Ectopic application of the repressive histone modification H3K9me2 establishes post-zygotic reproductive isolation in Arabidopsis thaliana.

Hybrid seed lethality as a consequence of interspecies or interploidy hybridizations is a major mechanism of reproductive isolation in plants. This mechanism is manifested in the endosperm, a dosage-sensitive tissue supporting embryo growth. Deregulated expression of imprinted genes such as ADMETOS (ADM) underpin the interploidy hybridization barrier in Arabidopsis thaliana; however, the mechanisms of their action remained unknown. In this study, we show that ADM interacts with the AT hook domain protein AHL10 and the SET domain-containing SU(VAR)3-9 homolog SUVH9 and ectopically recruits the heterochromatic mark H3K9me2 to AT-rich transposable elements (TEs), causing deregulated expression of neighboring genes. Several hybrid incompatibility genes identified in Drosophila encode for dosage-sensitive heterochromatin-interacting proteins, which has led to the suggestion that hybrid incompatibilities evolve as a consequence of interspecies divergence of selfish DNA elements and their regulation. Our data show that imbalance of dosage-sensitive chromatin regulators underpins the barrier to interploidy hybridization in Arabidopsis, suggesting that reproductive isolation as a consequence of epigenetic regulation of TEs is a conserved feature in animals and plants.

Differential CpG methylation at Nnat in the early establishment of beta cell heterogeneity.

AIMS/HYPOTHESIS: Beta cells within the pancreatic islet represent a heterogenous population wherein individual sub-groups of cells make distinct contributions to the overall control of insulin secretion. These include a subpopulation of highly connected 'hub' cells, important for the propagation of intercellular Ca2+ waves. Functional subpopulations have also been demonstrated in human beta cells, with an altered subtype distribution apparent in type 2 diabetes. At present, the molecular mechanisms through which beta cell hierarchy is established are poorly understood. Changes at the level of the epigenome provide one such possibility, which we explore here by focusing on the imprinted gene Nnat (encoding neuronatin [NNAT]), which is required for normal insulin synthesis and secretion. METHODS: Single-cell RNA-seq datasets were examined using Seurat 4.0 and ClusterProfiler running under R. Transgenic mice expressing enhanced GFP under the control of the Nnat enhancer/promoter regions were generated for FACS of beta cells and downstream analysis of CpG methylation by bisulphite sequencing and RNA-seq, respectively. Animals deleted for the de novo methyltransferase DNA methyltransferase 3 alpha (DNMT3A) from the pancreatic progenitor stage were used to explore control of promoter methylation. Proteomics was performed using affinity purification mass spectrometry and Ca2+ dynamics explored by rapid confocal imaging of Cal-520 AM and Cal-590 AM. Insulin secretion was measured using homogeneous time-resolved fluorescence imaging. RESULTS: Nnat mRNA was differentially expressed in a discrete beta cell population in a developmental stage- and DNA methylation (DNMT3A)-dependent manner. Thus, pseudo-time analysis of embryonic datasets demonstrated the early establishment of Nnat-positive and -negative subpopulations during embryogenesis. NNAT expression is also restricted to a subset of beta cells across the human islet that is maintained throughout adult life. NNAT+ beta cells also displayed a discrete transcriptome at adult stages, representing a subpopulation specialised for insulin production, and were diminished in db/db mice. 'Hub' cells were less abundant in the NNAT+ population, consistent with epigenetic control of this functional specialisation. CONCLUSIONS/INTERPRETATION: These findings demonstrate that differential DNA methylation at Nnat represents a novel means through which beta cell heterogeneity is established during development. We therefore hypothesise that changes in methylation at this locus may contribute to a loss of beta cell hierarchy and connectivity, potentially contributing to defective insulin secretion in some forms of diabetes. DATA AVAILABILITY: The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD048465.

The conservation of allelic DNA methylation and its relationship with imprinting in maize.

