Epigenomics

Maternal pancreatic {beta}-cells undergo functional and structural changes to adapt to increased metabolic demands during pregnancy. Lactogen signaling via the prolactin receptor (PRLR) contributes to these adaptations by increasing {beta}-cell mass, insulin transcription and glucose-stimulated insulin secretion[1-4]. In other lactogen-responsive tissues such as the mammary glands and specific hypothalamic nuclei, gestation induces epigenetic changes, some of which persist long after birth[5, 6]. We have previously found that prolactin treatment in islets regulates the expression of epigenetic modifiers[7, 8]. However, whether lactogen signaling in {beta}-cells mediates epigenetic changes to regulate chromatin accessibility has not been examined. Therefore, our objective was to determine whether PRLR signaling alters chromatin accessibility of {beta}-cells to facilitate transcriptional regulation. Using single-cell ATAC-sequencing, we identified differentially accessible regions (DARs) in {beta}-cells which had 718 overrepresented motifs following prolactin treatment of murine islets. Validating this approach, these included motifs bound by established PRLR signaling effectors such as the STAT family of transcription factors (TFs). Using RNA-sequencing we identified transcriptional changes in 41 TFs whose motifs were overrepresented in DARs, including several previously linked to PRLR signaling within {beta}-cells, including Myc, Mafb and Esr1. Importantly, we also identified TFs not previously associated with PRLR signaling, including OVOL2 an established regulator of epigenetic landscape within cells. OVOL2 is a transcription factor involved in EMT inhibition and energy homeostasis with unknown roles in pancreatic {beta}-cells. Here, we establish that OVOL2 acts as a negative regulator of lactogen-dependent effects on {beta}-cell proliferation, establishing a novel regulator of PRLR signaling.

Preeclampsia (PE) is a severe pregnancy complication that threatens maternal and neonatal health. Previous epigenome-wide association studies (EWAS) on PE have produced inconsistent results, possibly due to inadequate adjustment for confounders. Here, we analyzed DNA methylation changes in cord blood from newborns affected by PE, using a multi-ethnic cohort from Hawaii. We comprehensively adjusted for clinical variables (maternal age, BMI, parity) and estimated cell proportions. Additionally, we re-analyzed two public datasets with similar adjustments and conducted a meta-analysis combining all three datasets to increase statistical power. To further address confounding by gestational age, we also included idiopathic preterm samples as controls. After adjusting for cell type proportions and clinical characteristics, all previously reported significant CpG methylation changes associated with severe PE disappeared across our data, the two public datasets, and the meta-analysis. This result remained even after including idiopathic preterm samples. Instead, severe PE was associated with shifts in CD8T and natural killer (NK) cell proportions. We validated this lack of CpG changes using multiple published cord blood methylation datasets. Moreover, we observed that gestational progression itself is accompanied by significant changes in granulocyte, nRBC, CD8T, and B cell proportions. In summary, our study demonstrates that many previously reported DNA methylation changes in severe PE are artifacts caused by confounding factors such as cell type heterogeneity and gestational age. Severe PE is associated with changes in cell proportions rather than direct methylation alterations. These findings emphasize the importance of rigorous confounder adjustment in EWAS.

DNA methylation is extensively reprogrammed during early stage of mammalian development and is essential for normal embryogenesis. It is well established that mouse embryos acquire genome-wide DNA methylation during implantation, referred to as de novo DNA methylation, from globally hypomethylated blastocysts. However, the fact that the main de novo DNA methyltransferase 3B (DNMT3B) is initially expressed as early as the 8-cell stage, contradicts the current knowledge about timing of initiation of de novo DNA methylation. Here, we reported that a previously overlooked minor wave of de novo DNA methylation initially occurs during the transition from the 8-cell to blastocyst stage, before the well-known large-scale de novo DNA methylation during implantation. Functional analyses indicated that minor de novo DNA methylation regulates proliferation, lineage differentiation and metabolic homeostasis of preimplantation embryos, and is critical for embryonic developmental potential and pregnancy outcomes. Furthermore, bioinformatic and functional analyses indicated that minor de novo DNA methylation preferentially occurs on the X chromosome and co-regulates imprinted X-chromosome inactivation via the interaction between DNMT3B and polycomb repressive complexes 2 core components during blastocyst formation. Thus, our study updates the current knowledge of embryonic de novo DNA methylation, thereby providing a novel insight of early embryonic epigenetic reprogramming. Summary statementA minor wave of de novo DNA methylation has been initiated prior to blastocyst formation, but not during the implantation period, and co-regulates imprinted X-chromosome inactivation.

