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Introduction: Disturbed paternal epigenetic status of imprinted genes has been observed in infertility and recurrent spontaneous abortions. Shallow placentation has been associated with early-onset preeclampsia. Hence, the present study aimed to investigate the methylation patterns of imprinted genes involved in placental development, in the spermatozoa of partners of women experiencing preeclampsia., Methods: The study involved recruitment of couples into preeclampsia (n = 14) and control (n = 25) groups. Methylation analysis of imprinted gene differentially methylated regions (DMRs) and LINE1 repetitive element was carried out by pyrosequencing in the spermatozoa and placental villi. Global 5 mC levels in the spermatozoa were measured through ELISA. Expression of imprinted genes was quantified in the placental villi by real time qPCR. Association of birth weight with DNA methylation and gene expression was assessed., Results: KvDMR, PEG3 DMR, PEG10 DMR and DLK1-GTL2 IG-DMR were differentially methylated in the spermatozoa and placental villi of preeclampsia group. Global 5 mC content and LINE1 methylation levels did not differ between the spermatozoa of the two groups. Increased transcript levels of PEG3, IGF2, DLK1, PHLDA2 and CDKN1C were observed in the preeclamptic placental villi. Birth weight showed significant association with KvDMR, PEG10 DMR, DLK1-GTL2 IG-DMR and LINE1 methylation levels in the spermatozoa. DLK1 expression levels showed a negative association with birth weight., Discussion: The study highlighted the paternal contribution to early-onset preeclampsia, in the form of disrupted sperm DNA methylation patterns at imprinted gene loci. These loci, after further evaluation in future studies, could serve as sperm-based preeclampsia predictive markers, for couples planning pregnancy., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this article., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Context: Defects in MKRN3, DLK1, KISS1, and KISS1R and some disorders, such as Temple syndrome (TS14), cause central precocious puberty (CPP). Recently, pathogenic variants (PVs) in MECP2 have been reported to be associated with CPP., Objective: We aimed to clarify the contribution of (epi)genetic abnormalities to CPP and clinical and hormonal features in each etiology., Subjects and Methods: We conducted targeted sequencing for MKRN3, DLK1, MECP2, KISS1, and KISS1R and methylation analysis for screening of imprinting disorders such as TS14 associated with CPP in 90 patients with CPP (no history of brain injuries and negative brain MRI) and collected their clinical and laboratory data. We measured serum DLK1 levels in three patients with TS14 and serum MKRN3 levels in two patients with MKRN3 genetic defects, together with some etiology-unknown patients with CPP and controls., Results: We detected eight patients with TS14 (six, epimutation; one, mosaic maternal uniparental disomy chromosome 14; one, microdeletion) and three patients with MKRN3 genetic defects (one, PV; one, 13-bp deletion in the 5'-untranslated region (5'-UTR); one, microdeletion) with family histories of paternal early puberty. There were no patients with PVs identified in MECP2, KISS1, or KISS1R. We confirmed low serum MKRN3 level in the patient with a deletion in 5'-UTR. The median height at initial evaluation of TS14 patients was lower than that of all patients. Six patients with TS14 were born small for gestational age (SGA)., Conclusion: (Epi)genetic causes were identified in 12.2% of patients with CPP at our center. For patients with CPP born SGA or together with family histories of paternal early puberty, (epi)genetic testing for TS14 and MKRN3 genetic defects should be considered. (271/250)., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Endocrine Society. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com. See the journal About page for additional terms.)

Background: Imprinted genes play important functions in placentation and pregnancy; however, research on their roles in different placental diseases is limited. It is believed that epigenetic alterations, such as DNA methylation, of placental imprinting genes may contribute to the different pathological features of severe placental diseases, such as pre-eclampsia (PE) and placenta accreta spectrum disorders (PAS)., Results: In this study, we conducted a comparative analysis of the methylation and expression of placental imprinted genes between PE and PAS using bisulfite sequencing polymerase chain reaction (PCR) and quantitative PCR, respectively. Additionally, we assessed oxidative damage of placental DNA by determining 8-hydroxy-2'-deoxyguanosine levels and fetal growth by determining insulin-like growth factor 2 (IGF2) and cortisol levels in the umbilical cord blood using enzyme-linked immunosorbent assay. Our results indicated that methylation and expression of potassium voltage-gated channel subfamily Q member 1, GNAS complex locus, mesoderm specific transcript, and IGF2 were significantly altered in both PE and PAS placentas. Additionally, our results revealed that the maternal imprinted genes were significantly over-expressed in PE and significantly under-expressed in PAS compared with a normal pregnancy. Moreover, DNA oxidative damage was elevated and positively correlated with IGF2 DNA methylation in both PE and PAS placentas, and cortisol and IGF2 levels were significantly decreased in PE and PAS., Conclusions: This study suggested that DNA methylation and expression of imprinted genes are aberrant in both PE and PAS placentas and that PE and PAS have different methylation profiles, which may be linked to their unique pathogenesis., (© 2024. The Author(s).)

Adverse childhood experiences (ACEs) contribute to numerous negative health outcomes across the life course and across generations. Here, we extend prior work by examining the association of maternal ACEs, and their interaction with financial stress and discrimination, with methylation status within eight differentially methylated regions (DMRs) in imprinted domains in newborns. ACEs, financial stress during pregnancy, and experience of discrimination were self-reported among 232 pregnant women. DNA methylation was assessed at PEG10 / SGCE, NNAT, IGF2, H19, PLAGL1 , PEG3 , MEG3-IG , and DLK1/MEG3 regulatory sequences using pyrosequencing. Using multivariable linear regression models, we found evidence to suggest that financial stress was associated with hypermethylation of MEG3-IG in non-Hispanic White newborns; discrimination was associated with hypermethylation of IGF2 and NNAT in Hispanic newborns, and with hypomethylation of PEG3 in non-Hispanic Black newborns. We also found evidence that maternal ACEs interacted with discrimination to predict offspring PLAGL1 altered DMR methylation, in addition to interactions between maternal ACEs score and discrimination predicting H19 and SGCE/PEG10 altered methylation in non-Hispanic White newborns. However, these interactions were not statistically significant after multiple testing corrections. Findings from this study suggest that maternal ACEs, discrimination, and financial stress are associated with newborn aberrant methylation in imprinted gene regions.

