It was suggested that other forms of reproductive wastage, including diploid androgenetic moles, partial moles, polyploidy, recurrent spontaneous abortions and stillbirths of uncertain etiology, may be caused by NLRP7 or c6orf221 mutations in the mother. To elucidate which subpopulations of women with adverse reproductive outcomes should be screened for NLRP7/C6orf221 variants, we sequenced coding sequence and exon/intron boundaries of NLRP7 and C6orf221 in a well-defined group of 17 women with recurrent miscarriage and additional triploidy or complete hydatidiform moles. The major findings for this group were non-synonymous variants of NLRP7, rather than clearly pathogenic mutations. To assess the role of these variants, we genotyped them in a larger group including women with primary recurrent miscarriage (n = 39), paternal triploid conceptions (n = 22) and women with proven fertility after age 37 and no prior history of miscarriage or pregnancy complications (n = 52). No associations between non-synonymous NLRP7 variants and primary recurrent miscarriage or partial hydatidiform molar pregnancies were detected. Our findings suggest that neither mutations nor variants in NLRP7 and C6orf221 are major factors contributing to the risk of these types of pregnancy complications. Further studies in larger groups of patients and controls are needed to specify the impact of NLRP7 rare non-synonymous variants on genetic susceptibility to recurrent reproductive wastage.

The maternal and paternal copies of the genome are both required for mammalian development, and this is primarily due to imprinted genes, those that are monoallelically expressed based on parent-of-origin. Typically, this pattern of expression is regulated by differentially methylated regions (DMRs) that are established in the germline and maintained after fertilization. There are a large number of germline DMRs that have not yet been associated with imprinting, and their function in development is unknown. In this study, we developed a genome-wide approach to identify novel imprinted DMRs in the human placenta and investigated the dynamics of these imprinted DMRs during development in somatic and extraembryonic tissues. DNA methylation was evaluated using the Illumina HumanMethylation450 array in 134 human tissue samples, publicly available reduced representation bisulfite sequencing in the human embryo and germ cells, and targeted bisulfite sequencing in term placentas. Forty-three known and 101 novel imprinted DMRs were identified in the human placenta by comparing methylation between diandric and digynic triploid conceptions in addition to female and male gametes. Seventy-two novel DMRs showed a pattern consistent with placental-specific imprinting, and this monoallelic methylation was entirely maternal in origin. Strikingly, these DMRs exhibited polymorphic imprinted methylation between placental samples. These data suggest that imprinting in human development is far more extensive and dynamic than previously reported and that the placenta preferentially maintains maternal germline-derived DNA methylation.

Sensation seeking is the tendency to seek out new and thrilling experiences and to take risks for the sake of such experiences. A single-nucleotide polymorphism, -521 C/T (rs1800955) in the promoter region of the dopamine-4-receptor gene (DRD4), is associated with approach-related traits including novelty seeking and extraversion, in some, but not all studies. To our knowledge, no studies have been conducted on the genetics of risk-taking behavior in sports. Using a joint-analysis approach, we measured sensation seeking in two cohorts of experienced male and female skiers and snowboarders (n = 503) using a sports-specific tool developed for this study, the Contextual Sensation Seeking Questionnaire for Skiing and Snowboarding (CSSQ-S), and a more general trait measure, the Zuckerman-Kuhlman Personality Questionnaire impulsive sensation-seeking subscale. We detected, and then replicated a significant association between the DRD4 -521CC genotype and sports-specific sensation seeking as measured using the CSSQ-S (P < 0.001). These data suggest that the DRD4 -521 C/T polymorphism contributes to a "risk-taking phenotype" in skiers and snowboarders, but the variant was not associated with impulsive sensation seeking (P = 0.9).

