RNAseqCovarImpute: a multiple imputation procedure that outperforms complete case and single imputation differential expression analysis.

Missing covariate data is a common problem that has not been addressed in observational studies of gene expression. Here, we present a multiple imputation method that accommodates high dimensional gene expression data by incorporating principal component analysis of the transcriptome into the multiple imputation prediction models to avoid bias. Simulation studies using three datasets show that this method outperforms complete case and single imputation analyses at uncovering true positive differentially expressed genes, limiting false discovery rates, and minimizing bias. This method is easily implemented via an R Bioconductor package, RNAseqCovarImpute that integrates with the limma-voom pipeline for differential expression analysis.

Transcriptomic comparison of in vitro models of the human placenta.

Studying the human placenta through in vitro cell culture methods is necessary due to limited access and amenability of human placental tissue to certain experimental methods as well as distinct anatomical and physiological differences between animal and human placentas. Selecting an in vitro culture model of the human placenta is challenging due to representation of different trophoblast cell types with distinct biological roles and limited comparative studies that define key characteristics of these models. Therefore, the aim of this research was to create a comprehensive transcriptomic comparison of common in vitro models of the human placenta compared to bulk placental tissue from the CANDLE and GAPPS cohorts (N=1083). We performed differential gene expression analysis on publicly available RNA sequencing data from 6 common in vitro models of the human placenta (HTR-8/SVneo, BeWo, JEG-3, JAR, Primary Trophoblasts, and Villous Explants) and compared to CANDLE and GAPPS bulk placental tissue or cytotrophoblast, syncytiotrophoblast, and extravillous trophoblast cell types derived from bulk placental tissue. All in vitro placental models had a substantial number of differentially expressed genes (DEGs, FDR<0.01) compared to the CANDLE and GAPPS placentas (Average DEGs=10,873), and the individual trophoblast cell types (Average DEGs=5,346), indicating that there are vast differences in gene expression compared to bulk and cell-type specific human placental tissue. Hierarchical clustering identified 53 gene clusters with distinct expression profiles across placental models, with 22 clusters enriched for specific KEGG pathways, 7 clusters enriched for high-expression placental genes, and 7 clusters enriched for absorption, distribution, metabolism, and excretion genes. In vitro placental models were classified by fetal sex based on expression of Y-chromosome genes that identified HTR-8/SVneo cells as being of female origin, while JEG-3, JAR, and BeWo cells are of male origin. Overall, none of the models were a close approximation of the transcriptome of bulk human placental tissue, highlighting the challenges with model selection. To enable researchers to select appropriate models, we have compiled data on differential gene expression, clustering, and fetal sex into an accessible web application: "Comparative Transcriptomic Placental Model Atlas (CTPMA)" which can be utilized by researchers to make informed decisions about their selection of in vitro placental models.

Associations Between Prenatal Vitamin D and Placental Gene Expression.

Background: Vitamin D is a hormone regulating gene transcription. Prenatal vitamin D has been linked to immune and vascular function in the placenta, a key organ of pregnancy. To date, studies of vitamin D and placental gene expression have focused on a limited number of candidate genes. Transcriptome-wide RNA sequencing can provide a more complete representation of the placental effects of vitamin D. Objective: We investigated the association between prenatal vitamin D levels and placental gene expression in a large, prospective pregnancy cohort. Methods: Participants were recruited in Shelby County, Tennessee in the Conditions Affecting Neurocognitive Development and Learning in Early childhood (CANDLE) study. Vitamin D level (plasma total 25-hydroxyvitatmin D, [25(OH)D]) was measured at mid-pregnancy (16-28 weeks' gestation) and delivery. Placenta samples were collected at birth. RNA was isolated and sequenced. We identified differentially expressed genes (DEGs) using adjusted linear regression models. We also conducted weighted gene co-expression network analysis (WGCNA). Results: The median 25(OH)D of participants was 21.8 ng/mL at mid-pregnancy (N=774, IQR: 15.4-26.5 ng/mL) and 23.6 ng/mL at delivery (N=753, IQR: 16.8-29.1 ng/mL). Placental expression of 25 DEGs was associated with 25(OH)D at mid-pregnancy, but no DEG was associated with 25(OH)D at delivery. DEGs were related to energy metabolism, cytoskeletal function, and RNA transcription. Using WGCNA, we identified 2 gene modules whose expression was associated with 25(OH)D at mid-pregnancy and 1 module associated with 25(OH)D at delivery. These modules were enriched for genes related to mitochondrial and cytoskeletal function, and were regulated by transcription factors including ARNT2, BHLHE40, FOSL2, JUND, and NFKB1. Conclusions: Our results indicate that 25(OH)D during mid-pregnancy, but not at delivery, is associated with placental gene expression at birth. Future research is needed to investigate a potential role of vitamin D in programming placental mitochondrial metabolism, intracellular transport, and transcriptional regulation during pregnancy.

