Placental Gene Transcript Proportions are Altered in the Presence of In Utero Arsenic and Cadmium Exposures, Genetic Variants, and Birth Weight Differences.

Background: In utero arsenic and cadmium exposures are linked with reduced birth weight as well as alterations in placental molecular features. However, studies thus far have focused on summarizing transcriptional activity at the gene level and do not capture transcript specification, an important resource during fetal development to enable adaptive responses to the rapidly changing in utero physiological conditions. In this study, we conducted a genome-wide analysis of the placental transcriptome to evaluate the role of differential transcript usage (DTU) as a potential marker of in utero arsenic and cadmium exposure and fetal growth restriction. Methods: Transcriptome-wide RNA sequencing was performed in placenta samples from the Rhode Island Child Health Study (RICHS, n = 199). Arsenic and cadmium levels were measured in maternal toenails using ICP-MS. Differential transcript usage (DTU) contrasting small (SGA) and appropriate (AGA) for gestational age infants as well as above vs. below median exposure to arsenic and cadmium were assessed using the DRIMSeq R package. Genetic variants that influence transcript usage were determined using the sQTLseeker R package. Results: We identified 82 genes demonstrating DTU in association with SGA status at an FDR <0.05. Among these, one gene, ORMDL1, also demonstrated DTU in association with arsenic exposure, and fifteen genes (CSNK1E, GBA, LAMTOR4, MORF4L1, PIGO, PSG1, PSG3, PTMA, RBMS1, SLC38A2, SMAD4, SPCS2, TUBA1B, UBE2A, YIPF5) demonstrated DTU in association with cadmium exposure. In addition to cadmium exposure and SGA status, proportions of the LAMTOR4 transcript ENST00000474141.5 also differed by genetic variants (rs10231604, rs12878, and rs3736591), suggesting a pathway by which an in utero exposure and genetic variants converge to impact fetal growth through perturbations of placental processes. Discussion: We report the first genome-wide characterization of placental transcript usage and associations with intrauterine metal exposure and fetal growth restriction. These results highlight the utility of interrogating the transcriptome at finer-scale transcript-level resolution to identify novel placental biomarkers of exposure-induced outcomes.

Placental DNA methylation signatures of maternal smoking during pregnancy and potential impacts on fetal growth.

Maternal smoking during pregnancy (MSDP) contributes to poor birth outcomes, in part through disrupted placental functions, which may be reflected in the placental epigenome. Here we present a meta-analysis of the associations between MSDP and placental DNA methylation (DNAm) and between DNAm and birth outcomes within the Pregnancy And Childhood Epigenetics (PACE) consortium (N = 1700, 344 with MSDP). We identify 443 CpGs that are associated with MSDP, of which 142 associated with birth outcomes, 40 associated with gene expression, and 13 CpGs are associated with all three. Only two CpGs have consistent associations from a prior meta-analysis of cord blood DNAm, demonstrating substantial tissue-specific responses to MSDP. The placental MSDP-associated CpGs are enriched for environmental response genes, growth-factor signaling, and inflammation, which play important roles in placental function. We demonstrate links between placental DNAm, MSDP and poor birth outcomes, which may better inform the mechanisms through which MSDP impacts placental function and fetal growth.

Placental lncRNA expression associated with placental cadmium concentrations and birth weight.

Heavy metal exposures, such as cadmium, can have negative effects on infant birth weight (BW)-among other developmental outcomes-with placental dysfunction potentially playing a role in these effects. In this study, we examined how differential placental expression of long non-coding RNAs (lncRNAs) may be associated with cadmium levels in placenta and whether differences in the expression of those lncRNAs were associated with fetal growth. In the Rhode Island Child Health Study, we used data from Illumina HiSeq whole transcriptome RNA sequencing (n = 199) to examine association between lncRNA expression and measures of infant BW as well as placental cadmium concentrations controlled for appropriate covariates. Of the 1191 lncRNAs sequenced, 46 demonstrated associations (q < 0.05) with BW in models controlling for infant sex, maternal age, BMI, maternal education, and smoking during pregnancy. Furthermore, four of these transcripts were associated with placental cadmium concentrations, with MIR22HG and ERVH48-1 demonstrating increases in expression associated with increasing cadmium exposure and elevated odds of small for gestational age birth, while AC114763.2 and LINC02595 demonstrated reduced expression associated with cadmium, but elevated odds of large for gestational age birth with increasing expression. We identified relationships between lncRNA expression with both placental cadmium concentrations and BW. This study provides evidence that disrupted placental expression of lncRNAs may be a part of cadmium's mechanisms of reproductive toxicity.