Genomic imprinting refers to allele-specific expression of genes depending on parental origin, and it is regulated by epigenetic modifications. Intraspecific allelic variation for imprinting has been detected; however, the intraspecific genome-wide allelic epigenetic variation in maize and its correlation with imprinting variants remain unclear. Here, three reciprocal hybrids were generated by crossing Zea mays inbred lines CAU5, B73, and Mo17 in order to examine the intraspecific conservation of the imprinted genes in the kernel. The majority of imprinted genes exhibited intraspecific conservation, and these genes also exhibited interspecific conservation (rice, sorghum, and Arabidopsis) and were enriched in some specific pathways. By comparing intraspecific allelic DNA methylation in the endosperm, we found that nearly 15% of DNA methylation existed as allelic variants. The intraspecific whole-genome correlation between DNA methylation and imprinted genes indicated that DNA methylation variants play an important role in imprinting variants. Disruption of two conserved imprinted genes using CRISPR/Cas9 editing resulted in a smaller kernel phenotype. Our results shed light on the intraspecific correlation of DNA methylation variants and variation for imprinting in maize, and show that imprinted genes play an important role in kernel development.

The imprinted lncRNA Peg13 regulates sexual preference and the sex-specific brain transcriptome in mice.

Mammalian genomes include many maternally and paternally imprinted genes. Most of these are also expressed in the brain, and several have been implicated in regulating specific behavioral traits. Here, we have used a knockout approach to study the function of Peg13, a gene that codes for a fast-evolving lncRNA (long noncoding RNA) and is part of a complex of imprinted genes on chromosome 15 in mice and chromosome 8 in humans. Mice lacking the 3' half of the transcript look morphologically wild-type but show distinct behavioral differences. They lose interest in the opposite sex, instead displaying a preference for wild-type animals of the same sex. Further, they show a higher level of anxiety, lowered activity and curiosity, and a deficiency in pup retrieval behavior. Brain RNA expression analysis reveals that genes involved in the serotonergic system, formation of glutamatergic synapses, olfactory processing, and estrogen signaling-as well as more than half of the other known imprinted genes-show significant expression changes in Peg13-deficient mice. Intriguingly, these pathways are differentially affected in the sexes, resulting in male and female brains of Peg13-deficient mice differing more from each other than those of wild-type mice. We conclude that Peg13 is part of a developmental pathway that regulates the neurobiology of social and sexual interactions.

Haploid androgenetic development of bovine embryos reveals imbalanced WNT signaling and impaired cell fate differentiation†.

Haploid embryos have contributed significantly to our understanding of the role of parental genomes in development and can be applied to important biotechnology for human and animal species. However, development to the blastocyst stage is severely hindered in bovine haploid androgenetic embryos (hAE). To further our understanding of such developmental arrest, we performed a comprehensive comparison of the transcriptomic profile of morula-stage embryos, which were validated by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) of transcripts associated with differentiation in haploid and biparental embryos. Among numerous disturbances, results showed that pluripotency pathways, especially the wingless-related integration site (WNT) signaling, were particularly unbalanced in hAE. Moreover, transcript levels of KLF4, NANOG, POU5F1, SOX2, CDX2, CTNNBL1, AXIN2, and GSK3B were noticeably altered in hAE, suggesting disturbance of pluripotency and canonical WNT pathways. To evaluate the role of WNT on hAE competence, we exposed early Day-5 morula stage embryos to the GSK3B inhibitor CHIR99021. Although no alterations were observed in pluripotency and WNT-related transcripts, exposure to CHIR99021 improved their ability to reach the blastocysts stage, confirming the importance of the WNT pathway in the developmental outcome of bovine hAE.

H19 Promoter DNA Methylation is Lower Among Early Abortion Patients Undergoing IVF Embryo Transfer.