Timing of selective serotonin reuptake inhibitor use and risk for preterm birth and related adverse events: with a consideration of the COVID-19 pandemic period.

OBJECTIVE: There is uncertainty around the safety of SSRIs for treating depression during pregnancy. Nevertheless, the use of SSRIs has been gradually increasing, especially during the COVID-19 pandemic period. We aimed to (1) characterize maternal depression rate and use of SSRIs in a recent 10-year period, (2) address confounding by indication, as well as socioeconomic and environmental factors, and (3) evaluate associations of the timing of SSRI exposure in pregnancy with risk for preterm birth (PTB), low birthweight (LBW), and small for gestational age (SGA) infants among women with depression before pregnancy. METHODS: We conducted propensity score-adjusted regression to calculate odds ratios (ORs) of PTB, LBW, and SGA. We accounted for maternal/pregnancy characteristics, comorbidity, depression severity, time of delivery, social vulnerability, and rural residence. RESULTS: There were 50.3% and 40.3% increases in the prevalence rate of prenatal depression and prenatal SSRI prescription rate during the pandemic. We identified women with depression ≤180 days before pregnancy (n = 8406). Women with no SSRI order during pregnancy (n = 3760) constituted the unexposed group. The late SSRI exposure group consisted of women with an SSRI order after the first trimester (n = 3759). The early-only SSRI exposure group consisted of women with SSRI orders only in the first trimester (n = 887). The late SSRI exposure group had an increased risk of PTB of OR = 1.5 ([1.2,1.8]) and LBW of OR = 1.5 ([1.2,2.0]), relative to the unexposed group. Associations between late SSRI exposure and risk of PTB/LBW were similar among a subsample of patients who delivered during the pandemic. CONCLUSIONS: These findings suggest an association between PTB/LBW and SSRI exposure is dependent on exposure timing during pregnancy. Small for gestational age is not associated with SSRI exposure.

Ultra-processed and fast food consumption, exposure to phthalates during pregnancy, and socioeconomic disparities in phthalate exposures.

BACKGROUND: Consuming ultra-processed foods may increase exposure to phthalates, a group of endocrine disruptors prevalent in food contact materials. OBJECTIVES: Investigate associations between ultra-processed food intake and urinary phthalates during pregnancy, and evaluate whether ultra-processed foods mediate socioeconomic disparities in phthalate exposures. METHODS: In a socioeconomically diverse sample of 1031 pregnant women from the Conditions Affecting Neurocognitive Development and Learning in Early Childhood (CANDLE) Study in the urban South, the Block Food Frequency Questionnaire was administered and urinary phthalate metabolites were measured in the second trimester. Linear regressions modeled associations between phthalates and overall ultra-processed food consumption, individual ultra-processed foods, and exploratory factor analysis dietary patterns. Causal mediation analyses examined whether ultra-processed food intake mediates relationships between socioeconomic disparities and phthalate exposures. RESULTS: Ultra-processed foods constituted 9.8-59.0 % (mean = 38.6 %) of participants' diets. 10 % higher dietary proportion of ultra-processed foods was associated with 13.1 % (95 %CI: 3.4 %-22.9 %) higher molar sum concentrations of di(2-ethylhexyl) phthalate metabolites (ΣDEHP). 10 % higher consumption of minimally-processed foods was associated with lower ΣDEHP (10.8 %: 3.4 %-22.9 %). Ultra- and minimally-processed food consumption were not associated with non-DEHP metabolites. Standard deviation higher consumptions of hamburger/cheeseburger, French fries, soda, and cake were associated with 10.5 % (4.2 %-17.1 %), 9.2 % (2.6 %-16.2 %), 7.4 % (1.4 %-13.6 %), and 6.0 % (0.0 %-12.4 %), respectively, higher ΣDEHP. Exploratory factor analysis corroborated positive associations of processed food with ΣDEHP, and uncovered a healthy dietary pattern associated with lower urinary ΣDEHP, mono(2-ethyl-5-hydroxyhexyl) (MEHHP), mono(2-ethyl-5-carboxypentyl) (MECPP), mono(2-carboxymethylhexyl) (MCMHP), and mono-isononyl (MINP) phthalates. Significant indirect effects indicated that lower income and education levels were associated with 1.9 % (0.2 %-4.2 %) and 1.4 % (0.1 %-3.3 %) higher ΣDEHP, respectively, mediated via increased ultra-processed food consumption. CONCLUSIONS: Consumption of ultra-processed foods may increase exposure to phthalates. Policies to reduce dietary phthalate exposures from food packaging and processing are needed, as socioeconomic barriers can preclude dietary recommendations as a sole means to reduce phthalate exposures.