Background and aimsInflammatory Bowel Diseases (IBD) are chronic inflammatory conditions influenced heavily by environmental factors. DNA methylation is a form of epigenetic regulation linking environmental stimuli to gene expression changes and inflammation. Here, we investigated how DNA methylation of the TNF promoter differs between inflamed and uninflamed mucosa of IBD patients, including anti-TNF responders and non-responders. MethodsWe obtained mucosal biopsies from 200 participants (133 IBD and 67 controls) and analyzed TNF promoter methylation using bisulfite sequencing, comparing inflamed with uninflamed segments, in addition to paired inflamed/uninflamed samples from individual patients. We conducted similar analyses on purified intestinal epithelial cells from bowel resections. We also compared TNF methylation levels of inflamed and uninflamed mucosa from a separate cohort of 15 anti-TNF responders and 17 non-responders. Finally, we sequenced DNA methyltransferase genes to identify rare variants in IBD patients and functionally tested them using rescue experiments in a zebrafish genetic model of DNA methylation deficiency. ResultsTNF promoter methylation levels were decreased in inflamed mucosa of IBD patients and correlated with disease severity. Isolated IECs from inflamed tissue showed proportional decreases in TNF methylation. Anti-TNF non-responders showed lower levels of TNF methylation than responders in uninflamed mucosa. Our sequencing analysis revealed two missense variants in DNMT1, one of which had reduced function in vivo. ConclusionsOur study reveals an association of TNF promoter hypomethylation with mucosal inflammation, suggesting that IBD patients may be particularly sensitive to inflammatory environmental insults affecting DNA methylation. Together, our analyses indicate that TNF promoter methylation analysis may aid in the characterization of IBD status and evaluation of anti-TNF therapy response.

ObjectiveThis retrospective cohort investigated the role of leptins promoter methylation and microRNA targeting profile in developing adiposity and inflammation in neonates, using umbilical cord blood from preterm (n=67) and term (n=71) mothers. MethodsGlobal DNA methylation and leptin promoter methylation were performed. ELISA determined leptin and IGF1 levels. Real-time PCR measured mRNA levels. MicroRNA target prediction on the human leptin gene (LEP) was done in silico using network analysis. ResultsPreterm cord blood significantly reduced genome-wide (p<0.001) and LEP promoter methylation (p=0.001), increased LEP & LEPR expression (p=0.04), and circulatory leptin (p=0.41). Neonatal birth weight positively correlated with leptin and IGF1 levels in preterm (r=0.47, p=0.04) but not in the term. IL6 expression showed a positive correlation with circulatory leptin (r= 0.687, p=0.008), LEP (r= 0.763, p=0.009), and an inverse association with LEP promoter methylation (r= -0.636, p=0.04) in preterm. The obtained LEP targeting miRNAs showed their affinities for critical genes associated with body fat distribution, fat cell differentiation, and energy regulation, implicating a close association in the LEP-miRNA-obesity axis. ConclusionsThe strong correlation between LEP methylation and pro-inflammatory cytokine influences each other in developing chronic inflammation in preterm neonates, which might predispose them to obesity in later life. Study importanceWhat is already known? O_LILeptin communicates about the bodys fat deposits to the brain and aids in maintaining energy homeostasis and stable body weight. C_LIO_LIPreterm exhibit lower body weight and fat mass at birth than term neonates, who often show rapid compensatory catch-up growth. C_LI What does this study add? O_LILeptin gene (LEP) promoter methylation was reduced in preterm cord blood compared to term. C_LIO_LIHigher interleukin-6 (IL6) and tumour necrosis factor-alpha (TNF) expression in preterm but not in term. IL6 correlated positively with circulatory leptin and LEP expression while inversely associated with LEP-specific promoter methylation, indicating that a dysregulated epigenetic control can promote low-grade inflammation in preterm neonates. C_LIO_LILEP-targeting micro-RNAs showed affinities for critical genes associated with fat cell differentiation, energy regulation, and other processes. C_LI How might these results change the direction of research or the focus of clinical practice? O_LISince others observed dysregulated LEP methylation in the adipose tissue of obese subjects, these data imply that leptin could mediate the risk for obesity during preterm birth. C_LIO_LIWhile short-term outcomes of preterm birth are well addressed, its effect on long-term metabolic health is of concern as it might elevate the risk of obesity. C_LI Graphical AbstractMaternal factors leading to preterm birth and cord blood leptin dysregulation in predicting obesity. Elevated blood pressure, infection, and lower haemoglobin in preterm disrupted epigenetic control of leptin and activated inflammation that might induce leptin resistance. The latter is known to reduce satiety and increase body mass, elevating the risk of obesity. Solid arrows depict present data, and dotted lines indicate possible pathways. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=52 SRC="FIGDIR/small/24319077v1_ufig1.gif" ALT="Figure 1"> View larger version (11K): org.highwire.dtl.DTLVardef@1f3604corg.highwire.dtl.DTLVardef@13723b5org.highwire.dtl.DTLVardef@1094bfborg.highwire.dtl.DTLVardef@15b5ebc_HPS_FORMAT_FIGEXP M_FIG C_FIG