BACKGROUND: H19 is the first long noncoding RNA (lncRNA) found to be associated with gene imprinting. It is highly expressed in the embryonic stage and may have important regulatory effects on human embryonic development. We investigated the differences between the levels of H19 promoter DNA methylation in the chorionic villi of patients who experienced spontaneous abortion (SA) following in vitro fertilization embryo transfer (IVF-ET) and those of patients with a normal early pregnancy (NEP). We also analyzed the associated DNA methyltransferase (DNMT) activity. METHODS: Chorionic villus tissue from patients with SA and NEP were collected. The DNA methylation levels of two CpG islands in the promoter region of the H19 gene in the two groups were detected by bisulfite sequencing, and the mRNA expression of DNMTs was analyzed by real-time polymerase chain reaction. RESULTS: The sample size of each group was 32, and there were no significant differences in baseline data, including age, parity, and body mass index, between the two groups. Among the 7 CpG islands measured, the methylation rates of 3 CpG islands (CpG 1, 6, and 7) were significantly lower in the SA group than in the NEP group (P < 0.01). The methylation levels of the other 4 CpG islands were not significantly different between the two groups. There were no differences in the expression of DNMT1 between the two groups (P > 0.05), but DNMT3a and DNMT3b RNA levels were significantly lower in SA group than in the NEP group (P < 0.01). CONCLUSIONS: The lower H19 promoter DNA methylation levels found in the chorionic villi of patients with SA patients following IVF-ET may be explained by decreased expression of DNMT3a and DNMT3b.

Differential Expression of RNAseq Imprinted Genes from Bovine Females Before and After Puberty.

The productivity of beef cows depends on early reproduction traits such as puberty and has an economic impact on the efficiency of production system. Imprinted genes modulate many important endocrine processes such as growth, the onset of puberty and maternal reproductive and behavior. The role of imprinted genes in puberty is a challenging subject since they show the reciprocal role of maternal and paternal genomes in progeny. Although, there are evidences of the involvement of imprint genes in puberty in human, the role of this type of genes in the onset of puberty in cattle has not been studied yet. Here we examined the expression of 27 imprinted genes in pre and post puberty in a bovine model to find differentially expressed imprinted genes in maternal-paternal purebreds and reciprocal crosses across eight tissues and discussed the task of these genes in this crucial process of development and in onset of puberty. DLK1 and MKRN3 that previously described as cause of the central precocious puberty (CPP) in human were differentially expressed in this study. Functional annotation analysis of differentially imprinted genes in different tissues showed significant biological processes of cellular response to growth factor stimulus, response to growth factor, response to parathyroid hormone, developmental growth and the importance of alternative splicing. The results of this study have implications in understanding the role of imprinted genes in the onset of puberty in cattle.

ZFP57 regulates DNA methylation of imprinted genes to facilitate embryonic development of somatic cell nuclear transfer embryos in Holstein cows.

Aberrant epigenetic nuclear reprogramming, especially imprinting pattern disorders, is one of the major causes of failure of clone development from somatic cell nuclear transfer (SCNT). Previous studies showed that ZFP57 is a key protein required for imprint maintenance after fertilization. In this study, we found that imprinting control regions in several imprinted genes were significantly hypomethylated in cloned embryos compared with in vitro fertilization embryos, indicating a loss of imprinted gene methylation. The ZFP57 expression was capable of maintaining the correct degree of methylation at several imprinting control regions and correcting abnormal hypomethylation. Moreover, we successfully obtained bovine fetal fibroblasts overexpressing ZFP57, which were used as donors for SCNT. Our results demonstrated that overexpression of ZFP57 increased total and trophectoderm cell numbers and the ratio of inner cell mass to total cells, reduced the apoptosis rate and significantly enhanced the development of SCNT blastocysts in vitro, ultimately achieving a degree of methylation similar to that in in vitro fertilization embryos. We concluded that overexpression of ZFP57 in donor cells provided an effective method for enhancing nuclear reprogramming and developmental potential in SCNT embryos. The ZFP57 protein played a key role in maintaining the methylation of imprinted genes during early embryonic development, which may be effective for enhanced SCNT in cattle.

Diagnostic Utility of TSSC3 and RB1 Immunohistochemistry in Hydatidiform Mole.

