The antioxidant N-acetyl cysteine inhibits cytokine and prostaglandin release in human fetal membranes stimulated ex vivo with lipoteichoic acid or live group B streptococcus.

BACKGROUNDS: Infection during pregnancy is a significant public health concern due to the increased risk of adverse birth outcomes. Group B Streptococcus or Streptococcus agalactiae (GBS) stands out as a major bacterial cause of neonatal morbidity and mortality. We aimed to explore the involvement of reactive oxygen species (ROS) and oxidative stress pathways in pro-inflammatory responses within human fetal membrane tissue, the target tissue of acute bacterial chorioamnionitis. METHODS: We reanalyzed transcriptomic data from fetal membrane explants inoculated with GBS to assess the impact of GBS on oxidative stress and ROS genes/pathways. We conducted pathway enrichment analysis of transcriptomic data using the Database for Annotation, Visualization and Integrated Discovery (DAVID), a web-based functional annotation/pathway enrichment tool. Subsequently, we conducted ex vivo experiments to test the hypothesis that antioxidant treatment could inhibit pathogen-stimulated inflammatory responses in fetal membranes. RESULTS: Using DAVID analysis, we found significant enrichment of pathways related to oxidative stress or ROS in GBS-inoculated human fetal membranes, for example, "Response to Oxidative Stress" (FDR = 0.02) and "Positive Regulation of Reactive Oxygen Species Metabolic Process" (FDR = 2.6*10-4 ). There were 31 significantly changed genes associated with these pathways, most of which were upregulated after GBS inoculation. In ex vivo experiments with choriodecidual membrane explants, our study showed that co-treatment with N-acetylcysteine (NAC) effectively suppressed the release of pro-inflammatory cytokines (IL-6, IL-8, TNF-α) and prostaglandin PGE2, compared to GBS-treated explants (p < .05 compared to GBS-treated samples without NAC co-treatment). Furthermore, NAC treatment inhibited the release of cytokines and PGE2 stimulated by lipoteichoic acid (LTA) and lipopolysaccharide (LPS) in whole membrane explants (p < .05 compared to LTA or LPS-treated samples without NAC co-treatment). CONCLUSIONS: Our study sheds light on the potential roles of ROS in governing the innate immune response to GBS infection, offering insights for developing strategies to mitigate GBS-related adverse outcomes.

Group B streptococcus induces cellular senescence in human amnion epithelial cells through a partial interleukin-1-mediated mechanism.

Group B streptococcus (GBS) infection is a significant public health concern associated with adverse pregnancy complications and increased neonatal mortality and morbidity. However, the mechanisms underlying the impact of GBS on the fetal membrane, the first line of defense against pathogens, are not fully understood. Here, we propose that GBS induces senescence and inflammatory factors (IL-6 and IL-8) in the fetal membrane through interleukin-1 (IL-1). Utilizing the existing transcriptomic data on GBS-exposed human fetal membrane, we showed that GBS affects senescence-related pathways and genes. Next, we treated primary amnion epithelial cells with conditioned medium from the choriodecidual layer of human fetal membrane exposed to GBS (GBS collected choriodecidual [CD] conditioned medium) in the absence or presence of an IL-1 receptor antagonist (IL-1Ra). GBS CD conditioned medium significantly increased ß-galactosidase activity, IL-6 and IL-8 release from the amnion epithelial cells. Cotreatment with IL1Ra reduced GBS-induced ß-galactosidase activity and IL-6 and IL-8 secretion. Direct treatment with IL-1α or IL-1ß confirmed the role of IL-1 signaling in the regulation of senescence in the fetal membrane. We further showed that GBS CD conditioned medium and IL-1 decreased cell proliferation in amnion epithelial cells. In summary, for the first time, we demonstrate GBS-induced senescence in the fetal membrane and present evidence of IL-1 pathway signaling between the choriodecidua and amnion layer of fetal membrane in a paracrine manner. Further studies will be warranted to understand the pathogenesis of adverse pregnancy outcomes associated with GBS infection and develop therapeutic interventions to mitigate these complications.

Placental cell conditioned media modifies hematopoietic stem cell transcriptome invitro.