Placental transcriptomic signatures of prenatal and preconceptional maternal stress.

Prenatal exposure to maternal psychological stress is associated with increased risk for adverse birth and child health outcomes. Accumulating evidence suggests that preconceptional maternal stress may also be transmitted intergenerationally to negatively impact offspring. However, understanding of mechanisms linking these exposures to offspring outcomes, particularly those related to placenta, is limited. Using RNA sequencing, we identified placental transcriptomic signatures associated with maternal prenatal stressful life events (SLEs) and childhood traumatic events (CTEs) in 1 029 mother-child pairs in two birth cohorts from Washington state and Memphis, Tennessee. We evaluated individual gene-SLE/CTE associations and performed an ensemble of gene set enrichment analyses combing across 11 popular enrichment methods. Higher number of prenatal SLEs was significantly (FDR < 0.05) associated with increased expression of ADGRG6, a placental tissue-specific gene critical in placental remodeling, and decreased expression of RAB11FIP3, an endocytosis and endocytic recycling gene, and SMYD5, a histone methyltransferase. Prenatal SLEs and maternal CTEs were associated with gene sets related to several biological pathways, including upregulation of protein processing in the endoplasmic reticulum, protein secretion, and ubiquitin mediated proteolysis, and down regulation of ribosome, epithelial mesenchymal transition, DNA repair, MYC targets, and amino acid-related pathways. The directional associations in these pathways corroborate prior non-transcriptomic mechanistic studies of psychological stress and mental health disorders, and have previously been implicated in pregnancy complications and adverse birth outcomes. Accordingly, our findings suggest that maternal exposure to psychosocial stressors during pregnancy as well as the mother's childhood may disrupt placental function, which may ultimately contribute to adverse pregnancy, birth, and child health outcomes.

Placental single cell transcriptomics: Opportunities for endocrine disrupting chemical toxicology.

The placenta performs essential biologic functions for fetal development throughout pregnancy. Placental dysfunction is at the root of multiple adverse birth outcomes such as intrauterine growth restriction, preeclampsia, and preterm birth. Exposure to endocrine disrupting chemicals during pregnancy can cause placental dysfunction, and many prior human studies have examined molecular changes in bulk placental tissues. Placenta-specific cell types, including cytotrophoblasts, syncytiotrophoblasts, extravillous trophoblasts, and placental resident macrophage Hofbauer cells play unique roles in placental development, structure, and function. Toxicant-induced changes in relative abundance and/or impairment of these cell types likely contribute to placental pathogenesis. Although gene expression insights gained from bulk placental tissue RNA-sequencing data are useful, their interpretation is limited because bulk analysis can mask the effects of a chemical on individual populations of placental cells. Cutting-edge single cell RNA-sequencing technologies are enabling the investigation of placental cell-type specific responses to endocrine disrupting chemicals. Moreover, in situ bioinformatic cell deconvolution enables the estimation of cell type proportions in bulk placental tissue gene expression data. These emerging technologies have tremendous potential to provide novel mechanistic insights in a complex heterogeneous tissue with implications for toxicant contributions to adverse pregnancy outcomes.