Background: NLRP2 belongs to the subcortical maternal complex (SCMC) of mammalian oocytes and preimplantation embryos. This multiprotein complex, encoded by maternal-effect genes, plays a pivotal role in the zygote-to-embryo transition, early embryogenesis, and epigenetic (re)programming. The maternal inactivation of genes encoding SCMC proteins has been linked to infertility and subfertility in mice and humans. However, the underlying molecular mechanisms for the diverse functions of the SCMC, particularly how this cytoplasmic structure influences DNA methylation, which is a nuclear process, are not fully understood., Results: We undertook joint transcriptome and DNA methylome profiling of pre-ovulatory germinal-vesicle oocytes from Nlrp2 -null, heterozygous (Het), and wild-type (WT) female mice. We identified numerous differentially expressed genes (DEGs) in Het and Nlrp2 -null when compared to WT oocytes. The genes for several crucial factors involved in oocyte transcriptome modulation and epigenetic reprogramming, such as DNMT1, UHRF1, KDM1B and ZFP57 were overexpressed in Het and Nlrp2 -null oocytes. Absence or reduction of Nlrp2 , did not alter the distinctive global DNA methylation landscape of oocytes, including the bimodal pattern of the oocyte methylome. Additionally, although the methylation profile of germline differentially methylated regions (gDMRs) of imprinted genes was preserved in oocytes of Het and Nlrp2 -null mice, we found altered methylation in oocytes of both genotypes at a small percentage of the oocyte-characteristic hyper- and hypomethylated domains. Through a tiling approach, we identified specific DNA methylation differences between the genotypes, with approximately 1.3% of examined tiles exhibiting differential methylation in Het and Nlrp2 -null compared to WT oocytes., Conclusions: Surprisingly, considering the well-known correlation between transcription and DNA methylation in developing oocytes, we observed no correlation between gene expression differences and gene-body DNA methylation differences in Nlrp2 -null versus WT oocytes or Het versus WT oocytes. We therefore conclude that post-transcriptional changes in the stability of transcripts rather than altered transcription is primarily responsible for transcriptome differences in Nlrp2 -null and Het oocytes., Competing Interests: Competing interests: The authors declare that they have no competing interests.

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 Ca 2+ 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 Ca 2+ 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., (© 2024. The Author(s).)

Introduction: Imprinted genes are expressed from one parental allele as a consequence of epigenetic processes initiated in the germline. Consequently, their ability to influence phenotype depends on their parent-of-origin. Recent research suggests that the sex of the individual expressing the imprinted gene is also important. We have previously reported that genetically wildtype (WT) dams carrying and caring for pups mutant for PEG3 exhibit anxiety-like behaviours and their mutant pups show a reduction in ultrasonic vocalisation when separated from their mothers. Sex-specificity was not examined., Methods: WT female mice were mated with WT, heterozygous Peg3 -/+ or homozygous Peg3 -/- studs to generate all WT (control), 50:50 mixed or 100% mutant litters, respectively, followed by behavioural assessment of both dams and their pups., Results: We reproduced our original finding that WT dams carrying and caring for 100% mutant litters exhibit postpartum anxiety-like symptoms and delayed pup retrieval. Additionally, these WT dams were found to allocate less time to pup-directed care behaviours relative to controls. Male Peg3 -deficient pups demonstrated significantly reduced vocalisation with a more subtle communication deficit in females. Postweaning, male mutants exhibited deficits across a number of key social behaviours as did WT males sharing their environment with mutants. Only modest variations in social behaviour were detected in experimental females., Discussion: We have experimentally demonstrated that Peg3 deficiency confined to the offspring causes anxiety in mouse mothers and atypical behaviour including deficits in communication in their male offspring. A male-specific reduction in expression PEG3 in the fetally-derived placenta has previously been associated with maternal depression in human pregnancy. Maternal mood disorders such as depression and anxiety are associated with delays in language development and neuroatypical behaviour more common in sons. Peg3 deficiency could drive the association of maternal and offspring behavioural disorders reported in humans., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Tyson, Harrison, Higgs, Isles and John.)

Objective: To investigate alterations of the global DNA methylation profile in placenta, cord blood, and neonatal buccal smears in infants conceived using in vitro maturation (IVM) with a prematuration step (capacitation-IVM [CAPA-IVM]) vs. in vitro fertilization (IVF)., Design: Analysis of data from the offspring of participants in a randomized controlled trial., Setting: Private clinic., Patients: Forty-six women with polycystic ovary syndrome and/or high antral follicle count and their offspring (58 newborns)., Intervention(s): Women with polycystic ovary syndrome and/or a high antral follicle count participating in the clinical trial were randomized to undergo CAPA-IVM or conventional IVF., Main Outcome Measure(s): At delivery, biological samples including cord blood, placental tissue, and a neonatal buccal smear were collected. Genome-wide DNA methylation was determined using the Illumina Infinium MethylationEPIC BeadChip. Variability in methylation was also considered, and mean variances for the two treatment categories were compared., Results: In neonatal buccal smears, there were no significant differences between the CAPA-IVM and conventional IVF groups on the basis of the CpG probe after linear regression analysis using a significant cut-off of false-discovery rate <0.05 and |Δβ|≥0.05. In cord blood, only one CpG site showed a significant gain of methylation in the CAPA-IVM group. In the placenta, CAPA-IVM was significantly associated with changes in methylation at five CpG sites. Significantly more variable DNA methylation was found in five probes in the placenta, 54 in cord blood, and two in buccal smears after IVM of oocytes. In cord blood samples, 20 CpG sites had more variable methylation in the conventional IVF vs. IVM group. Isolated CpG sites showing differences in methylation in cord blood were not associated with changes in gene expression of the overlapping genes., Conclusion(s): Capacitation-IVM appeared to be associated with only a small amount of epigenetic variation in cord blood, placental tissue, and neonate buccal smears., Clinical Trial Registration Number: NCT03405701 (www., Clinicaltrials: gov)., (Copyright © 2023 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

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 Author(s) 2023. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