INTRODUCTION: Hematopoietic stem cells are cells that differentiate into blood cell types. Although the placenta secretes hormones, proteins and other factors important for maternal/fetal health, cross-talk between placental and hematopoietic stem cells is poorly understood. Moreover, toxicant impacts on placental-hematopoietic stem cell communication is understudied. The goals of this study were to determine if factors secreted from placental cells alter transcriptomic responses in hematopoietic stem cells and if monoethylhexyl phthalate (MEHP), the bioactive metabolite of the pollutant diethylhexyl phthalate, modifies these effects. METHODS: We used K-562 and BeWo cells as in vitro models of hematopoietic stem cells and placental syncytiotrophoblasts, respectively. We treated K-562 cells with medium conditioned by incubation with BeWo cells, medium conditioned with BeWo cells treated with 10 µM MEHP for 24 h, or controls treated with unconditioned medium. We extracted K-562 cell RNA, performed RNA sequencing, then conducted differential gene expression and pathway analysis. RESULTS: Relative to controls, K-562 cells treated with BeWo cell conditioned medium differentially expressed 173 genes (FDR<0.05 and fold-change>2.0), including 2.4-fold upregulatation of tropomyosin 4 (TPM4, a cytoskeletal regulator involved in processes such as cell morphology and migration) and 3.3-fold upregulatation of sphingosine-1-phosphate receptor 3 (S1PR3, a mediator of myeloid cell differentiation and inflammatory responses). Upregulated genes were enriched for pathways including stem cell maintenance, cell proliferation and immune processes. Downregulated genes were enriched for terms involved in protein translation and transcriptional regulation. MEHP treatment differentially expressed eight genes (FDR<0.05), including genes involved in lipid metabolism (e.g., Perilipin 2, fold-change: 1.4; Carnitine Palmitoyltransferase 1A, fold-change: 1.4). DISCUSSION: K-562 cells, a model of hematopoietic stem cells, are responsive to media conditioned by placental cells, potentially impacting pathways like stem cell maintenance.

N-Acetyl-L-cysteine and aminooxyacetic acid differentially modulate toxicity of the trichloroethylene metabolite S-(1,2-dichlorovinyl)-L-cysteine in human placental villous trophoblast BeWo cells.

Trichloroethylene (TCE) is a known human carcinogen with toxicity attributed to its metabolism. S-(1,2-Dichlorovinyl)-L-cysteine (DCVC) is a metabolite of TCE formed downstream in TCE glutathione (GSH) conjugation and is upstream of several toxic metabolites. Despite knowledge that DCVC stimulates reactive oxygen species (ROS) generation and apoptosis in placental cells, the extent to which these outcomes are attributable to DCVC metabolism is unknown. The current study used N-acetyl-L-cysteine (NAC) at 5 mM and aminooxyacetic acid (AOAA) at 1 mM as pharmacological modifiers of DCVC metabolism to investigate DCVC toxicity at concentrations of 5-50 µM in the human placental trophoblast BeWo cell model capable of forskolin-stimulated syncytialization. Exposures of unsyncytialized BeWo cells, BeWo cells undergoing syncytialization, and syncytialized BeWo cells were studied. NAC pre/co-treatment with DCVC either failed to inhibit or exacerbated DCVC-induced H2O2 abundance, PRDX2 mRNA expression, and BCL2 mRNA expression. Although NAC increased mRNA expression of CYP3A4, which would be consistent with increased generation of the toxic metabolite N-acetyl-DCVC sulfoxide (NAcDCVCS), a CYP3A4 inhibitor ketoconazole did not significantly alter BeWo cell responses. Moreover, AOAA failed to inhibit cysteine conjugate ß-lyase (CCBL), which bioactivates DCVC, and did not affect the percentage of nuclei condensed or fragmented, a measure of apoptosis, in all BeWo cell models. However, syncytialized cells had higher CCBL activity compared to unsyncytialized cells, suggesting that the former may be more sensitive to DCVC toxicity. Together, although neither NAC nor AOAA mitigated DCVC toxicity, differences in CCBL activity and potentially CYP3A4 expression dictated the differential toxicity derived from DCVC.

Trichloroethylene Metabolite S-(1,2-Dichlorovinyl)-l-cysteine Stimulates Changes in Energy Metabolites and Amino Acids in the BeWo Human Placental Trophoblast Model during Syncytialization.