BackgroundEpithelial-mesenchymal transition (EMT) is a phenomenon in which epithelial cells acquire mesenchymal traits. It contributes to organogenesis and tissue homeostasis, as well as stem cell differentiation. Emerging evidence indicates that heterogeneous expression of EMT gene markers presents in sub-populations of germline stem cells (GSCs). However, the functional implications of such heterogeneity are largely elusive. ResultsWe unravelled an EMT-like process in GSCs by in vitro extracellular matrix (ECM) model and single-cell genomics approaches. We found that histone methyltransferase G9a regulated an EMT-like program in GSC in vitro and contributed to neonatal germ cell migration in vivo. Through modulating ECM, we demonstrated that GSCs exist in interconvertible epithelial-like and mesenchymal-like cell states. GSCs gained higher migratory ability after transition to a mesenchymal-like cell state, which was largely mediated by the TGF-{beta} signaling pathway. Dynamics of epigenetic regulation at the single-cell level was also found to align with the EMT-like process. Chromatin accessibility profiles generated by single-cell sequencing assay for transposase-accessible chromatin (scATAC-seq) clustered GSCs into epithelial-like and mesenchymal-like states, which were associated with differentiation status. The high-resolution data revealed regulators in the EMT-like process, including transcription factors Zeb1. We further identified putative enhancer-promoter interactions and cis-co-accessibility networks at loci such as Tgfb1, Notch1 and Lin28a. Lastly, we identified HES1 as the putative target underlying G9as regulation. ConclusionOur work provides the foundation for understanding the EMT-like process and a comprehensive resource for future investigation of epigenetic regulatory networks in GSCs.

The phenotype of human placental extravillous trophoblast (EVT) at the end of pregnancy reflects both first trimester differentiation from villous cytotrophoblast (CTB) and later gestational changes, including loss of proliferative and invasive capacity. Invasion abnormalities are central to two major placental pathologies, preeclampsia and placenta accreta spectrum, so characterization of the corresponding normal processes is crucial. In this report, our gene expression analysis, using purified human CTB and EVT cells, highlights an epithelial- mesenchymal transition (EMT) mechanism underlying CTB-EVT differentiation and provides a trophoblast-specific EMT signature. In parallel, DNA methylation profiling shows that CTB cells, already hypomethylated relative to non-trophoblast cell lineages, show further genome- wide hypomethylation in the transition to EVT. However, a small subgroup of genes undergoes gains of methylation (GOM) in their regulatory regions or gene bodies, associated with differential mRNA expression (DE). Prominent in this GOM-DE group are genes involved in the EMT, including multiple canonical EMT markers and the EMT-linked transcription factor RUNX1, for which we demonstrate a functional role in modulating the migratory and invasive capacities of JEG3 trophoblast cells. This analysis of DE associated with locus-specific GOM, together with functional studies of an important GOM-DE gene, highlights epigenetically regulated genes and pathways acting in human EVT differentiation and invasion, with implications for obstetric disorders in which these processes are dysregulated.

SATB homeobox proteins are important regulators of developmental gene expression. Among the stem cell lineages determined during early embryonic development, trophoblast stem (TS) cells exhibit robust SATB expression. Both SATB1 and SATB2 act to maintain trophoblast stem-state. However, the molecular mechanisms that regulate TS-specific Satb expression are not yet known. We identified Satb1 variant 2 as the predominant transcript in trophoblasts. Histone marks, and RNA polymerase II occupancy in TS cells indicated active state of the promoter. A novel cis-regulatory region with active histone marks was identified [~]21kbp upstream of variant 2 promoter. CRISPR/Cas9 mediated disruption of this sequence decreased Satb1 expression in TS cells and chromatin conformation capture confirmed looping of this regulatory region into the promoter. Scanning position weight matrices across the enhancer predicted two ELF5 binding sites in close vicinity of SATB1 sites, which were confirmed by chromatin immunoprecipitation. Knockdown of ELF5 downregulated Satb1 expression in TS cells and overexpression of ELF5 increased the enhancer-reporter activity. Interestingly, ELF5 interacts with SATB1 in TS cells, and the enhancer activity was upregulated following SATB overexpression. Our findings indicate that trophoblast-specific Satb1 expression is regulated by long-range chromatin looping of an enhancer that interacts with ELF5 and SATB proteins.