DNA methylation is influenced by various exogenous factors such as nutrition, temperature, toxicants, and stress. Bulls from the Pacific Northwest region of the United States and other northern areas are exposed to extreme cold temperatures during winter. However, the effects of cold exposure on the methylation patterns of bovine sperm remain unclear. To address, DNA methylation profiles of sperm collected during late spring and winter from the same bulls were analyzed using whole genome bisulfite sequencing (WGBS). Bismark (0.22.3) were used for mapping the WGBS reads and R Bioconductor package DSS was used for differential methylation analysis. Cold exposure induced 3,163 differentially methylated cytosines (DMCs) with methylation difference ≥10% and a q-value < 0.05. We identified 438 differentially methylated regions (DMRs) with q-value < 0.05, which overlapped with 186 unique genes. We also identified eight unique differentially methylated genes (DMGs) ( Pax6, Macf1, Mest, Ubqln1, Smg9, Ctnnb1, Lsm4, and Peg10 ) involved in embryonic development, and nine unique DMGs ( Prmt6, Nipal1, C21h15orf40, Slc37a3, Fam210a, Raly, Rgs3, Lmbr1, and Gan) involved in osteogenesis. Peg10 and Mest , two paternally expressed imprinted genes, exhibited >50% higher methylation. The differential methylation patterns of six distinct DMRs: Peg10, Smg9 and Mest related to embryonic development and Lmbr1, C21h15orf40 and Prtm6 related to osteogenesis, were assessed by methylation-specific PCR (MS-PCR), which confirmed the existence of variable methylation patterns in those locations across the two seasons. In summary, cold exposure induces differential DNA methylation patterns in genes that appear to affect embryonic development and osteogenesis in the offspring. Our findings suggest the importance of replicating the results of the current study with a larger sample size and exploring the potential of these changes in affecting offspring development., Competing Interests: Author MM was employed by ABS Global. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Hossain, Gao, Hatfield, de Avila, McClure and Du.)

DNA methylation is closely related to folate levels and acts as a mechanism linking developmental disorders to chronic diseases. Folic acid supplementation can impact DNA methylation levels of imprinted genes crucial for neonatal development. Imprinted genes are vital for regulating embryonic and postnatal fetal growth. This review summarizes imprinted genes, DNA methylation, folic acid's influence on growth and development and their correlation. It aims to provide a comprehensive overview of research advancements on imprinted genes, DNA methylation and folic acid regulation concerning growth and development.

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., (© The Author(s) 2023. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Previously, we showed that DNA methylation defects in spermatozoa from male partners of couples undergoing recurrent pregnancy loss (RPL) could be a contributing paternal factor. In the present study, we aimed to determine whether the methylation levels of selected imprinted genes can be used as diagnostic markers to identify epigenetically abnormal spermatozoa sample in these cases. The methylation levels of selected imprinted genes in spermatozoa, which were previously found to be differentially methylated, were combined into a probability score (between 0-1) using multiple logistic regression. Different combinations of these genes were investigated using Receiver Operating Characteristic analysis, and the threshold values were experimentally validated in an independent cohort of 38 control and 45 RPL spermatozoa samples. Among the different combinations investigated, a combination of five imprinted genes comprising IGF2-H19 DMR, IG-DMR, ZAC, KvDMR, and PEG3 (AUC = 0.88) with a threshold value of 0.61 was selected with a specificity of 90.41% and sensitivity of 70%. The results from the validation study indicated that 97% of the control samples had probability scores below this threshold, whereas 40% of the RPL samples were above this threshold with a post-hoc power of 97.8%. Thus, this combination can correctly classify control samples and potentially identify epigenetically abnormal spermatozoa samples in the male partners of couples undergoing RPL. We propose that the combined DNA methylation levels of these imprinted genes can be used as a diagnostic tool to identify spermatozoa samples with epigenetic defects which could contribute to the pathophysiology of RPL and the couple could be counselled appropriately.

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., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Cadmium (Cd) is a toxic metal ubiquitous in the environment. In utero , Cd is inefficiently transported to the foetus but causes foetal growth restriction (FGR), likely through impairment of the placenta where Cd accumulates. However, the underlying molecular mechanisms are poorly understood. Cd can modulate the expression of imprinted genes, defined by their transcription from one parental allele, which play critical roles in placental and foetal growth. The expression of imprinted genes is governed by DNA methylation at Imprinting Control Regions (ICRs), which are susceptible to environmental perturbation. The imprinted gene Cdkn1c/CDKN1C is a major regulator of placental development, is implicated in FGR, and shows increased expression in response to Cd exposure in mice. Here, we use a hybrid mouse model of in utero Cd exposure to determine if the increase in placental Cdkn1c expression is caused by changes to ICR DNA methylation and loss of imprinting (LOI). Consistent with prior studies, Cd causes FGR and impacts placental structure and Cdkn1c expression at late gestation. Using polymorphisms to distinguish parental alleles, we demonstrate that increased Cdkn1c expression is not driven by changes to DNA methylation or LOI. We show that Cdkn1c is expressed primarily in the placental labyrinth which is proportionally increased in size in response to Cd. We conclude that the Cd-associated increase in Cdkn1c expression can be fully explained by alterations to placental structure. These results have implications for understanding mechanisms of Cd-induced placental dysfunction and, more broadly, for the study of FGR associated with increased Cdkn1c/CDKN1C expression.