Associations of prenatal exposure to NO2 and near roadway residence with placental gene expression.

INTRODUCTION: Traffic-related air pollution (TRAP), a common exposure, potentially impacts pregnancy through altered placental function. We investigated associations between prenatal TRAP exposure and placental gene expression. METHODS: Whole transcriptome sequencing was performed on placental samples from CANDLE (Memphis, TN) (n = 776) and GAPPS (Seattle and Yakima, WA) (n = 205), cohorts of the ECHO-PATHWAYS Consortium. Residential NO2 exposures were computed via spatiotemporal models for full-pregnancy, each trimester, and the first/last months of pregnancy. Individual cohort-specific, covariate-adjusted linear models were fit for 10,855 genes and respective exposures (NO2 or roadway proximity [≤150 m]). Infant-sex/exposure interactions on placental gene expression were tested with interaction terms in separate models. Significance was based on false discovery rate (FDR<0.10). RESULTS: In GAPPS, final-month NO2 exposure was positively associated with MAP1LC3C expression (FDR p-value = 0.094). Infant-sex interacted with second-trimester NO2 on STRIP2 expression (FDR interaction p-value = 0.011, inverse and positive associations among male and female infants, respectively) and roadway proximity on CEBPA expression (FDR interaction p-value = 0.045, inverse among females). In CANDLE, infant-sex interacted with first-trimester and full-pregnancy NO2 on RASSF7 expression (FDR interaction p-values = 0.067 and 0.013, respectively, positive among male infants and inverse among female infants). DISCUSSION: Overall, pregnancy NO2 exposure and placental gene expression associations were primarily null, with exception of final month NO2 exposure and placental MAP1LC3C association. We found several interactions of infant sex and TRAP exposures on placental expression of STRIP2, CEBPA, and RASSF7. These highlighted genes suggest influence of TRAP on placental cell proliferation, autophagy, and growth, though additional replication and functional studies are required for validation.

Transcriptomic Signatures of Single-Suture Craniosynostosis Phenotypes.

Craniosynostosis is a birth defect where calvarial sutures close prematurely, as part of a genetic syndrome or independently, with unknown cause. This study aimed to identify differences in gene expression in primary calvarial cell lines derived from patients with four phenotypes of single-suture craniosynostosis, compared to controls. Calvarial bone samples (N = 388 cases/85 controls) were collected from clinical sites during reconstructive skull surgery. Primary cell lines were then derived from the tissue and used for RNA sequencing. Linear models were fit to estimate covariate adjusted associations between gene expression and four phenotypes of single-suture craniosynostosis (lambdoid, metopic, sagittal, and coronal), compared to controls. Sex-stratified analysis was also performed for each phenotype. Differentially expressed genes (DEGs) included 72 genes associated with coronal, 90 genes associated with sagittal, 103 genes associated with metopic, and 33 genes associated with lambdoid craniosynostosis. The sex-stratified analysis revealed more DEGs in males (98) than females (4). There were 16 DEGs that were homeobox (HOX) genes. Three TFs (SUZ12, EZH2, AR) significantly regulated expression of DEGs in one or more phenotypes. Pathway analysis identified four KEGG pathways associated with at least one phenotype of craniosynostosis. Together, this work suggests unique molecular mechanisms related to craniosynostosis phenotype and fetal sex.

SLC20a1/PiT-1 is required for chorioallantoic placental morphogenesis.