Placental Expression of Imprinted Genes, Overall and in Sex-Specific Patterns, Associated with Placental Cadmium Concentrations and Birth Size.

BACKGROUND: Prenatal cadmium (Cd) exposure has been recognized to restrict growth, and male and female fetuses may have differential susceptibility to the developmental toxicity of Cd. Imprinted genes, which exhibit monoallelic expression based on parent of origin, are highly expressed in placental tissues. The function of these genes is particularly critical to fetal growth and development, and some are expressed in sex-specific patterns. OBJECTIVES: We aimed to examine whether prenatal Cd associates with the expression of imprinted placental genes, overall or in fetal sex-specific patterns, across two independent epidemiologic studies. METHODS: We tested for Cd­sex interactions in association with gene expression, then regressed the placental expression levels of 74 putative imprinted genes on placental log-Cd concentrations while adjusting for maternal age, sex, smoking history, and educational attainment. These models were performed within study- and sex-specific strata in the New Hampshire Birth Cohort Study (NHBCS; [Formula: see text]) and the Rhode Island Child Health Study (RICHS; [Formula: see text]). We then used fixed-effects models to estimate the sex-specific and overall associations across strata and then examine heterogeneity in the associations by fetal sex. RESULTS: We observed that higher Cd concentrations were associated with higher expression of distal-less homeobox 5 (DLX5) ([Formula: see text]), and lower expression of h19 imprinted maternally expressed transcript (H19) ([Formula: see text]) and necdin, MAGE family member (NDN) ([Formula: see text]) across study and sex-specific strata, while three other genes [carboxypeptidase A4 (CPA4), growth factor receptor bound protein 10 (GRB10), and integrin-linked kinase (ILK)] were significantly associated with Cd concentrations, but only among female placenta ([Formula: see text]). Additionally, the expression of DLX5, H19, and NDN, the most statistically significant Cd-associated genes, were also associated with standardized birth weight z-scores. DISCUSSION: The differential regulation of a set of imprinted genes, particularly DLX5, H19, and NDN, in association with prenatal Cd exposure may be involved in overall developmental toxicity, and some imprinted genes may respond to Cd exposure in a manner that is specific to infant gender. https://doi.org/10.1289/EHP4264.

Maternal circadian disruption is associated with variation in placental DNA methylation.

Circadian disruption is a common environmental and occupational exposure with public health consequences, but not much is known about whether circadian disruption affects in utero development. We investigated whether maternal circadian disruption, using night shift work as a proxy, is associated with variations in DNA methylation patterns of placental tissue in an epigenome-wide association study (EWAS) of night shift work. Here, we compared cytosine-guanosine dinucleotide (CpG) specific methylation genome-wide of placental tissue (measured with the Illumina 450K array) from participants (n = 237) in the Rhode Island Child Health Study (RICHS) who did (n = 53) and did not (n = 184) report working the night shift, using robust linear modeling and adjusting for maternal age, pre-pregnancy smoking, infant sex, maternal adversity, and putative cell mixture. Statistical analyses were adjusted for multiple comparisons and results presented with Bonferroni or Benjamini and Hochberg (BH) adjustment for false discovery rate. Night shift work was associated with differential methylation in placental tissue, including CpG sites in the genes NAV1, SMPD1, TAPBP, CLEC16A, DIP2C, FAM172A, and PLEKHG6 (Bonferroni-adjusted p<0.05). CpG sites within NAV1, MXRA8, GABRG1, PRDM16, WNT5A, and FOXG1 exhibited the most hypomethylation, while CpG sites within TDO2, ADAMTSL3, DLX2, and SERPINA1 exhibited the most hypermethylation (BH q<0.10). Functional analysis indicated GO-terms associated with cell-cell adhesion and enriched GWAS results for psoriasis. Night shift work was associated with differential methylation of the placenta, which may have implications for fetal health and development. This is the first study to examine the epigenetic impacts of night shift exposure, as a proxy for circadian disruption, on placental methylation in humans, and, while results should be interpreted with caution, suggests circadian disruption may have epigenetic impacts.