A previous study reported positive associations of maternal urinary concentrations of triclosan, a synthetic phenol with widespread exposure in the general population, with placental DNA methylation of male fetuses. Given the high number of comparisons performed in -omic research, further studies were needed to validate and extend on these findings. Using a cohort of male and female fetuses with repeated maternal urine samples to assess exposure, we studied the associations between triclosan and placental DNA methylation. We assessed triclosan concentrations in two pools of 21 urine samples collected among 395 women from the SEPAGES cohort. We used Infinium Methylation EPIC arrays to measure DNA methylation in placental biopsies collected at delivery. We performed a candidate study restricted to a set of candidate CpGs (n = 500) identified in a previous work as well as an exploratory epigenome-wide association study to investigate the associations between triclosan and differentially methylated probes and regions. Analyses were conducted on the whole population and stratified by child's sex. Mediation analysis was performed to test whether heterogeneity of placental tissue may mediate the observed associations. In the candidate approach, we confirmed 18 triclosan-associated genes when both sexes were considered. After stratification for child's sex, triclosan was associated with 72 genes in females and three in males. Most of the associations were positive and several CpGs mapped to imprinted genes: FBRSL1, KCNQ1, RHOBTB3, and SMOC1. A mediation effect by placental tissue heterogeneity was identified for most of the observed associations. In the exploratory analysis, we identified a few isolated associations in the sex-stratified analysis. In line with a previous study on male placentas, our approach revealed several positive associations between triclosan exposure and placental DNA methylation. Several identified loci mapped to imprinted genes., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Genomic imprinting, an epigenetic mechanism that regulates gene expression from parental chromosomes, holds substantial relevance in multiple cancers, including hematopoietic malignancies. In the present study, the expression of a panel of 16 human imprinted genes in bone marrow samples from 64 patients newly diagnosed with cytogenetically normal-acute myeloid leukemia (CN-AML) were examined alongside peripheral blood samples from 85 healthy subjects. The validated findings of the present study revealed significant upregulation of seven genes [COPI coat complex subunit gamma 2 ( COPG2 ), H19 imprinted maternally expressed transcript ( H19 ), insulin like growth factor 2 ( IGF2 ), PEG3 antisense RNA 1 ( PEG3-AS1 ), DNA primase subunit 2 ( PRIM2 ), solute carrier family 22 member 3 SLC22A3 and Zinc finger protein 215 ( ZNF215 )] in patients with CN-AML (P<0.001). Notably, the expression level of H19 exhibited an inverse association with the survival duration of the patients (P=0.018), establishing it as a predictive marker for two- and five-year survival in patients with CN-AML. Kaplan-Meier analysis demonstrated that patients with lower H19 expression had superior two- and five-year survival rates compared with those with higher H19 expression. The results of the present study highlighted the association between loss of imprinting and leukemogenesis in CN-AML, underscoring the significance of H19 imprinting loss as a prognostic indicator for unfavorable two- and five-year survival in CN-AML patients., Competing Interests: The authors declare that they have no competing interests., (Copyright: © Yang et al.)

With the increasing maturity and widespread application of assisted reproductive technology (ART), more attention has been paid to the health outcomes of offspring following ART. It is well established that children born from ART treatment are at an increased risk of imprinting errors and imprinting disorders. The disturbances of genetic imprinting are attributed to the overlap of ART procedures and important epigenetic reprogramming events during the development of gametes and early embryos, but the detailed mechanisms are hitherto obscure. In this review, we summarized the DNA methylation-dependent and independent mechanisms that control the dynamic epigenetic regulation of imprinted genes throughout the life cycle of a mammal, including erasure, establishment, and maintenance. In addition, we systematically described the dysregulation of imprinted genes in embryos conceived through ART and discussed the corresponding underlying mechanisms according to findings in animal models. This work is conducive to evaluating and improving the safety of ART.

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.

Immature teratomas are a subset of ovarian teratomas, and the pathogenic relationship between mature and immature ovarian teratomas is unclear. Mature ovarian teratomas are parthenogenetic tumors that arise from a single oocyte/ovum, whereas the origin of immature ovarian teratomas has not been extensively investigated. Since parthenogenetic tumors contain only maternal genomes, genome imprinting in these tumors usually follows a maternal pattern. DNA methylation is among the most important mechanisms of genome imprinting. Therefore, we analyzed the methylation profile of imprinted genes by performing methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) of 25 imprinting control regions (ICRs) in 10 imprinted genes/gene clusters from formalin-fixed, paraffin-embedded samples obtained from 4 immature ovarian teratomas, 8 mature ovarian teratomas, and 4 ovarian yolk sac tumors (YSTs). Both the immature and mature components showed similar methylation levels in each ICR in immature teratomas. Overall, immature ovarian teratomas showed maternal methylation patterns of imprinted genes in concordance with their parthenogenetic origin. However, they also showed aberrant methylation levels in a few imprinted genes, suggesting that genome imprinting in immature teratomas may partially differ from that in mature teratomas. Microscopic foci of YST were seen in one immature teratoma; the YST component also showed a maternal methylation pattern, unlike the pure YSTs that showed irregular patterns. Thus, teratoma-associated YST and pure YST may have different pathogenic mechanisms., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Background: In their attempt to fulfill the wish of having children, women who suffer from fertility issues often undergo assisted reproductive technologies such as ovarian stimulation, which has been associated with adverse health outcomes and imprinting disorders in children. However, given the crucial role of exogenous hormone stimulation in improving human infertility treatments, a more comprehensive analysis of the potential impacts on DNA methylation in embryos following ovarian stimulation is needed. Here, we provide genome-wide DNA methylation profiles of blastocysts generated after superovulation of prepubertal or adult mice, compared with blastocysts derived from non-stimulated adult mice. Additionally, we assessed the impact of the in vitro growth and maturation of oocytes on methylation in blastocysts., Results: Neither hormone stimulation nor sexual maturity had an impact on the low global methylation levels characteristic of the blastocyst stage or was associated with extensive DNA methylation alterations. However, we found hormone- and age-associated changes at specific positions but dispersed throughout the genome. In particular, we detected anomalous methylation at a limited number of CpG islands. Additionally, superovulation in adult mice was associated with alterations at the Sgce and Zfp777 imprinted genes. On the other hand, in vitro culture of follicles from the early pre-antral stage was associated with globally reduced methylation and increased variability at imprinted loci in blastocysts., Conclusions: Our results indicate a minimal effect of ovarian stimulation of adult and prepubertal mice on the DNA methylation landscape attained at the blastocyst stage, but potentially greater impacts of in vitro growth and maturation of oocytes. These findings have potential significance for the improvement of assisted reproductive techniques, in particular for those related to treatments in prepubertal females, which could be crucial for improving human fertility preservation strategies., (© 2023. The Author(s).)

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., (The Authors. Published by Elsevier Inc. and Fass Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

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., Competing Interests: The authors declare no conflicts of interest.