background: Changes in DNA methylation may play an important role in the deleterious reproductive effects reported in association with exposure to environmental pollutants. In this pilot study, we identify candidate methylation changes associated with exposure to pollutants in women undergoing in vitro fertilization (IVF). methods: Blood and urine were collected from women on the day of oocyte retrieval. Whole blood was analyzed for mercury and lead, and urine for cadmium using inductively coupled plasma mass spectrometry. Unconjugated bisphenol A (BPA) was analyzed in serum using high-performance liquid chromatography with Coularray detection. Participants were dichotomized as higher or lower exposure groups by median concentrations. Using the Illumina GoldenGate Methylation Cancer Panel I, DNA methylation in whole blood from 43 women was assessed at 1505 CpG sites for association with exposure levels of each pollutant. Candidate CpG sites were identified using a Diff Score .|13| (P , 0.05) and an absolute difference .10% which were confirmed using bisulfite pyrosequencing. results: Methylation of the GSTM1/5 promoter was increased for women with higher mercury exposure (P ¼ 0.04); however, no correlation was observed (r ¼ 0.17, P ¼ 0.27). Reduced methylation was detected in the COL1A2 promoter in women with higher exposure to lead (P ¼ 0.004), and an inverse correlation was observed (r ¼ 2 0.45, P ¼ 0.03). Lower methylation of a promoter CpG site at the TSP50 gene was detected in women with higher BPA exposure (P ¼ 0.005), and again an inverse correlation was identified (r ¼ 2 0.51, P ¼ 0.001). conclusions: Altered DNA methylation at various CpG sites was associated with exposure to mercury, lead or BPA, providing candidates to be investigated using a larger study sample, as the results may reflect an independently associated predictor (e.g. socioeconomic status, diet, genetic variants, altered blood cell composition). Further studies accommodating variations in these factors will be needed to confirm these associations and identify their underlying causes.

Background Recurrent miscarriage affects 1-2% of couples trying to conceive, and is idiopathic in nearly half. Female fertility is controlled by the hypothalamus-pituitary-ovarian (HPO) axis and we hypothesize that genetic polymorphisms affecting the function of genes involved in regulating the HPO axis will be associated with recurrent miscarriage. Methods Whole peripheral blood DNA from 227 women with recurrent miscarriage and 130 control women was obtained for this study. Using the Sequenom iPlex assay for fragment analysis, 31 single-nucleotide polymorphisms (SNPs) and 4 short tandem repeat (STR) polymorphisms in 20 candidate genes were evaluated for genetic association with recurrent miscarriage. Results Several candidate associations were identified with an uncorrected P-value of 0.05. Genotype distribution at an SNP (rs37389) in the prolactin receptor gene (P = 0.03), and allele distributions at an SNP (rs41423247) in the glucocorticoid receptor gene (P = 0.04) and an STR polymorphism in the estrogen receptor β gene (P = 0.03) were associated with recurrent miscarriage. The T allele of an SNP (rs2033962) within the activin receptor type 1 gene (ACVR1) was associated with increased number of miscarriages in an additive manner (P = 0.02). These candidate associations were not statistically significant after correcting for multiple analyses. Conclusions Candidate associations were identified between recurrent miscarriage and genetic variation within ESR2, PRLR, GCCR and ACVR1 genes. Independent confirmation of these results is needed, as limitations of this study include the heterogeneous etiology of recurrent miscarriage, limited sample size, partial availability of reproductive history of the control group and investigation of only a subset of the genetic variation within each gene.

Objective To assess the role of hormone receptor/binding protein variants in genetic predisposition to premature ovarian failure (POF). Design Case-control study. Setting Academic. Patient(s) Fifty-five POF patients, 107 control women from the general population, and 27 control women who had proven fertility after age 37. Intervention(s) None. Main Outcome Measure(s) Allele distributions in cases and controls were assessed for genetic association. Result(s) Allele distributions of polymorphisms at the androgen receptor (AR) gene, estrogen receptor β (ESR2) gene, sex hormone-binding globulin (SHBG ) gene, and FSH receptor (FSHR) gene did not differ between patients and controls. At a repeat in a promoter of the estrogen receptor α (ESR1) gene, POF patients had fewer ( P =.004 vs. combined controls). Genotypes consisting of two short alleles were found in 36.4% of control women but only 5.5% of POF patients ( P ESR1 repeat may confer risk for POF in a simple dominant manner in which carriers of a long repeat have a relative risk of 9.7 (95% CI=2.6 – 35.6). Conclusion(s) Polymorphisms at the ESR1 gene are associated with POF in this patient population, while those in AR , ESR2 , SHBG , and FSHR showed no association. Further studies are necessary to confirm these findings in larger patient samples and to identify the specific predisposing lesion.