The placenta mediates the transport of nutrients, such as inorganic phosphate (Pi), between the maternal and fetal circulatory systems. The placenta itself also requires high levels of nutrient uptake as it develops to provide critical support for fetal development. This study aimed to determine placental Pi transport mechanisms using in vitro and in vivo models. We observed that Pi (P33) uptake in BeWo cells is sodium dependent and that SLC20A1/Slc20a1 is the most highly expressed placental sodium-dependent transporter in mouse (microarray), human cell line (RT-PCR) and term placenta (RNA-seq), supporting that normal growth and maintenance of the mouse and human placenta requires SLC20A1/Slc20a1. Slc20a1 wild-type (Slc20a1+/+) and knockout (Slc20a1-/-) mice were produced through timed intercrosses and displayed yolk sac angiogenesis failure as expected at E10.5. E9.5 tissues were analyzed to test whether placental morphogenesis requires Slc20a1. At E9.5, the developing placenta was reduced in size in Slc20a1-/-. Multiple structural abnormalities were also observed in the Slc20a1-/-chorioallantois. We determined that monocarboxylate transporter 1 protein (MCT1+) cells were reduced in developing Slc20a1-/-placenta, confirming that Slc20a1 loss reduced trophoblast syncytiotrophoblast 1 (SynT-I) coverage. Next, we examined the cell type-specific Slc20a1 expression and SynT molecular pathways in silico and identified Notch/Wnt as a pathway of interest that regulates trophoblast differentiation. We further observed that specific trophoblast lineages express Notch/Wnt genes that associate with endothelial cell tip-and-stalk cell markers. In conclusion, our findings support that Slc20a1 mediates the symport of Pi into SynT cells, providing critical support for their differentiation and angiogenic mimicry function at the developing maternal-fetal interface.

Mono(2-ethylhexyl) phthalate induces transcriptomic changes in placental cells based on concentration, fetal sex, and trophoblast cell type.

Phthalates are ubiquitous plasticizer chemicals found in consumer products. Exposure to phthalates during pregnancy has been associated with adverse pregnancy and birth outcomes and differences in placental gene expression in human studies. The objective of this research was to evaluate global changes in placental gene expression via RNA sequencing in two placental cell models following exposure to the phthalate metabolite mono(2-ethylhexyl) phthalate (MEHP). HTR-8/SVneo and primary syncytiotrophoblast cells were exposed to three concentrations (1, 90, 180 µM) of MEHP for 24 h with DMSO (0.1%) as a vehicle control. mRNA and lncRNAs were quantified using paired-end RNA sequencing, followed by identification of differentially expressed genes (DEGs), significant KEGG pathways, and enriched transcription factors (TFs). MEHP caused gene expression changes across all concentrations for HTR-8/SVneo and primary syncytiotrophoblast cells. Sex-stratified analysis of primary cells identified different patterns of sensitivity in response to MEHP dose by sex, with male placentas being more responsive to MEHP exposure. Pathway analysis identified 11 KEGG pathways significantly associated with at least one concentration in both cell types. Four ligand-inducible nuclear hormone TFs (PPARG, PPARD, ESR1, AR) were enriched in at least three treatment groups. Overall, we demonstrated that MEHP differentially affects placental gene expression based on concentration, fetal sex, and trophoblast cell type. This study confirms prior studies, as enrichment of nuclear hormone receptor TFs were concordant with previously published mechanisms of phthalate disruption, and generates new hypotheses, as we identified many pathways and genes not previously linked to phthalate exposure.

Placental transcriptomic signatures of prenatal exposure to Hydroxy-Polycyclic aromatic hydrocarbons.

BACKGROUND: Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous pollutants originating from petrogenic and pyrogenic sources. PAH compounds can cross the placenta, and prenatal PAH exposure is linked to adverse infant and childhood health outcomes. OBJECTIVE: In this first human transcriptomic assessment of PAHs in the placenta, we examined associations between prenatal PAH exposure and placental gene expression to gain insight into mechanisms by which PAHs may disrupt placental function. METHODS: The ECHO PATHWAYS Consortium quantified prenatal PAH exposure and the placental transcriptome from 629 pregnant participants enrolled in the CANDLE study. Concentrations of 12 monohydroxy-PAH (OH-PAH) metabolites were measured in mid-pregnancy urine using high performance liquid chromatography tandem mass spectrometry. Placental transcriptomic data were obtained using paired-end RNA sequencing. Linear models were fitted to estimate covariate-adjusted associations between maternal urinary OH-PAHs and placental gene expression. We performed sex-stratified analyses to evaluate whether associations varied by fetal sex. Selected PAH/gene expression analyses were validated by treating HTR-8/SVneo cells with phenanthrene, and quantifying expression via qPCR. RESULTS: Urinary concentrations of 6 OH-PAHs were associated with placental expression of 8 genes. Three biological pathways were associated with 4 OH-PAHs. Placental expression of SGF29 and TRIP13 as well as the vitamin digestion and absorption pathway were positively associated with multiple metabolites. HTR-8/SVneo cells treated with phenanthrene also exhibited 23 % increased TRIP13 expression compared to vehicle controls (p = 0.04). Fetal sex may modify the relationship between prenatal OH-PAHs and placental gene expression, as more associations were identified in females than males (45 vs 28 associations). DISCUSSION: Our study highlights novel genes whose placental expression may be disrupted by OH-PAHs. Increased expression of DNA damage repair gene TRIP13 may represent a response to double-stranded DNA breaks. Increased expression of genes involved in vitamin digestion and metabolism may reflect dietary exposures or represent a compensatory mechanism to combat damage related to OH-PAH toxicity. Further work is needed to study the role of these genes in placental function and their links to perinatal outcomes and lifelong health.