Cryopreservation has important application in assisted reproductive technology (ART). The vitrification technique has been widely used in the cryopreservation of oocytes and embryos, as a large number of clinical results and experimental studies have shown that vitrification can achieve a higher cell survival rate and preimplantation development rate and better pregnancy outcomes. Ovarian tissue vitrification is an alternative method to slow freezing that causes comparatively less damage to the original follicular DNA. At present, sperm preservation mainly adopts slow freezing or rapid freezing (LN2 vapor method), although the vitrification method can achieve higher sperm motility after warming. However, due to the use of high-concentration cryoprotectants and ultra-rapid cooling, vitrification may cause strong stress to gametes, embryos and tissue cells, resulting in potentially adverse effects. Imprinted genes are regulated by epigenetic modifications, including DNA methylation, and show single allele expression. Their accurate regulation and correct expression are very important for the placenta, fetal development and offspring health. Considering that genome imprinting is very sensitive to changes in the external environment, we comprehensively summarized the effect of cryopreservation-especially the vitrification method in ART-on imprinted genes. Animal studies have found that the vitrification of oocytes and embryos can have a significant impact on some imprinted genes and DNA methylation, but the few studies in humans have reported almost no influence, which need to be further explored. This review provides useful information for the safety assessment and further optimization of the current cryopreservation techniques in ART., (© 2022. The Author(s).)

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., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

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., (© The Author(s) 2022. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

To investigate the influences of fresh embryo transfer (ET) and frozen embryo transfer (FET) on neonatal birthweight and the expression of imprinted genes PEG10 and L3MBTL1 in the placenta after in vitro fertilization-embryo transfer (IVF-ET), we analyzed the neonatal birthweight between fresh ET and FET transfer cycles. Then, we collected placentas delivered by fresh ET and FET, and real-time quantitative PCR, Western blotting and immunohistochemistry were used to detect the expression of PEG10 and L3MBTL1. The mean neonatal birthweight of fresh ET was lower than that of FET(3348.48 ± 521.05 vs. 3450.34 ± 524.13, P < 0.001). The risks of low birthweight (LBW) and small-for-gestational age (SGA) were lower after FET (3.9 % vs. 5.4 %; 7.2 % vs. 10.3 %), with adjusted rate ratios of 0.74 (95 % CI, 0.59-0.93; P = 0.003) and 0.70 (95 % CI, 0.59-0.84; P < 0.001), respectively. FET resulted in higher frequencies of macrosomia and large-for-gestational age (LGA) (14.2 % vs. 10.3; 11.0 % vs. 7.1 %) than fresh ET, with adjusted rate ratios of 1.45 (95 % CI, 1.26-1.68; P < 0.001) and 1.62 (95 % CI, 1.37-1.91; P < 0.001), respectively. We also observed PEG10 mRNA and protein expression levels in placentas delivered by fresh ET and FET were significantly different, but there were no significant differences in L3MBTL1 between the two groups. Fresh ET may affect the expression of the imprinted gene PEG10 in the placenta and adverse to placental implantation and development, resulting to increasing incidences of LBW and SGA., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

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 .

This dataset represents genes that are dysregulated in the postnatal day 12 (P12) mouse testis when ATRX is specifically inactivated in Sertoli cells ( ScAtrxKO mice). The differentially expressed genes included in the dataset may play important roles in the testicular phenotypes observed in the ScAtrxKO mice, which were first reported in our previous work [1]. In fetal ScAtrxKO mice, Sertoli cells undergo apoptosis due to cell cycle defects, resulting in smaller testes with reduced tubule volume [1]. Adult ScAtrxKO mice show a wide range of spermatogenesis defects probably due to a failure of the dysfunctional ATRX protein to interact with the androgen receptor (AR) [1]. ATRX, a chromatin remodeling protein, is widely expressed in the human testis including Sertoli cells [2,3]. In XY individuals, the loss of ATRX leads to ATR-X (alpha thalassemia, mental retardation, X-linked) syndrome associated with a wide range of genital abnormalities such as hypospadias, ambiguous genitalia, and small testes with reduced tubule volume [4], [5], [6], [7], [8]. Our dataset contributes towards understanding the mechanism underlying ATRX regulation of testis development and spermatogenesis., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2022 The Author(s).)

Black tea was reported to alter the microbiome populations and metabolites in diet-induced obese mice and displays properties that prevent obesity, but the underlying mechanism of the preventative effect of black tea on high-fat diet (HFD) induced obesity has not been elucidated. Epigenetic studies are a useful tool for determining the relationship between obesity and environment. Here, we show that the water extract of black tea (Lapsang souchong, LS) reverses HFD-induced gut dysbiosis, alters the tissue gene expression, changes the level of a major epigenetic modification (DNA methylation), and prevents obesity in HFD feeding mice. The anti-obesity properties of black tea are due to alkaloids, which are the principal active components. Our data indicate that the anti-obesity benefits of black tea are transmitted via fecal transplantation, and the change of tissue gene expression and the preventative effects on HFD-induced obesity in mice of black tea are dependent on the gut microbiota. We further show that black tea could regulate the DNA methylation of imprinted genes in the spermatozoa of high-fat diet mice. Our results show a mechanistic link between black tea, changes in the gut microbiota, epigenetic processes, and tissue gene expression in the modulation of diet-induced metabolic dysfunction.

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., (Copyright © 2022 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.)

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.

Based on prior knowledge and with the support of new methodology, solid progress in the understanding of seed life has taken place over the few last years. This update reflects recent advances in three key traits of seed life (i.e., preharvest sprouting, genomic imprinting, and stored-mRNA). The first breakthrough refers to cloning of the mitogen-activated protein kinase-kinase 3 (MKK3) gene in barley and wheat. MKK3, in cooperation with ABA signaling, controls seed dormancy. This advance has been determinant in producing improved varieties that are resistant to preharvest sprouting. The second advance concerns to uniparental gene expression (i.e., imprinting). Genomic imprinting primarily occurs in the endosperm. Although great advances have taken place in the last decade, there is still a long way to go to complete the puzzle regarding the role of genomic imprinting in seed development. This trait is probably one of the most important epigenetic facets of developing endosperm. An example of imprinting regulation is polycomb repressive complex 2 (PRC2). The mechanism of PRC2 recruitment to target endosperm with specific genes is, at present, robustly studied. Further progress in the knowledge of recruitment of PRC2 epigenetic machinery is considered in this review. The third breakthrough referred to in this update involves stored mRNA. The role of the population of this mRNA in germination is far from known. Its relations to seed aging, processing bodies (P bodies), and RNA binding proteins (RBPs), and how the stored mRNA is targeted to monosomes, are aspects considered here. Perhaps this third trait is the one that will require greater experimental dedication in the future. In order to make progress, herein are included some questions that are needed to be answered.