Life history theory (LHT) predicts a trade-off between reproductive effort and the pace of biological aging. Energy invested in reproduction is not available for tissue maintenance, thus having more offspring is expected to lead to accelerated senescence. Studies conducted in a variety of non-human species are consistent with this LHT prediction. Here we investigate the relationship between the number of surviving children born to a woman and telomere length (TL, a marker of cellular aging) over 13 years in a group of 75 Kaqchikel Mayan women. Contrary to LHT's prediction, women who had fewer children exhibited shorter TLs than those who had more children (p = 0.045) after controlling for TL at the onset of the 13-year study period. An "ultimate" explanation for this apparently protective effect of having more children may lay with human's cooperative-breeding strategy. In a number of socio-economic and cultural contexts, having more chilren appears to be linked to an increase in social support for mothers (e.g., allomaternal care). Higher social support, has been argued to reduce the costs of further reproduction. Lower reproductive costs may make more metabolic energy available for tissue maintenance, resulting in a slower pace of cellular aging. At a "proximate" level, mechanisms involved may include the actions of the gonadal steroid estradiol, which increases dramatically during pregnancy. Estradiol is known to protect TL from the effects of oxidative stress as well as increase telomerase activity, an enzyme that maintains TL. Future research should explore the potential role of social support as well as that of estradiol and other potential biological pathways in the trade-offs between reproductive effort and the pace of cellular aging within and among human as well as in non-human populations.

Background The presence of unique copy number variations (CNVs) in miscarriages suggests that their integral genes have a role in maintaining early pregnancy. In our previous work, we identified 19 unique CNVs in ~40% of studied euploid miscarriages, which were predominantly familial in origin. In our current work, we assessed their relevance to miscarriage by expression analysis of 14 genes integral to CNVs in available miscarriage chorionic villi. As familial CNVs could cause miscarriage due to imprinting effect, we investigated the allelic expression of one of the genes (TIMP2) previously suggested to be maternally expressed in placenta and involved in placental remodelling and embryo development. Results Six out of fourteen genes had detectable expression in villi and for three genes the RNA and protein expression was altered due to maternal CNVs. These genes were integral to duplication on Xp22.2 (TRAPPC2 and OFD1) or disrupted by a duplication mapping to 17q25.3 (TIMP2). RNA and protein expression was increased for TRAPPC2 and OFD1 and reduced for TIMP2 in carrier miscarriages. The three genes have roles in processes important for pregnancy development such as extracellular matrix homeostasis (TIMP2 and TRAPPC2) and cilia function (OFD1). TIMP2 allelic expression was not affected by the CNV in miscarriages in comparison to control elective terminations. Conclusion We propose that functional studies of CNVs could help determine if and how the miscarriage CNVs affect the expression of integral genes. In case of parental CNVs, assessment of the function of their integral genes in parental reproductive tissues should be also considered in the future, especially if they affect processes relevant for pregnancy development and support. Electronic supplementary material The online version of this article (doi:10.1186/s13039-015-0109-8) contains supplementary material, which is available to authorized users.

Drug, hormonal, and nutritional exposures in pregnancy can cause birth defects, or have more subtle influences that may affect adult health through epigenetic developmental programming. Characterizing these epigenetic changes may provide valuable insight into the mechanism by which our health is influenced by the environment and provide biomarkers of exposure; however, currently there are few data from human studies directly addressing this. We review data on three types of exposures-nutritional, glucocorticoid, and endocrine-disrupting chemicals-and discuss how these may be associated with epigenetic changes in the fetus and the placenta, a key mediator of in utero environment. In addition, we outline some considerations for investigation of epigenetic effects in the fetus and placenta that are important to consider in the design of studies for the future.

Mutations within the coding regions of the synaptonemal complex gene SYCP3 have previously been reported in women with recurrent miscarriage. The present study found no mutations in any of the coding exons or the intron/exon boundaries among 50 recurrent miscarriage patients with at least one documented trisomic miscarriage, suggesting that mutations in SYCP3 do not contribute significantly to risk for recurrent miscarriage through maternal meiotic nondisjunction.