Intrauterine multi-metal exposure is associated with reduced fetal growth through modulation of the placental gene network.

BACKGROUND: Intrauterine metal exposures and aberrations in placental processes are known contributors to being born small for gestational age (SGA). However, studies to date have largely focused on independent effects, failing to account for potential interdependence among these markers. OBJECTIVES: We evaluated the inter-relationship between multi-metal indices and placental gene network modules related to SGA status to highlight potential molecular pathways through which in utero multi-metal exposure impacts fetal growth. METHODS: Weighted quantile sum (WQS) regression was performed using a panel of 16 trace metals measured in post-partum maternal toe nails collected from the Rhode Island Child Health Study (RICHS, n = 195), and confirmation of the derived SGA-related multi-metal index was conducted using Bayesian kernel machine regression (BKMR). We leveraged existing placental weighted gene coexpression network data to examine associations between the SGA multi-metal index and placental gene expression. Expression of select genes were assessed using RT-PCR in an independent birth cohort, the New Hampshire Birth Cohort Study (NHBCS, n = 237). RESULTS: We identified a multi-metal index, predominated by arsenic (As) and cadmium (Cd), that was positively associated with SGA status (Odds ratio = 2.73 [1.04, 7.18]). This index was also associated with the expression of placental gene modules involved in "gene expression" (ß = -0.02 [-0.04, -0.01]) and "metabolic hormone secretion" (ß = 0.02 [0.00, 0.05]). We validated the association between cadmium exposure and the expression of GRHL1 and INHBA, genes in the "metabolic hormone secretion" module, in NHBCS. CONCLUSION: We present a novel approach that integrates the application of advanced bioinformatics and biostatistics methods to delineate potential placental pathways through which trace metal exposures impact fetal growth.

Placental imprinting variation associated with assisted reproductive technologies and subfertility.

Infertility affects one in 6 couples in developed nations, resulting in an increasing use of assisted reproductive technologies (ART). Both ART and subfertility appear to be linked to lower birth weight outcomes, setting infants up for poor long-term health. Prenatal growth is, in part, regulated via epigenetically-controlled imprinted genes in the placenta. Although differences in DNA methylation between ART and control infants have been found, it remains unclear whether these differences are due to the ART procedures or to the underlying parental subfertility and how these methylation differences affect imprinted gene expression. In this study, we examined the expression of 108 imprinted genes in placental tissues from infants born to subfertile parents (n = 79), matched naturally-conceived controls (n = 158), and infants conceived using in vitro fertilization (IVF, n = 18). Forty-five genes were identified as having significantly different expression between the subfertile infants and controls, whereas no significant differences were identified between the IVF and control groups. The expression of 4 genes-IGF2, NAPIL5, PAX8-AS1, and TUBGCP5-was significantly downregulated in the IVF compared with the subfertile group. Three of the 45 genes significantly dysregulated between subfertile and control placentae-GRB10, NDN, and CD44 -were found to have a significant positive correlation between expression and birth weight. Methylation levels for these 3 genes and 4 others-MKRN3, WRB, DHCR24, and CYR61-were significantly correlated with expression. Our findings indicate that epigenetic differences in placentas resulting from IVF pregnancies may be related to the underlying subfertility in parents using IVF rather than the IVF procedure itself.