The endosperm is a vital storage tissue in plant seeds. It provides nutrients to the embryos or the seedlings during seed development and germination. Although the genetic information in the endosperm cannot be passed directly to the next generation, its inherited epigenetic marks affect gene expression and its development and, consequently, embryo and seed growth. DNA methylation is a major form of epigenetic modification that can be investigated to understand the epigenome changes during reproductive development. Therefore, it is of great significance to explore the effects of endosperm DNA methylation on crop yield and traits. In this review, we discuss the changes in DNA methylation and the resulting imprinted gene expression levels during plant endosperm development, as well as their effects on seed development., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Lu, Zhai and Xi.)

Zfp57 has both maternal and zygotic functions in mouse. It maintains genomic imprinting at most known imprinted regions and controls allelic expression of the target imprinted genes in mouse embryos. The DNA methylation imprint at many imprinting control regions (ICRs) is lost when both maternal and zygotic Zfp57 are absent in Zfp57 maternal-zygotic mutant mouse embryos. Interestingly, we found that DNA methylation at a few ICRs was partially lost without maternal Zfp57 in Zfp57 heterozygous mouse embryos derived from Zfp57 homozygous female mice. This suggests that maternal Zfp57 is essential for the maintenance of DNA methylation at a small subset of imprinted regions in mouse embryos. This maternal effect of Zfp57 was applied to allelic expression switch as well as expression levels of the corresponding imprinted genes. It is rather surprising that DNA methylation imprint was affected differently at Rasgrf1 and AK008011 imprinted regions in the female or male Zfp57 maternal-zygotic mutant embryos, with more significant loss of DNA methylation observed in the male mutant embryos. Loss of ZFP57 resulted in gender-specific differences in allelic expression switch and expression level changes of some imprinted genes in female or male mutant embryos. These results indicate maternal and sexually dimorphic effects of ZFP57 on genomic imprinting in mouse., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Xu, Shi, Zhang, Liu, Zhao, Chen, Song, Geng, Xie, Wu, Bai, Yang and Li.)

Assisted oocyte activation (AOA) has been proposed as an effective technique to overcome the problem of impaired fertilization after intracytoplasmic sperm injection (ICSI) but the safety of AOA remains a concern. We aimed to investigate if AOA induces imprinting effects on embryos. We used 13 cleavage embryos, nine blastocysts, and eight placentas from 15 patients. The subjects were divided into six groups by tissue type and with or without AOA. The methylation levels of imprinted genes (H19, paternally expressed gene [PEG3] and small nuclear ribonucleoprotein polypeptide N [SNRPN]) were tested by pyrosequencing. We observed different methylation levels among cleavage embryos. The variability was much more remarkable between cleavage embryos than blastocysts and placenta tissues. The methylation levels were especially higher in SNRPN and lower in the H19 gene in AOA embryos than those without AOA. No significant difference was found either among blastocysts or among placenta tissues regardless of AOA. The methylation levels of the three genes in blastocysts were very similar to those in the placenta. Compared to conventional ICSI, AOA changed imprinting methylation rates at H19 and SNRPN in cleavage embryos but not in the blastocyst stage and placenta. We recommend that blastocyst transfer should be considered for patients undergoing AOA during in vitro fertilization., Competing Interests: Conflict of interest: None., (© 2022 Rong Liang et al., published by De Gruyter.)

Despite several decades of research and prevention efforts, fetal alcohol spectrum disorders (FASD) remain the most common preventable cause of neurodevelopmental disabilities worldwide. Animal and human studies have implicated fetal alcohol-induced alterations in epigenetic programming as a chief mechanism in FASD. Several studies have demonstrated fetal alcohol-related alterations in methylation and expression of imprinted genes in placental, brain, and embryonic tissue. Imprinted genes are epigenetically regulated in a parent-of-origin-specific manner, in which only the maternal or paternal allele is expressed, and the other allele is silenced. The chief functions of imprinted genes are in placental development, somatic growth, and neurobehavior-three domains characteristically affected in FASD. In this review, we summarize the growing body of literature characterizing prenatal alcohol-related alterations in imprinted gene methylation and/or expression and discuss potential mechanistic roles for these alterations in the teratogenic effects of prenatal alcohol exposure. Future research is needed to examine potential physiologic mechanisms by which alterations in imprinted genes disrupt development in FASD, which may, in turn, elucidate novel targets for intervention. Furthermore, mechanistic alterations in imprinted gene expression and/or methylation in FASD may inform screening assays that identify individuals with FASD neurobehavioral deficits who may benefit from early interventions., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Intrauterine growth restriction (IUGR) due to uteroplacental insufficiency results in a placenta that is unable to provide adequate nutrients and oxygen to the fetus. These growth-restricted babies have an increased risk of hypertension and chronic kidney disease later in life. In rats, both male and female growth-restricted offspring have nephron deficits but only males develop kidney dysfunction and high blood pressure. In addition, there is transgenerational transmission of nephron deficits and hypertension risk. Therefore, epigenetic mechanisms may explain the sex-specific programming and multigenerational transmission of IUGR-related phenotypes. Expression of DNA methyltransferases (Dnmt1and Dnmt3a) and imprinted genes (Peg3, Snrpn, Kcnq1, and Cdkn1c) were investigated in kidney tissues of sham and IUGR rats in F1 (embryonic day 20 (E20) and postnatal day 1 (PN1)) and F2 (6 and 12 months of age, paternal and maternal lines) generations (n = 6-13/group). In comparison to sham offspring, F1 IUGR rats had a 19% decrease in Dnmt3a expression at E20 (P < 0.05), with decreased Cdkn1c (19%, P < 0.05) and increased Kcnq1 (1.6-fold, P < 0.01) at PN1. There was a sex-specific difference in Cdkn1c and Snrpn expression at E20, with 29% and 34% higher expression in IUGR males compared to females, respectively (P < 0.05). Peg3 sex-specific expression was lost in the F2 IUGR offspring, only in the maternal line. These findings suggest that epigenetic mechanisms may be altered in renal embryonic and/or fetal development in growth-restricted offspring, which could alter kidney function, predisposing these offspring to kidney disease later in life.