Previous studies have suggested an association between rates of biological and reproductive aging. Short telomere length is a marker for cellular aging and may occur more frequently in women with poor oocyte quality and a history of premature reproductive senescence. Telomere length, however, has been both positively and negatively associated with different measures of reproductive aging in a number of studies. In the present study, the investigators tested the hypothesis that women with a history of premature reproductive aging have a shorter average telomere length than women without such a history. Telomere length in peripheral blood leukocytes was measured in 2 groups of women with evidence of premature reproductive aging: Group 1 (n = 34) was comprised of women with idiopathic premature ovarian failure (POF) who experienced menopause before 40 years of age and group 2 (n = 95) contained women with a history of recurrent miscarriage (RM). The 2 control groups were healthy women of reproductive age from the general population (C1, n = 108) and women who had had a healthy pregnancy after 37 years of age (C2, n = 46). Telomere length was measured using telomere-specific quantitative PCR. The age-adjusted mean telomere length was shorter among women in the RM group than among those in both control groups (8.46 kb for RM vs. 8.92 kb for C1, [P = 0.0004] and 9.11 kb for C2, [P = 0.02]), respectively. However, this effect was not specifically confined to women known to have experienced trisomic pregnancies. Contrary to expectation, women in the POF group did not have a shorter age-adjusted mean telomere length than those in the controls: Telomere length in POF women was longer compared to C1 (9.58 vs. 8.92 kb, P = 0.01) and not significantly different than C2. These findings suggest that both the opposing association of RM and POF with telomere length, and lack of an association of trisomic pregnancy―as manifestations of different types of reproductive aging―may result from unique physiological factors. The results require confirmation in studies with larger and more defined patient populations.

Background Chromosomal abnormalities are a frequent cause of miscarriage; however the aetiology of karyotypically normal miscarriages remains unknown in many cases. Objective Our objective was to find new genetic causes of idiopathic miscarriages by evaluating miscarriage CNVs and sequence variants detected by whole exome sequencing (WES). Methods We used a) bioinformatics to assess genomic characteristics of CNVs (size, gene content) reported in 4 recent studies of 101 miscarriages; b) RNA and protein expression to determine the function of their integral genes in miscarriage cells and c) exome sequencing to look for mutations associated with recurrent pregnancy loss. Results The rare and common CNVs reported in 101 miscarriages were comparable in size (median size of ˜0.18 and 0.14 Mb, respectively), however, rare CNVs showed a trend towards higher gene density with 46 genes/Mb in rare and 21 genes/Mb in common CNVs (p = 0.06). For 3/14 genes integral to CNVs the RNA and protein expression was abnormal in miscarriages. These genes have a role in processes required for successful pregnancy development: TIMP2 and TRAPPC2 in tissue remodelling, cell adhesion and migration and OFD1 in cilia function. WES of recurrent miscarriages from 4 couples revealed 11 genes with rare and pathogenic compound heterozygous variants. It is of interest that 4/11 variants occurred in dynein genes, known to be involved in cilia function, and required for early organogenesis. Compound heterozygous mutations in one of these genes, DYNC2H1, were recently reported in a family with recurrent fetal loss. Conclusion Our study represents a comprehensive assessment of genomic changes in miscarriage and has the potential to identify novel genetic causes of pregnancy loss.asta.

About 1 in 100 women enter menopause before age 40; they are generally diagnosed as having premature ovarian failure (POF). The only gene that has consistently been associated with POF is FMR1 (Fragile X Mental Retardation 1), but the finding that only about 14% of women with familial POF carry an FMR1 premutation suggests that other genes may be associated with POF. This case-control study examined the role, if any, of hormone receptor or binding protein variants in genetic predisposition to POF. Fifty-five women diagnosed as having POF were compared with 107 control women from the general population and with 27 others who were proved fertile after age 37 years. Participants were from a predominantly Caucasian population in western Canada with significant Asian (mainly Chinese) admixture. Allele frequencies for repeat polymorphisms and single nucleotide polymorphisms were examined at several candidate gene sites. No significant differences were found between POF patients and control women in allele distributions of polymorphisms at the androgen receptor (AR) gene, estrogen receptor β (ESR2) gene, sex hormone-binding globulin (SHBG) gene, or follicle-stimulating hormone (FSH) receptor (FSHR) gene. In contrast, at a repeat in a promoter of the estrogen receptor a (ESR1) gene, women with POF had fewer (less than 18) short repeat alleles than control women (P = 0.004 versus the combined control groups). A genotype consisting of 2 short alleles was identified in 36.4% of control women but only 5.5% of those with POF (P < 0.0001 versus combined controls). Calculations showed that the ESR1 repeat may create a risk of POF in a simple dominant manner in which carriers of a long repeat have a relative risk of POF of 9.7, with a 95% confidence interval of 2.6-35.6.