The general notion of complete hydatidiform moles is that most of them consist entirely of paternal DNA; hence, they do not express p57, a paternally imprinted gene. This forms the basis for the diagnosis of hydatidiform moles. There are about 38 paternally imprinted genes. The aim of this study is to determine whether other paternally imprinted genes could also assist in the diagnostic approach of hydatidiform moles. This study comprised of 29 complete moles, 15 partial moles and 17 non-molar abortuses. Immunohistochemical study using the antibodies of paternal-imprinted (RB1, TSSC3 and DOG1) and maternal-imprinted (DNMT1 and GATA3) genes were performed. The antibodies' immunoreactivity was evaluated on various placental cell types, namely cytotrophoblasts, syncytiotrophoblasts, villous stromal cells, extravillous intermediate trophoblasts and decidual cells. TSSC3 and RB1 expression were observed in all cases of partial moles and non-molar abortuses. In contrast, their expression in complete moles was identified in 31% (TSSC3) and 10.3% (RB1), respectively (p < 0.0001). DOG1 was consistently negative in all cell types in all cases. The expressions of maternally imprinted genes were seen in all cases, except for one case of complete mole where GATA3 was negative. Both TSSC3 and RB1 could serve as a useful adjunct to p57 for the discrimination of complete moles from partial moles and non-molar abortuses, especially in laboratories that lack comprehensive molecular service and in cases where p57 staining is equivocal.

Advanced molecular approaches in male infertility diagnosis†.

In the recent years a special attention has been given to a major health concern namely to male infertility, defined as the inability to conceive after 12 months of regular unprotected sexual intercourse, taken into account the statistics that highlight that sperm counts have dropped by 50-60% in recent decades. According to the WHO, infertility affects approximately 9% of couples globally, and the male factor is believed to be present in roughly 50% of cases, with exclusive responsibility in 30%. The aim of this article is to present an evidence-based approach for diagnosing male infertility that includes finding new solutions for diagnosis and critical outcomes, retrieving up-to-date studies and existing guidelines. The diverse factors that induce male infertility generated in a vast amount of data that needed to be analyzed by a clinician before a decision could be made for each individual. Modern medicine faces numerous obstacles as a result of the massive amount of data generated by the molecular biology discipline. To address complex clinical problems, vast data must be collected, analyzed, and used, which can be very challenging. The use of artificial intelligence (AI) methods to create a decision support system can help predict the diagnosis and guide treatment for infertile men, based on analysis of different data as environmental and lifestyle, clinical (sperm count, morphology, hormone testing, karyotype, etc.), and "omics" bigdata. Ultimately, the development of AI algorithms will assist clinicians in formulating diagnosis, making treatment decisions, and predicting outcomes for assisted reproduction techniques.

Does underlying infertility in natural conception modify the epigenetic control of imprinted genes and transposable elements in newborns?

RESEARCH QUESTION: Does the epigenetic control of imprinted genes and transposable elements at birth differ according to time to conception in natural conception and after intrauterine insemination (IUI)? DESIGN: A total of 144 singletons were included in four groups: 50 natural pregnancies obtained within 6 months after stopping contraception (group 1); 34 natural pregnancies with infertility period between 6 and 12 months (group 2); 36 pregnancies with an infertility period of more than 12 months (group 3) and 24 pregnancies obtained after IUI (group 4). RESULTS: The placental DNA methylation levels of H19/IGF2 and KCNQ1OT1 were lower in groups 2, 3 and 4 than in group 1 (P = 0.025 in the overall comparison). The DNA methylation rate for LINE-1 was higher in placentas from group 2 than in group 1 (P = 0.022). In cord blood, DNA methylation levels were not significantly different between groups except for H19/IGF2 for which the DNA methylation levels were higher in group 2 than in group 1 (H19/IGF2-seq1 and seq2: P = 0.023 and P = 0.002, respectively). In placenta tissue, compared with group 1, relative expression for SNRPN and for LINE-1 was significantly higher in group 2 (P = 0.002 and P < 0.001, respectively). The relative expression of KCNQ1 in placenta was lower in group 4 than in group 1 (P = 0.013). In cord blood, compared with group 1, the relative expression for H19 was significantly higher in group 3 (P = 0.026), and the relative expression of LINE-1 was higher in groups 2 and 3 and in group 4 (P < 0.001). CONCLUSIONS: Infertility itself, and not only ART techniques, could contribute to potential epigenetic risks for children.

Methylation profile of imprinted genes provides evidence for teratomatous origin of a subset of mucinous ovarian tumours.