1.0Preeclampsia (PE) is a hypertensive disorder of pregnancy characterized by immune dysregulation and significant risks to maternal and fetal health. While current management relies on high-risk patient monitoring and early diagnosis, these methods are costly and burdensome, especially for low-risk pregnancies. There is a pressing need for non-invasive tools to predict and monitor PE. DNA methylation (DNAm) is a type of DNA modification that influences gene expression, and has been associated with immune cell dynamics and PE pathogenesis. This study explores whether DNAm-based immune cell composition profiling can provide insights into PE-related immune dysregulation. We conducted a search in the Gene Expression Omnibus (GEO) for DNA methylation datasets using Illumina 27K, 450K, and EPIC arrays from maternal blood in both healthy and PE pregnancies. We found two studies that met our criteria, involving a total of 24 healthy pregnancies and 14 with PE. To estimate the composition of immune cells (including CD8T, CD4T, Monocytes, Natural Killer, Neutrophils, Eosinophils, and B cells) based on DNA methylation data, we employed the R package EpiDISH. We used a linear model to compare statistical differences in the proportions of immune cells between PE cases and the control group. Longitudinal trends were also examined to capture immune cell shifts from pregnancy to postpartum. We found that monocyte proportions were significantly reduced in preeclamptic pregnancies compared to normotensive pregnancies (p=0.013). No significant differences were observed in other immune cell types, including T cells, B cells, neutrophils, eosinophils, and natural killer cells. Longitudinal analyses revealed substantial immune cell shifts in the postpartum period, including increased monocytes, B cells, CD4+ T cells, and CD8+ T cells, emphasizing the importance of gestational age in immune dynamics. These findings support DNAm profiling as a valuable tool for understanding immune cell dynamics in PE. Reduced monocyte proportions in PE highlight the role of immune dysregulation in its pathogenesis. Longitudinal sampling provides additional insights into the evolution of immune changes throughout pregnancy and postpartum, offering potential for developing predictive and monitoring tools for PE. Future studies with larger, more diverse cohorts are essential to refine the utility of DNAm in pregnancy complications.

BackgroundA growing body of literature has reported molecular and histological changes in the human placenta in association with preeclampsia (PE). Placental DNA methylation (DNAme) and transcriptomic patterns have revealed molecular subgroups of PE that are associated with placental histopathology and clinical phenotypes of the disease. However, the heterogeneity of PE both across and within subtypes, whether defined clinically or molecularly, complicates the study of this disease. PE is most strongly associated with placental pathology and adverse fetal and maternal outcomes when it develops early in pregnancy. We focused on placentae from pregnancies affected by preeclampsia that were delivered before 34 weeks of gestation to develop eoPred, a predictor of the DNAme signature associated with the placental phenotype of early-onset preeclampsia (EOPE). ResultsPublic data from 83 placental samples (HM450K), consisting of 42 EOPE and 41 normotensive preterm birth (nPTB) cases, was used to develop eoPred - a supervised model that relies on a highly discriminative 45 CpG DNAme signature of EOPE in the placenta. The performance of eoPred was assessed using cross-validation (AUC=0.95) and tested in an independent validation cohort (n=49, AUC=0.725). A subset of fetal growth restriction (FGR) and late-PE cases showed a similar DNAme profile at the 45 predictive CpGs, consistent with the overlap in placental pathology between these conditions. The relationship between the EOPE probability generated by eoPred and various phenotypic variables was also assessed, revealing that it is associated with gestational age, and it is not driven by cell composition differences. ConclusionseoPred relies on a 45 CpG DNAme signature to predict EOPE, and it can be used in a discrete or continuous manner. Using this classifier should 1) improve the consistency of future placental DNAme studies of PE and placental insufficiency, 2) facilitate identifying cases of EOPE in public data sets and 3) importantly, standardize the placental diagnosis to allow better cross-cohort comparisons. Lastly, classification of cases with eoPred should be useful for testing associations between placental pathology and genetic or environmental variables.