Objective: To determine whether the epigenetic control of imprinted genes (IGs) and transposable elements (TEs) differs at birth between fresh or frozen embryo transfers and natural conceptions., Design: Prospective study., Setting: University hospital., Patient(s): A total of 202 singleton births were divided into three groups: 84 natural pregnancies (controls), 66 in vitro fertilization/intracytoplasmic sperm injection with fresh embryo transfers, and 52 vitro fertilization/intracytoplasmic sperm injection with frozen embryo transfers., Intervention(s): None., Main Outcome Measure(s): Pyrosequencing was used to assess the DNA methylation profiles of three IGs (H19/IGF2:IG-DMR [two sequences], KCNQ1OT1:TSS-DMR, and SNURF:TSS-DMR) and two TEs (LINE-1 and HERV-FRD) in cord blood and placenta. The quantitative reverse transcriptase polymerase chain reaction was used to study the transcription of three IGs (H19, KCNQ1, and SNRPN) and two TEs (LINE-1 and ORF2)., Result(s): After adjustment, the placental DNA methylation levels of H19/IGF2 were lower in the fresh embryo transfer group than in the control (H19/IGF2-seq1) and frozen embryo transfer (H19/IGF2-seq2) groups. The DNA methylation rate for LINE-1 was lower in placentas from the fresh embryo transfer group than in placentas from the control and frozen embryo transfer groups and for HERV-FRD compared with controls. In cord blood, DNA methylation levels were not significantly associated with the mode of conception. The relative expression of LINE-1 and ORF2 was decreased in both cord blood and placental tissues from fresh embryo transfer conceptions compared with natural conceptions and frozen embryo transfer conceptions., Conclusion(s): Compared with natural conceptions and frozen embryo transfers, fresh embryo transfers were associated with methylation and/or transcription changes in some TEs and IGs, mostly in placental samples, which could indicate altered placental epigenetic regulation resulting from ovarian stimulation protocols., (Copyright © 2021 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Background: Uniparental disomy (UPD) is defined as an inheritance of two chromosomes from only one of the parents with no representative copy from the other. Paternal-origin UPD of chromosome 3 is a very rare condition, with only five cases of paternal UPD(3) reported., Case Presentation: Here, we report a prenatal case that is only the second confirmed paternal UPD(3) reported with no apparent disease phenotype. The fetus had a normal karyotype and normal ultrasound features throughout gestation. Copy neutral regions of homozygosity on chromosome 3 were identified by single nucleotide polymorphism (SNP) array. Subsequent SNP array data of parent-child trios showed that the fetus carried complete paternal uniparental isodisomy (isoUPD) of chromosome 3. The parents decided to continue with the pregnancy after genetic counseling, and the neonate had normal physical findings at birth and showed normal development after 1.5 years., Conclusions: These findings provided further evidence to confirm that there were no important imprinted genes on paternal chromosome 3 that caused serious diseases and a reference for the prenatal diagnosis and genetic counseling of UPD(3) in the future., (© 2021. The Author(s).)

Traditionally, Cornelia de Lange Syndrome (CdLS) is considered a cohesinopathy caused by constitutive mutations in cohesin complex genes. Cohesin is a major regulator of chromatin architecture, including the formation of chromatin loops at the imprinted IGF2 / H19 domain. We used 3C analysis on lymphoblastoid cells from CdLS patients carrying mutations in NIPBL and SMC1A genes to explore 3D chromatin structure of the IGF2 / H19 &nbsp; locus and evaluate the influence of cohesin alterations in chromatin architecture. We also assessed quantitative expression of imprinted loci and WNT pathway genes, together with DMR methylation status of the imprinted genes. A general impairment of chromatin architecture and the emergence of new interactions were found. Moreover, imprinting alterations also involved the expression and methylation levels of imprinted genes, suggesting an association among cohesin genetic defects, chromatin architecture impairment, and imprinting network alteration. The WNT pathway resulted dysregulated: canonical WNT, cell cycle, and WNT signal negative regulation were the most significantly affected subpathways. Among the deregulated pathway nodes, the key node of the frizzled receptors was repressed. Our study provides new evidence that mutations in genes of the cohesin complex have effects on the chromatin architecture and epigenetic stability of genes commonly regulated by high order chromatin structure.