Mucinous ovarian tumours are sometimes associated with mature teratomas. It is suggested that the mucinous tumours in this setting are derived from teratomas, but there remains the possibility of collision or metastasis from extra-ovarian sites. Because mature ovarian teratomas are considered to be parthenogenetic tumours that arise from a single oocyte/ovum, they have only a maternal genome and therefore show maternal genome imprinting. If mucinous ovarian tumours originate from teratomas, their genome imprinting is theoretically maternal. One of the most important mechanisms of genome imprinting is DNA methylation. In the present study, we analysed a total of 28 mucinous ovarian tumours (7 with teratomas, 21 without teratomas; 14 malignant, 14 borderline) to clarify the methylation profiles of their imprinted genes using methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) of 21 imprinting control regions (ICRs) of nine imprinted genes/gene clusters using formalin-fixed, paraffin-embedded samples. All cases lacked evidence of an extra-ovarian primary mucinous tumour. In all seven mucinous tumours with teratomas, the overall methylation profile of mucinous tumours was comparable to that of teratomas, although some ICRs showed aberrant methylation. In contrast, all but one of the mucinous tumours without teratomas showed somatic or irregular methylation patterns. Morphologically, there was little teratomatous tissue in some mucinous tumours carrying teratoma-type methylation profiles, suggesting that mucinous tumours overwhelmed ancestral teratomas. In conclusion, the methylation profile of imprinted genes provides evidence that a subset of mucinous ovarian tumours originated from mature teratomas. Genome imprinting-based analysis is a promising strategy to verify the teratomatous origin of human tumours. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Effects of oocyte vitrification on gene expression in the liver and kidney tissues of adult offspring.

Oocyte vitrification is an important assisted reproductive technology (ART) that preserves the fertility of unmarried patients with malignant tumors, and promotes the development of the oocyte donation program. In recent years, the effects of ART, including the vitrification of oocytes and embryos on the health of offspring, have attracted much attention; however, it is difficult to conduct long-term follow-up and biochemical evaluation in humans. In this study, we detected the effect of oocyte vitrification on gene expression in the organs of adult mice offspring by RNA sequencing for the first time. Our results showed that only a small amount of gene expression was significantly affected. Seven genes (Tpm3, Hspe1-rs1, Ntrk2, Cyp4a31, Asic5, Cyp4a14, Retsat) were abnormally expressed in the liver, and ten genes (Lbp, Hspe1-rs1, Prxl2b, Pfn3, Gm9008, Bglap3, Col8a1, Hmgcr, Ero1lb, Ifi44l) were abnormal in the kidney. Several genes were related to metabolism and disease occurrence in the liver or kidney. Besides, we paid special attention to the expression of known imprinted genes and DNA methylation-related genes in adult organs, which are susceptible to oocyte cryopreservation in the preimplantation stage. As a result, some of these transcripts were detected in adult organs, but they were not affected by oocyte vitrification. In conclusion, we first report that oocyte vitrification did not significantly change the global gene expression in offspring organs; nonetheless, it can still influence the transcription of a few functional genes. The potential adverse effects caused by oocyte vitrification need attention and further study.

Conservation Study of Imprinted Genes in Maize Triparental Heterozygotic Kernels.

Genomic imprinting is a classic epigenetic phenomenon related to the uniparental expression of genes. Imprinting variability exists in seeds and can contribute to observed parent-of-origin effects on seed development. Here, we conducted allelic expression of the embryo and endosperm from four crosses at 11 days after pollination (DAP). First, the F1 progeny of B73(♀) × Mo17(♂) and the inducer line CAU5 were used as parents to obtain reciprocal crosses of BM-C/C-BM. Additionally, the F1 progeny of Mo17(♀) × B73(♂) and CAU5 were used as parents to obtain reciprocal crosses of MB-C/C-MB. In total, 192 and 181 imprinted genes were identified in the BM-C/C-BM and MB-C/C-MB crosses, respectively. Then, by comparing the allelic expression of these imprinted genes in the reciprocal crosses of B73 and CAU5 (BC/CB), fifty-one Mo17-added non-conserved genes were identified as exhibiting imprinting variability. Fifty-one B73-added non-conserved genes were also identified by comparing the allelic expression of imprinted genes identified in BM-C/C-BM, MB-C/C-MB and MC/CM crosses. Specific Gene Ontology (GO) terms were not enriched in B73-added/Mo17-added non-conserved genes. Interestingly, the imprinting status of these genes was less conserved across other species. The cis-element distribution, tissue expression and subcellular location were similar between the B73-added/Mo17-added conserved and B73-added/Mo17-added non-conserved imprinted genes. Finally, genotypic and phenotypic analysis of one non-conserved gene showed that the mutation and overexpression of this gene may affect embryo and kernel size, which indicates that these non-conserved genes may also play an important role in kernel development. The findings of this study will be helpful for elucidating the imprinting mechanism of genes involved in maize kernel development.