Placental transcriptomic signatures of spontaneous preterm birth.

BACKGROUND: Spontaneous preterm birth accounts for most preterm births and leads to significant morbidity in the newborn and childhood period. This subtype of preterm birth represents an increasing proportion of all preterm births when compared with medically indicated preterm birth, yet it is understudied in omics analyses. The placenta is a key regulator of fetal and newborn health, and the placental transcriptome can provide insight into pathologic changes that lead to spontaneous preterm birth. OBJECTIVE: This analysis aimed to identify genes for which placental expression was associated with spontaneous preterm birth (including early preterm and late preterm birth). STUDY DESIGN: The ECHO PATHWAYS consortium extracted RNA from placental samples collected from the Conditions Affecting Neurocognitive Development and Learning in Early Childhood and the Global Alliance to Prevent Prematurity and Stillbirth studies. Placental transcriptomic data were obtained by RNA sequencing. Linear models were fit to estimate differences in placental gene expression between term birth and spontaneous preterm birth (including gestational age subgroups defined by the American College of Obstetricians and Gynecologists). Models were adjusted for numerous confounding variables, including labor status, cohort, and RNA sequencing batch. This analysis excluded patients with induced labor, chorioamnionitis, multifetal gestations, or medical indications for preterm birth. Our combined cohort contained gene expression data for 14,023 genes in 48 preterm and 540 term samples. Genes and pathways were considered statistically significantly different at false discovery rate-adjusted P value of <.05. RESULTS: In total, we identified 1728 genes for which placental expression was associated with spontaneous preterm birth with more differences in expression in early preterm samples than late preterm samples when compared with full-term samples. Of those, 9 genes were significantly decreased in both early and late spontaneous preterm birth, and the strongest associations involved placental expression of IL1B, ALPL, and CRLF1. In early and late preterm samples, we observed decreased expression of genes involved in immune signaling, signal transduction, and endocrine function. CONCLUSION: This study provides a comprehensive assessment of the differences in the placental transcriptome associated with spontaneous preterm birth with robust adjustment for confounding. Results of this study are in alignment with the known etiology of spontaneous preterm birth, because we identified multiple genes and pathways for which the placental and chorioamniotic membrane expression was previously associated with prematurity, including IL1B. We identified decreased expression in key signaling pathways that are essential for placental growth and function, which may be related to the etiology of spontaneous preterm birth. We identified increased expression of genes within metabolic pathways associated exclusively with early preterm birth. These signaling and metabolic pathways may provide clinically targetable pathways and biomarkers. The findings presented here can be used to understand underlying pathologic changes in premature placentas, which can inform and improve clinical obstetrics practice.

Prenatal exposure to particulate matter and placental gene expression.