In the United States, cardiovascular disease is the leading cause of death, and the rate of maternal mortality remains among the highest of any industrialized nation. Maternal cardiometabolic health throughout gestation and postpartum is representative of placental health and physiology. Both proper placental functionality and placental microRNA expression are essential to successful pregnancy outcomes, and both are highly sensitive to genetic and environmental sources of variation. While placental pathologies, such as preeclampsia, are associated with maternal cardiovascular health and may contribute to the developmental programming of cardiovascular disease, the role of more subtle alterations to placental function and microRNA expression in this relationship remains poorly understood. To develop a more comprehensive understanding of how cardiometabolic health influences placental microRNA expression, and how this shapes placental functionality, we performed small RNA sequencing to investigate microRNA in the placentae from the Rhode Island Child Health Study (n=230). We modeled microRNA counts on maternal family history of cardiovascular disease using negative binomial generalized linear models, and identified microRNAs that were differential expressed (DEmiRs) at a false discovery rate (FDR) less than 0.10. Utilizing parallel mRNA sequencing data and bioinformatic target prediction software, we identified potential mRNA targets of these DEmiRs. We identified 9 DEmiRs, with predicted targets of those miRNA enriched overwhelmingly in the TGF{beta} signaling pathway but also in pathways involving cellular metabolism and immunomodulation. Overall, we identified a robust association existing between familial cardiovascular disease and placental microRNA expression which may be implicated in both placental insufficiencies and the developmental programming of cardiovascular disease.

BackgroundEpigenetic alterations during fetal development have been proposed as key factors explaining associations between maternal lifestyle during pregnancy and later health outcomes in the offspring, pertaining to the developmental origin of health and disease (DOHAD) hypothesis. ObjectivesTo assess the association of maternal lifestyle with offsprings birth weight and underlying epigenetic mediatory mechanisms in the NorthPop prospective birth cohort. MethodsA three-step analytic pipeline was applied. In 722 mother-child pairs, overall associations between 10 maternal lifestyle factors and the offsprings standardized birth weight were first evaluated by multiple linear regression. Three high dimensional mediation methods (HDMA, HIMA, and HIMA2) were then applied on the beta methylation matrix to identify candidate CpG mediators in cord blood driving the significant overall associations. Finally, robust- and ordinary least squares-regression-based classical mediation, including single- and multiple-(parallel and serial) mediator models were assessed. ResultsGestational weight gain (GWG) ({beta}-adj = 0.03; p = 2x10-5) and maternal BMI at the beginning of pregnancy ({beta}-adj = 0.036; p = 1x10-4) were significantly associated with the offsprings standardized birth weight. High dimensional mediation analyses identified pooled sets of four (cg19242268; cg08461903; cg14798382; cg21516291) and five (cg17040807; cg19242268; cg26552621; cg04457572; cg06457011) candidate CpG mediators related to GWG and BMI at the beginning of pregnancy, respectively. For both exposures, classical mediation analyses revealed a range of significant single- and multiple (both serial and parallel) mediator models via both robust- and OLS-regression based approaches. These indicated the likely presence of individual-, causally linked multiple-, and causally independent multiple mediatory pathways underlying the two significant overall associations. ConclusionsOur findings support the hypothesis that neonatal health effects related to maternal lifestyle may be partly mediated by epigenetic alterations. Findings also suggest the possible involvement of multiple DNA methylation sites via various mediatory pathways.

BackgroundMicroRNAs (miRNAs) are a class of small non-coding RNAs regulating gene expression. They are involved in many biological processes, including adaptation to pregnancy. The identification of genetic variants associated with gene expression, known as expression quantitative trait loci (eQTL), helps to understand the underlying molecular mechanisms and determinants of complex diseases. Using data from the prospective pre-birth Gen3G cohort, we investigated associations between maternal genotypes and plasmatic miRNA levels measured during the first trimester of pregnancy of 369 women. ResultsAssessing the associations between about 2 million SNPs and miRNA proximal pairs using best practices from the GTEx consortium, a total of 22,140 significant eQTLs involving 147 unique miRNAs were identified. Elastic-net regressions were applied to select the most relevant SNPs to build genetic risk scores (GRS) for each of these 147 miRNAs. For about half of the circulating miRNAs, the GRS captured >10% of the variance abundance. As a demonstration of the usefulness of the identified eQTLs and derived GRS, we used the GRSs as instrumental variables to test for association between the circulating levels of miRNAs quantified before the 16th week of pregnancy and the development of pregnancy complications (gestational diabetes [GDM] or pre-eclampsia [PE]) developing more than three months later on average. Using predicted miRNA levels derived from instrumental variables, we found 18 significant associations of miRNAs with potential support of causal inference for GDM or PE. ConclusionsOur results represent a valuable resource to understand miRNA regulation and highlight the potential of genetic instruments in predicting circulating miRNA levels and their possible contribution in disease development.