Objective: The current study addresses the cellular complexity and plasticity of subcutaneous (inguinal) white adipose tissue (iWAT) in mice during the critical periods of perinatal growth and establishment., Methods: We performed a large-scale single cell transcriptomic (scRNA-seq) and epigenomic (snATAC-seq) characterization of cellular subtypes (adipose stromal cells (ASC) and adipocyte nuclei) during inguinal WAT (subcutaneous; iWAT) development in mice, capturing the early postnatal period (postnatal days (PND) 06 and 18) through adulthood (PND56)., Results: Perinatal and adult iWAT contain 3 major ASC subtypes that can be independently identified by RNA expression profiles and DNA transposase accessibility. Furthermore, the transcriptomes and enhancer landscapes of both ASC and adipocytes dynamically change during postnatal development. Perinatal ASC (PND06) are highly enriched for several imprinted genes (IGs; e.g., Mest, H19, Igf2) and extracellular matrix proteins whose expression then declines prior to weaning (PND18). By comparison, adult ASC (PND56) are more enriched for transcripts associated with immunoregulation, oxidative stress, and integrin signaling. Two clusters of mature adipocytes, identified through single nuclei RNA sequencing (snRNA-seq), were distinctive for proinflammatory/immune or metabolic gene expression patterns that became more transcriptionally diverse in adult animals. Single nuclei assay for transposase-accessible chromatin (snATAC-seq) revealed that differences in gene expression were associated with developmental changes in chromatin accessibility and predicted transcription factor motifs (e.g., Plagl1, Ar) in both stromal cells and adipocytes., Conclusions: Our data provide new insights into transcriptional and epigenomic signaling networks important during iWAT establishment at a single cell resolution, with important implications for the field of metabolic programming., (Copyright © 2021 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Embryos that are produced in vitro frequently present epigenetic modifications. However, maternal supplementation with folic acid (FA) may improve oocyte maturation and embryo development, preventing epigenetic errors in the offspring. We sought to evaluate the influence of FA supplementation during in vitro maturation of grade I (GI) and grade III (GIII) bovine oocytes on embryo production rate and the expression of IGF2 and KCNQ1OT1 genes. The oocytes were matured in vitro with different concentrations of FA (0, 10, 30 and 100 μM), followed by in vitro fertilization and embryo culture. On the seventh day (D7) of culture, embryo production was evaluated and gene expression was measured using real-time qPCR. Supplementation with 10 μM of FA did not affect embryo production for GI and GIII oocytes. Moderate supplementation (30 μM) seemed to be a positive influence, increasing embryo production for GIII (P = 0.012), while the highest dose (100 μM) reduced embryo production (P = 0.010) for GI, and IGF2 expression was not detected. In GIII, only embryos whose oocyte maturation was not supplemented with FA demonstrated detected IGF2 expression. The lowest concentration of FA (10 μM) reduced KCNQ1OT1 expression (P = 0.05) on embryos from GIII oocytes. Different FA concentrations induced different effects on bovine embryo production and gene expression that was related to oocyte quality. Despite the epigenetic effects of FA, supplementation seems to be a promising factor to improve bovine embryo production if used carefully, as concentration is an important factor, especially in oocytes with impaired quality.

Parthenogenesis activation (PA), as an important artificial breeding method, can stably preserve the dominant genotype of a species. However, the delayed development of PA embryos is still overly severe and largely leads to pre-implantation failure in pigs. The mechanisms underlying the deficiencies of PA embryos have not been completely understood. For further understanding of the molecular mechanism behind PA embryo failure, we performed transcriptome analysis among pig oocytes (meiosis II, MII) and early embryos at three developmental stages (zygote, morula, and blastocyst) in vitro fertilization (IVF) and PA group. Totally, 11,110 differentially expressed genes (DEGs), 4694 differentially expressed lincRNAs (DELs) were identified, and most DEGs enriched the regulation of apoptotic processes. Through cis- and trans-manner functional prediction, we found that hub lincRNAs were mostly involved in abnormal parthenogenesis embryonic development. In addition, twenty DE imprinted genes showed that some paternally imprinted genes in IVF displayed higher expression than that in PA. Notably, we identified that three DELs of imprinted genes ( MEST , PLAGL1 , and DIRAS3 ) were up regulated in IVF, and there was no significant change in PA group. Disordered expression of key genes for embryonic development might play key roles in abnormal parthenogenesis embryonic development. Our study indicates that embryos derived from different production techniques have varied in vitro development to the blastocyst stage, and they also affect the transcription level of corresponding genes, such as imprinted genes. This work will help future research on these genes and molecular-assisted breeding for pig parthenotes.

Maternal periconceptional diets have known associations with proper offspring neurodevelopment. Mechanisms for such associations include improper energy/nutrient balances between mother and fetus, as well as altered offspring epigenetics during development due to maternal nutrient and inflammatory status. Using a comprehensive food frequency questionnaire and assessing offspring temperament with the Infant-Toddler Social and Emotional Assessment ( n = 325, mean age = 13.9 months), we sought to test whether a maternal periconceptional diet characterized by high glycemic loading (MGL) would affect offspring temperament using adjusted ordinal regression. After limiting false discovery to 10%, offspring born to mothers in tertile 3 of glycemic loading (referent = tertile 1) were more likely to be in the next tertile of anxiety [OR (95% CI) = 4.51 (1.88-11.07)] and inhibition-related behaviors [OR (95% CI) = 3.42 (1.49-7.96)]. Male offspring were more likely to exhibit impulsive [OR (95% CI) = 5.55 (1.76-18.33)], anxiety [OR (95% CI) = 4.41 (1.33-15.30)], sleep dysregulation [OR (95% CI) = 4.14 (1.34-13.16)], empathy [6.68 (1.95-24.40)], and maladaptive behaviors [OR (95% CI) = 9.86 (2.81-37.18)], while females were more likely to exhibit increased anxiety-related behaviors [OR (95% CI) = 15.02 (3.14-84.27)]. These associations persisted when concurrently modeled with the maternal-Mediterranean dietary pattern. In a subset ( n = 142), we also found MGL associated with increased mean methylation of the imprint control region of SGCE/PEG10 . In conclusion, these findings highlight the importance of maternal dietary patterns on offspring neurodevelopment, offering avenues for prevention options for mothers.

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., (Copyright © 2021 Elsevier Inc. All rights reserved.)

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., (© 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.)

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Rotondo, Lanzillotti, Mazziotta, Tognon and Martini.)

The placenta develops from the outer trophoblastic layer following the differentiation of the fertilized ovum and is therefore more susceptible to epigenetic regulatory changes caused by environmental interventions and influences during assisted reproductive technology. Furthermore, the placenta regulates the development of the fetal heart, brain, kidneys, bones, and other tissues and organs [1]. Placental dysplasia leads to poor perinatal outcomes as well as long-term health risks later in life, including neurodevelopmental disorders, tumors, and adult metabolic syndrome [2,3]. In view of the decisive role of the placenta during intrauterine fetal development, Graham J. Burton, an expert in placentology from the University of Cambridge, formally proposed the theory of "placenta-derived chronic diseases" in 2018 based on embryonic-derived diseases [4]. In this review, we summarized the changes in placental morphology and structure, growth dynamics, imprinted and non-imprinted genes, and other aspects attributable to assisted reproduction technology. Our review provides a theoretical basis for further research on placental changes caused by assisted reproductive technology that are most strongly associated with an increased risk of neonatal long-term diseases., Competing Interests: Declaration of Competing Interest All the authors declare that they have no conflict of interest and nothing to disclose., (Copyright © 2021 Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn. Published by Elsevier B.V. All rights reserved.)