BACKGROUND: While strong evidence supports adverse maternal and offspring consequences of air pollution, mechanisms that involve the placenta, a key part of the intrauterine environment, are largely unknown. Previous studies of air pollution and placental gene expression were small candidate gene studies that rarely considered prenatal windows of exposure or the potential role of offspring sex. We examined overall and sex-specific associations of prenatal exposure to fine particulate matter (PM2.5) with genome-wide placental gene expression. METHODS: Participants with placenta samples, collected at birth, and childhood health outcomes from CANDLE (Memphis, TN) (n = 776) and GAPPS (Seattle, WA) (n = 205) cohorts of the ECHO-PATHWAYS Consortium were included in this study. PM2.5 exposures during trimesters 1, 2, 3, and the first and last months of pregnancy, were estimated using a spatiotemporal model. Cohort-specific linear adjusted models were fit for each exposure window and expression of >11,000 protein coding genes from paired end RNA sequencing data. Models with interaction terms were used to examine PM2.5-offspring sex interactions. False discovery rate (FDR < 0.10) was used to correct for multiple testing. RESULTS: Mean PM2.5 estimate was 10.5-10.7 µg/m3 for CANDLE and 6.0-6.3 µg/m3 for GAPPS participants. In CANDLE, expression of 13 (11 upregulated and 2 downregulated), 20 (11 upregulated and 9 downregulated) and 3 (2 upregulated and 1 downregulated) genes was associated with PM2.5 in the first trimester, second trimester, and first month, respectively. While we did not find any statistically significant association, overall, between PM2.5 and gene expression in GAPPS, we found offspring sex and first month PM2.5 interaction for DDHD1 expression (positive association among males and inverse association among females). We did not observe PM2.5 and offspring sex interactions in CANDLE. CONCLUSION: In CANDLE, but not GAPPS, we found that prenatal PM2.5 exposure during the first half of pregnancy is associated with placental gene expression.

The Placental Epigenome as a Molecular Link Between Prenatal Exposures and Fetal Health Outcomes Through the DOHaD Hypothesis.

PURPOSE OF REVIEW: The developmental origins of health and disease (DOHaD) hypothesis posits that the perinatal environment can impact fetal and later life health. The placenta is uniquely situated to assess prenatal exposures in the context of DOHaD because it is an essential ephemeral fetal organ that manages the transport of oxygen, nutrients, waste, and endocrine signals between the mother and fetus. The purpose of this review is to summarize recent studies that evaluated the DOHaD hypothesis in human placentas using epigenomics, including DNA methylation and transcriptomic studies of mRNA, lncRNA, and microRNAs. RECENT FINDINGS: Between 2016 and 2021, 28 articles evaluated associations between prenatal exposures and placental epigenomics across broad exposure categories including maternal smoking, psychosocial stressors, chemicals, air pollution, and metals. Sixteen of these studies connected exposures to health outcome such as birth weight, fetal growth, or infant neurobehavior through mediation analysis, identification of shared associations between exposure and outcome, or network analysis. These aspects of infant and childhood health serve as a foundation for future studies that aim to use placental epigenetics to understand relationships between the prenatal environment and perinatal complications (such as preterm birth or fetal growth restriction) or later life childhood health. Placental DNA methylation and RNA expression have been linked to numerous prenatal exposures, such as PM2.5 air pollution, metals, and maternal smoking, as well as infant and childhood health outcomes, including fetal growth and birth weight. Placental epigenomics provides a unique opportunity to expand the DOHaD premise, particularly if research applies novel methodologies such as multi-omics analysis, sequencing of non-coding RNAs, mixtures analysis, and assessment of health outcomes beyond early childhood.

A Comprehensive Assessment of Associations between Prenatal Phthalate Exposure and the Placental Transcriptomic Landscape.

BACKGROUND: Phthalates are commonly used endocrine-disrupting chemicals that are ubiquitous in the general population. Prenatal phthalate exposure may alter placental physiology and fetal development, leading to adverse perinatal and childhood health outcomes. OBJECTIVE: We examined associations between prenatal phthalate exposure in the second and third trimesters and the placental transcriptome at birth, including genes and long noncoding RNAs (lncRNAs), to gain insight into potential mechanisms of action during fetal development. METHODS: The ECHO PATHWAYs consortium quantified 21 urinary phthalate metabolites from 760 women enrolled in the CANDLE study (Shelby County, TN) using high-performance liquid chromatography-tandem mass spectrometry. Placental transcriptomic data were obtained using paired-end RNA sequencing. Linear models were fitted to estimate separate associations between maternal urinary phthalate metabolite concentration during the second and third trimester and placental gene expression at birth, adjusted for confounding variables. Genes were considered differentially expressed at a Benjamini-Hochberg false discovery rate (FDR) p<0.05. Associations between phthalate metabolites and biological pathways were identified using self-contained gene set testing and considered significantly altered with an FDR-adjusted p<0.2. RESULTS: We observed significant associations between second-trimester phthalate metabolites mono (carboxyisooctyl) phthalate (MCIOP), mono-2-ethyl-5-carboxypentyl phthalate, and mono-2-ethyl-5-oxohexyl phthalate and 18 genes in total, including four lncRNAs. Specifically, placental expression of NEAT1 was associated with multiple phthalate metabolites. Third-trimester MCIOP and mono-isobutyl phthalate concentrations were significantly associated with placental expression of 18 genes and two genes, respectively. Expression of genes within 27 biological pathways was associated with mono-methyl phthalate, MCIOP, and monoethyl phthalate concentrations. DISCUSSION: To our knowledge, this is the first genome-wide assessment of the relationship between the placental transcriptome at birth and prenatal phthalate exposure in a large and diverse birth cohort. We identified numerous genes and lncRNAs associated with prenatal phthalate exposure. These associations mirror findings from other epidemiological and in vitro analyses and may provide insight into biological pathways affected in utero by phthalate exposure. https://doi.org/10.1289/EHP8973.