BackgroundThe human placenta is an essential organ for fetal development and pregnancy success. Across gestation, blastocyst-derived trophoblasts facilitate the major functions of the placenta. Specifically, invasive trophoblast subtypes, called extravillous trophoblasts (EVT), play central roles in coordinating nutrient accessibility and maternal immunomodulation to semi-allogeneic fetal and placental tissues. Like the fetus, these trophoblasts can be chromosomally male (XY) or female (XX). While male and female trophoblasts are associated with distinct placental gene signatures, specific differences between male and female EVT have not been defined across the first trimester. MethodsTo understand how male and female EVT differ, we subjected male and female first trimester EVT cell preparations to bulk RNA sequencing. Concurrently, publicly available single-cell RNA sequencing datasets of first trimester placental and decidual tissues were utilized to resolve EVT differentiation and EVT subtype-specific sex differences. Candidate genes were then selected and immuno-localized to specific regions and cell populations in male and female placentas. ResultsWe found that before week 10 of gestation, both male and female EVT lineage cells increase expression of transcripts associated with cell proliferation. Sex-related gene differences within this early developmental time-point are restricted to genes residing on sex chromosomes. Following week 10 of gestation, there is a broad up-regulation of genes linked to immunoregulation in male and female EVT. However, within this later developmental period, autosomal gene differences appear in relation to biological sex. We go on to show that these sex-dependent autosomal gene differences influence EVT-maternal cell signalling within the uterus whereby pregnancies exposed to a male placenta demonstrate more complex MIF and CD99 as well as angiogenesis-associated VEGF cell-cell signals between male EVT and the female maternal immune and non-immune cells found throughout the uterus. ConclusionsThese findings resolve early first trimester EVT lineage trophoblast sex differences and highlight a developmental timepoint that is critical to male and female autosomal gene expression. HIGHLIGHTSO_LIFirst trimester male and female placenta cells were compared using bulk and single-cell transcriptomics C_LIO_LIGestational age and placental sex are the leading drivers of variation in gene expression C_LIO_LISex-related differences in autosomal genes generally arise on week 10 of gestation C_LIO_LISex-related differences in trophoblast and uterine cell crosstalk are driven by autosomal gene differences arising after week 10 of gestation C_LI PLAIN ENGLISH SUMMARYThe human placenta is a temporary organ that forms during pregnancy. Importantly, the placenta acts as a surrogate for not yet functioning fetal organ systems (i.e., the heart, lungs, and kidneys) while they mature within the fetus. Because the placenta develops from cells of the early embryo, it can be biologically male or female. Male and female placentas are genetically different from each other, where these differences may differentially influence placental functions and pregnancy health through unknown mechanisms. Therefore, this study aimed to understand how male and female placenta cells differ at the level of gene expression. We find that on week 10 of gestation, but not before, female placenta cells express a different repertoire of genes compared to male placenta cells. We show that these differences potentially affect how cells of the placenta and uterus interact with each other. Taken together, these results identify a developmental timepoint in pregnancy where the biological sex of the placenta may instruct subtle differences in how male or female placenta cells communicate with the maternal compartment in pregnancy. Summary StatementBulk and single-cell RNA sequencing provides comprehensive comparison between uncultured male and female HLA-G-purified trophoblasts derived from first-trimester human placentas.

BackgroundUnderstanding the causes of the exceptional rate of evolution of the mammalian placenta is likely to aid the understanding of placental development and the aetiology of the human-specific pregnancy disorder pre-eclampsia (PE). As retroelements (REs) are often lineage-specific and known to be co-opted for placental functioning, here we consider the RE binding of GATA3 and DLX5, these transcription factors being dysregulated in PE, and their downstream consequences. MethodsMultiomics analyses identified the retroviral regulatory sequence LTR8B in the PSG gene array, as a contributor to expression diversification in the placenta. To characterize this genomic domain, we performed copy number variation analysis and whole-genome sequencing. Multiomics data was employed to identify loci that might act as an active chromatin loop boundary around the PSG region. CRISPR-Cas9 knockouts with aligned RNAseq and epigenetic mark data tested for trophoblast-specific cis-regulatory elements (CREs-enhancer and/or promoter sequences) of resulting loci. Functional assays were employed to characterize the phenotypic effects of a candidate locus. Structural analysis of PSG family members also identified an additional RE, MER65-int. RNA-seq and antibody staining was employed to consider polyadenylation and functional diversification. ResultsThe LTR8B CRE facilitates the binding of transcription factors (e.g., GATA3, DLX5, TFAP2A/C), resulting in a diversified PSG gene expression pattern within a primate-specific genomic region that exhibits high intraspecies variability. The LTR8B/PSG9 regulatory element influences other PSG family members. PSG9, unique among PSGs, produces both secreted and membrane-anchored isoforms, MER65-int providing alternative polyA signals, enabling the evolution of secreted PSG variants through the truncation of the ancestral CEACAM proteins transmembrane domain. The LTR8B/PSG9 locus regulates the differentiation of multinucleated trophoblasts (syncytialization) and, like chorionic gonadotropin and syncytin1, determines the identity of syncytiotrophoblasts. Notably, PSG9 is the most upregulated PSG in PE, with levels correlated with GATA3 and DLX5 levels. ConclusionsREs contribute to the structural and expression evolution of PSG genes, facilitating lineage-specific placental evolution. The LTR8B/PSG9 regulatory network plays a central role in syncytiotrophoblast differentiation. Given the association between DLX5/GATA3 dysregulation and elevated PSG9 levels, along with PSG9s expression in the first trimester, PSG9 shows potential as a predictive biomarker for PE.