Transgenerational male reproductive effect of prenatal arsenic exposure: abnormal spermatogenesis with Igf2/H19 epigenetic alteration in CD1 mouse.

Developmental exposure to environmental toxicants can induce transgenerational reproductive disease phenotypes through epigenetic mechanisms. We treated pregnant CD-1 (F0) mice with drinking water containing sodium arsenite (85 ppm) from days 8 to 18 of gestation. Male offspring were bred with untreated female mice until the F3 generation was produced. Our results revealed that F0 transient exposure to arsenic can cause decreased sperm quality and histological abnormalities in the F1 and F3. The overall methylation status of Igf2 DMR2 and H19 DMR was significantly lower in the arsenic-exposed group than that of the control group in both F1 and F3. The relative mRNA expression levels of Igf2 and H19 in arsenic-exposed males were significantly increased in both F1 and F3. This study indicates that ancestral exposure to arsenic may result in transgenerational inheritance of an impaired spermatogenesis phenotyping involving both epigenetic alterations and the abnormal expression of Igf2 and H19.

Alteration of imprinted genes and offspring organ development caused by environmental factors.

Imprinted genes are a special subset of about 100 genes, which are mainly expressed in the form of parental monoallelic genes, and play important roles in the growth and development of embryos. In recent years, it has been found that epigenetic modification of imprinted genes induced by environmental factors can cause fetal multi-organ dysplasia and even susceptibility to multiple diseases in adulthood, which also exhibit multi-generational inheritance. In this review, we summarize the effects of expression changes of imprinted genes on ontogenetic development and organ functions in late stage of life, and propose that abnormal epigenetic modification and expression of imprinted genes caused by environmental deleterious factors are important mechanisms for explaining the multi-organ dysplasia in offspring. Such mechanisms are greatly significant for understanding the phenotypic changes caused by alteration of imprinted gene expression during ontogeny and exploring early prevention and treatment strategies of diseases.

Loss of ZNF215 imprinting is associated with poor five-year survival in patients with cytogenetically abnormal-acute myeloid leukemia.

Genomic imprinting is a form of epigenetic regulation and imprinted genes are silenced in a parental-specific manner. Imprinting is associated with various human diseases and cancers, but its roles in leukemogenesis remains elusive. In this study, the expression of a panel of 16 human imprinted genes was investigated using real-time quantitative polymerase chain reaction and 8 of them were further validated in 114 patients newly diagnosed with cytogenetically abnormal-acute myeloid leukemia (CA-AML) and 85 healthy subjects. Our results demonstrated upregulated expression of 8 imprinted genes (C15orf2, COPG2, H19, IGF2, PEG3-AS1, PRIM2, SLC22A3 and ZNF215) was observed in patients with CA-AML (p < 0.001). Patients' survival days were negatively correlated with the expression levels of H19 (p = 0.024), PGE3-AS1 (p = 0.038), and ZNF215 (p = 0.012). Multivariate logistic regression analysis further revealed the expression level ZNF215 can be used as a predictor for five-year survival for patients with CA-AML (p = 0.009) with a hazard ratio of 0.870 (95.0% confident interval: 0.784-0.965). Our results demonstrated that loss of imprinting of imprinted genes is critical for the leukemogenesis of AML under CA condition, and loss of ZNF215 imprinting is associated with poor five-year survival of patients with CA-AML.

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

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.

Do assisted reproductive technologies and in vitro embryo culture influence the epigenetic control of imprinted genes and transposable elements in children?