Deep phenotyping during pregnancy for predictive and preventive medicine.

Deep phenotyping during pregnancy offers an opportunity to define the antecedents of lifelong health and wellness, and to improve pregnancy outcomes.

MicroRNA-transcriptome networks in whole blood and monocytes of women undergoing preterm labour.

Preterm birth is attributed to neonatal morbidity as well as cognitive and physiological challenges. We have previously identified significant differences in mRNA expression in whole blood and monocytes, as well as differences in miRNA concentration in blood plasma, extracellular vesicles (EV) and EV-depleted plasma in women undergoing spontaneous preterm labour (sPTL). The goal of this analysis was to identify differences in miRNA expression within whole blood (WB) and peripheral monocytes (PM) from the same population of women undergoing sPTL compared with non-labouring controls matched by gestational age. We performed single-end small RNA sequencing in whole blood and peripheral monocytes from women undergoing sPTL with active contractions (24-34 weeks of gestation, N = 15) matched for gestational age to healthy pregnant non-labouring controls (>37 weeks gestation, N = 30) who later delivered at term as a part of the Ontario Birth Study (Toronto, Ontario CA). We identified significant differences in expression of 16 miRNAs in PMs and nine miRNAs in WB in women undergoing sPTL. In PMs, these miRNAs were predicted targets of 541 genes, including 28 previously associated with sPTL. In WB, miRNAs were predicted to target 303 genes, including nine previously associated with sPTL. These genes were involved in a variety of immune pathways, including interleukin-2 signalling. This study is the first to identify changes in miRNA expression in WB and PMs of women undergoing sPTL. Our results shed light on potential mechanisms by which miRNAs may play a role in mediating systemic inflammatory response in pregnant women that deliver prematurely.

Distinct communication patterns of trophoblastic miRNA among the maternal-placental-fetal compartments.

INTRODUCTION: The placenta produces microRNAs (miRNA) that may traffic to the maternal or fetal compartments and influence the physiology of pregnancy. The trafficking patterns of miRNA expressed from the large human chromosome 19 and chromosome 14 clusters (C19MC and C14MC), remains unclear. We interrogated the cross-sectional landscape of miRNA expression within the human placenta, fetal and maternal plasma to elucidate miRNA trafficking. We hypothesized that C19MC and C14MC miRNAs have similar expression patterns across the maternal-fetal compartments. METHODS: Placental biopsies, maternal and fetal venous plasma were collected from 25 pregnancies, and RNA was quantified using next generation sequencing. We identified expression and correlations differences among the compartments, and uncovered distinct miRNA expression patterns using consensus clustering. RESULTS: We found that the placenta exhibits the highest total abundance, average miRNA expression and lowest variance of both C19MC and C14MC miRNAs. The C19MC miRNAs had a comparable expression and variance in fetal and maternal plasma and higher expression in the placenta. In contrast, the C14MC miRNAs had comparable expression between the placenta and fetal plasma, which was higher than the maternal plasma. We also identified 5 distinct groups of trophoblastic miRNAs with different expression patterns in each compartment. DISCUSSION: This is the first comprehensive analysis of C19MC and C14MC miRNA expression patterns in the human placental, maternal and fetal compartments. Our findings suggest that C14MC miRNAs are produced by both the fetus and placenta, but C19MC miRNAs are produced primarily in the placenta and are trafficked to the fetal and maternal compartments.