BackgroundPlacental-originated cell-free DNA (cfDNA) provides unique opportunities to study (epi)genetic placental programming remotely, but studies investigating the cfDNA methylome are scarce and usually technologically challenging. Methylated DNA sequencing (MeD-seq) is well-compatible with low cfDNA concentrations and has a high genome-wide coverage. We therefore aim to investigate the feasibility of genome-wide methylation profiling of first trimester maternal cfDNA using MeD-seq, by identifying placental-specific methylation marks in cfDNA. MethodsWe collected cfDNA from non-pregnant controls (female n=6, male n=12) and pregnant women (n=10), first trimester placentas (n=10), and paired preconceptional and first trimester buffy coats (total n=20). Differentially methylated regions (DMRs) were identified between pregnant and non-pregnant women. We investigated placental-specific markers in maternal cfDNA, including RASSF1 promoter and Y-chromosomal methylation, and studied overlap with placental and buffy coat DNA methylation. ResultsWe identified 436 DMRs between cfDNA from pregnant and non-pregnant women which were validated using male cfDNA. RASSF1 promoter methylation was higher in maternal cfDNA (fold change 2.87, unpaired t-test p<0.0001). Differential methylation of Y-chromosomal sequences could determine fetal sex. DMRs in maternal cfDNA showed large overlap with DNA methylation of these regions in placentas and buffy coats, indicating a placental and immune-cell contribution to the pregnancy-specific cfDNA methylation signature. Sixteen DMRs in maternal cfDNA were specifically found only in placentas. These novel potential placental-specific DMRs were more prominent than RASSF1. ConclusionsMeD-seq can detect (novel) genome-wide placental DNA methylation marks and determine fetal sex in maternal cfDNA. This study supports future research into maternal cfDNA methylation using MeD-seq. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=134 SRC="FIGDIR/small/610227v1_ufig1.gif" ALT="Figure 1"> View larger version (51K): org.highwire.dtl.DTLVardef@b8fd55org.highwire.dtl.DTLVardef@ffe942org.highwire.dtl.DTLVardef@12afbf4org.highwire.dtl.DTLVardef@1036a6a_HPS_FORMAT_FIGEXP M_FIG C_FIG Studies investigating the maternal cell-free DNA (cfDNA) methylome are scarce and generally technologically challenging. We identified 436 autosomal differentially methylated regions (DMRs) between cfDNA from pregnant and non-pregnant women, using the innovative methylated DNA sequencing (MeD-seq) technique. Y-chromosomal methylation could determine fetal sex, we show hypermethylation of the placental-marker RASSF1, and identify 16 novel placental-specific markers in maternal cfDNA including DMRs related to TMEM240, DHRS3, and PCMTD2. This pilot study supports future research into the maternal cfDNA methylome using MeD-seq.

BackgroundGenomic imprinting is an epigenetic phenomenon that results in parent-of-origin-specific gene expression and has been extensively characterized in mice and humans. However, in pigs, imprinting has been investigated primarily through analyses of orthologs of known imprinted genes in mice and humans. The objective of this study was to examine DNA methylation status and gene expression at a porcine locus containing newly identified imprinted calcitonin receptor-stimulating peptide (CRSP)-encoding genes, to compare orthologous loci in mice and humans, and to investigate a potential underlying mechanism. ResultsAnalyses of differentially methylated regions (DMRs) between porcine parthenogenetic embryos and biparental controls revealed multiple parental DMRs at a locus we term the CRSP complex locus, which harbors CRSP-encoding genes that likely arose through gene duplication. In contrast, orthologous genomic intervals in mice and humans exhibited unmethylated promoters and lacked evidence of imprinting. Consistently, CRSP-encoding genes in pigs showed parent-of-origin-specific monoallelic expression, whereas genes within the orthologous locus in mice and humans were biallelically expressed. Further analysis indicated that porcine CRSP promoters are embedded within oocyte-expressed alternative transcripts and co-occurred with DNA methylation, suggesting a transcription-dependent imprinting mechanism. ConclusionsOur comparative analyses identified CRSP-encoding genes at the porcine CRSP complex locus as novel imprinted genes, indicating species-specific evolution of this imprinted domain. The results further suggest that lineage-specific gene duplication may have contributed to the emergence of imprinting at this locus.