STUDY QUESTION: Do assisted reproductive technologies (ART) and in vitro embryo culture influence the epigenetic control of imprinted genes (IGs) and transposable elements (TEs) in children? SUMMARY ANSWER: Significant differences in the DNA methylation of IGs or transposon families were reported between ART and naturally conceived children, but there was no difference between culture media. WHAT IS KNOWN ALREADY: There is concern that ART may play a role in increasing the incidence of adverse health outcomes in children, probably through epigenetic mechanisms. It is crucial to assess epigenetic control, especially following non-optimal in vitro culture conditions and to compare epigenetic analyses from ART-conceived and naturally conceived children. STUDY DESIGN, SIZE, DURATION: This follow-up study was based on an earlier randomized study comparing in vitro fertilization outcomes following the use of two distinct culture media. We compared the epigenetic profiles of children from the initial randomized study according to the mode of conception [i.e. ART singletons compared with those of a cohort of naturally conceived singleton children (CTL)], the type of embryo culture medium used [global medium (LifeGlobal) and single step medium (Irvine Scientific)] and the mode of in vitro fertilization (i.e. IVF versus ICSI). PARTICIPANTS/MATERIALS, SETTING, METHODS: A total of 57 buccal smears were collected from 7- to 8-year-old children. The DNA methylation profiles of four differentially methylated regions (DMRs) of IGs (H19/IGF2: IG-DMR, KCNQ1OT1: TSS-DMR, SNURF: TSS-DMR, and PEG3: TSS-DMR) and two TEs (AluYa5 and LINE-1) were first assessed by pyrosequencing. We further explored IGs and TEs' methylation changes through methylation array (Human MethylationEPIC BeadChip referred as EPIC array, Illumina). MAIN RESULTS AND THE ROLE OF CHANCE: Changes in the IGs' DNA methylation levels were found in ART children compared to controls. DNA methylation levels of H19/IGF2 DMR were significantly lower in ART children than in CTL children [52% versus 58%, P = 0.003, false discovery rate (FDR) P = 0.018] while a significantly higher methylation rate was observed for the PEG3 DMR (51% versus 48%, P = 0.007, FDR P = 0.021). However, no differences were found between the culture media. After observing these targeted modifications, analyses were performed at wider scale. Again, no differences were detected according to the culture media, but imprinted-related DMRs overlapping promoter region near the genes major for the development (MEG3, BLCAP, and DLX5) were detected between the ART and CTL children. LIMITATIONS, REASONS FOR CAUTION: The sample size could seem relatively small, but the high consistency of our results was ensured by the homogeneity of the cohort from the initial randomized study, the standardized laboratory techniques and the robust statistical analyses accounting for multiple testing. WIDER IMPLICATIONS OF THE FINDINGS: Although this study did not report DNA methylation differences depending on the culture medium, it sheds light on epigenetic changes that could be observed in some children conceived by ART as compared to CTL children. The clinical relevance of such differences remains largely unknown, and it is still unclear whether such changes are due to some specific ART procedures and/or to parental infertility. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by funding from the Agence Nationale pour la Recherche ('CARE'-ANR JCJC 2017). The authors have no conflicts of interest. TRIAL REGISTRATION NUMBER: Not concerned.

Tet3 regulates cellular identity and DNA methylation in neural progenitor cells.

TET enzymes oxidize 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (5hmC), a process thought to be intermediary in an active DNA demethylation mechanism. Notably, 5hmC is highly abundant in the brain and in neuronal cells. Here, we interrogated the function of Tet3 in neural precursor cells (NPCs), using a stable and inducible knockdown system and an in vitro neural differentiation protocol. We show that Tet3 is upregulated during neural differentiation, whereas Tet1 is downregulated. Surprisingly, Tet3 knockdown led to a de-repression of pluripotency-associated genes such as Oct4, Nanog or Tcl1, with concomitant hypomethylation. Moreover, in Tet3 knockdown NPCs, we observed the appearance of OCT4-positive cells forming cellular aggregates, suggesting de-differentiation of the cells. Notably, Tet3 KD led to a genome-scale loss of DNA methylation and hypermethylation of a smaller number of CpGs that are located at neurogenesis-related genes and at imprinting control regions (ICRs) of Peg10, Zrsr1 and Mcts2 imprinted genes. Overall, our results suggest that TET3 is necessary to maintain silencing of pluripotency genes and consequently neural stem cell identity, possibly through regulation of DNA methylation levels in neural precursor cells.