BackgroundDNA methylation (DNAm) may play an important role in birth outcomes. Material and MethodsGenome wide DNAm was analysed in peripheral blood DNA of women at multiple time points during gestation. A novel empirical method was used to identify CpG sites with high temporal variance in methylation associating with preterm birth. ResultsHigh variability at 1296 CpG sites from the promoter regions of 1197 genes significantly associated with PTB. These genes belonged to pathways involved in signalling by platelet derived growth factor, platelet homeostasis, collagen degradation, extracellular matrix and circadian clock. ConclusionsThe findings provide novel information which might help in development of predictive biomarkers of preterm birth outcome.

BackgroundPregnancy requires a delicate balance between the maternal immune system and inflammatory responses. Elevated maternal body mass index (BMI) significantly compromises the immune system and increases systemic inflammation. High maternal BMI is associated with adverse pregnancy outcomes, including an increased risk of both pre-eclampsia and preterm birth, which may be mediated through immune-related blood cell changes. MethodsThis study used Mendelian randomisation (MR) to investigate the causal relationship between maternal BMI and pregnancy outcomes, including birth weight, placental weight, gestational duration, and pre-eclampsia. We applied two-step MR to assess whether immune-related blood counts, such as neutrophils, lymphocytes, and platelets, mediate these relationships. Single nucleotide polymorphism (SNP) effect estimates for maternal BMI and pregnancy outcomes were sourced from publicly available genome-wide association studies (GWAS), with pregnancy outcomes partitioned into maternal genetic effects to proxy genetic effects on the intrauterine environment. ResultsWe found that elevated maternal BMI causally increased placental weight ({beta}IVW = 0.164sd, P = 2.92 x 10-7) and risk of pre-eclampsia (ORiVW 1.75, P = 6.3 x 10-30). The effect of maternal BMI on placental weight was larger than its effect on birth weight. Mediation analysis found no evidence of the involvement of immune-related blood counts in these relationships. ConclusionsMaternal BMI has a significant impact on pregnancy outcomes, particularly by increasing placental weight and the risk of pre-eclampsia. These findings highlight BMI-driven placental adaptations as key contributors to pregnancy complications.

BACKGROUNDDysregulated myometrial contractility contributes to obstetric complications. Sphingosine-1-phosphate (S1P) is an important inflammatory regulator in the myometrium and decidua, yet its metabolic dynamics during pregnancy are poorly characterized. This study aimed to profile the expression of S1P metabolic enzymes and receptors, and quantify sphingolipid metabolism in human gestational tissues across pregnancy. METHODSMyometrium, decidua parietalis, and chorioamnion were collected from women undergoing cesarean sections at term ([&ge;]37 weeks gestation) without labor (TNL), at term with labor (TL), and preterm (<37 weeks gestation) without labor (PTNL). Messenger RNA (mRNA) expression of S1P metabolic enzymes and receptors was assessed using quantitative polymerase chain reaction, while sphingolipids were quantified using targeted liquid chromatography-tandem mass spectrometry. RESULTSS1P metabolic enzymes and receptors were differentially expressed across gestational tissues. At TNL, SPHK1 expression was significantly higher in the decidua parietalis than in the chorioamnion and myometrium. The myometrium exhibited the highest mRNA expression of S1P receptors (S1PR1-4) compared to the decidua and chorioamnion. At term, S1P was more abundant in the myometrium than in the decidua parietalis and chorioamnion. Both SPHK1 and S1P were significantly increased in TL compared to TNL myometrium. S1P levels were higher in the myometrium at TNL compared to PTNL, while no significant differences were observed in the decidua and chorioamnion. Overall, sphingolipid metabolism was highest in the decidua and myometrium and lowest in the chorioamnion at term. CONCLUSIONThese findings reveal tissue-specific regulation of S1P metabolism and signaling in human gestational tissues, suggesting a therapeutic role of S1P in modulating myometrial contractility.