Syncytialization, the fusion of cytotrophoblasts into an epithelial barrier that constitutes the maternal-fetal interface, is a crucial event of placentation. This process is characterized by distinct changes to amino acid and energy metabolism. A metabolite of the industrial solvent trichloroethylene (TCE), S-(1,2-dichlorovinyl)-l-cysteine (DCVC), modifies energy metabolism and amino acid abundance in HTR-8/SVneo extravillous trophoblasts. In the current study, we investigated DCVC-induced changes to energy metabolism and amino acids during forskolin-stimulated syncytialization in BeWo cells, a human villous trophoblastic cell line that models syncytialization in vitro. BeWo cells were exposed to forskolin at 100 µM for 48 h to stimulate syncytialization. During syncytialization, BeWo cells were also treated with DCVC at 0 (control), 10, or 20 µM. Following treatment, the targeted metabolomics platform, "Tricarboxylic Acid Plus", was used to identify changes in energy metabolism and amino acids. DCVC treatment during syncytialization decreased oleic acid, aspartate, proline, uridine diphosphate (UDP), UDP-d-glucose, uridine monophosphate, and cytidine monophosphate relative to forskolin-only treatment controls, but did not increase any measured metabolite. Notable changes stimulated by syncytialization in the absence of DCVC included increased adenosine monophosphate and guanosine monophosphate, as well as decreased aspartate and glutamate. Pathway analysis revealed multiple pathways in amino acid and sugar metabolisms that were altered with forskolin-stimulated syncytialization alone and DCVC treatment during syncytialization. Analysis of ratios of metabolites within the pathways revealed that DCVC exposure during syncytialization changed metabolite ratios in the same or different direction compared to syncytialization alone. Building off our oleic acid findings, we found that extracellular matrix metalloproteinase-2, which is downstream in oleic acid signaling, underwent the same changes as oleic acid. Together, the metabolic changes stimulated by DCVC treatment during syncytialization suggest changes in energy metabolism and amino acid abundance as potential mechanisms by which DCVC could impact syncytialization and pregnancy.

Placental Cell Conditioned Media Modifies Hematopoietic Stem Cell Transcriptome In Vitro.

Background: Hematopoietic stem cells are cells that differentiate into all blood cell types. Although the placenta secretes hormones, proteins and other factors important for maternal and fetal health, cross-talk between placental cells and hematopoietic stem cells is poorly understood. Moreover, toxicant impacts on placental-hematopoietic stem cell communication is understudied. The goals of this study were to determine if factors secreted from placental cells alter transcriptomic responses in hematopoietic stem cells and if monoethylhexyl phthalate (MEHP), the bioactive metabolite of the pollutant diethylhexyl phthalate, modifies these effects. Methods: We used K-562 and BeWo cells as in vitro models of hematopoietic stem cells and placental syncytiotrophoblasts, respectively. We treated K-562 cells with medium conditioned by incubation with BeWo cells, medium conditioned with BeWo cells treated with 10 µM MEHP for 24 hours, or controls treated with unconditioned medium. We extracted K-562 cell RNA, performed RNA sequencing, then conducted differential gene expression and pathway analysis by treatment group. Results: Relative to controls, K-562 cells treated with BeWo cell conditioned medium differentially expressed 173 genes (FDR<0.05 and fold-change>2.0), including 2.4 fold upregulatation of TPM4 and 3.3 fold upregulatation of S1PR3. Upregulated genes were enriched for pathways including stem cell maintenance, cell proliferation and immune processes. Downregulated genes were enriched for terms involved in protein translation and transcriptional regulation. MEHP treatment differentially expressed eight genes (FDR<0.05), including genes involved in lipid metabolism (PLIN2, fold-change: 1.4; CPT1A, fold-change: 1.4). Conclusion: K-562 cells, a model of hematopoietic stem cells, are responsive to media conditioned by placental cells, potentially impacting pathways like stem cell maintenance and proliferation.

Sexually concordant and dimorphic transcriptional responses to maternal trichloroethylene and/or N-acetyl cysteine exposure in Wistar rat placental tissue.

Numerous Superfund sites are contaminated with the volatile organic chemical trichloroethylene (TCE). In women, exposure to TCE in pregnancy is associated with reduced birth weight. Our previous study reported that TCE exposure in pregnant rats decreased fetal weight and elevated oxidative stress biomarkers in placentae, suggesting placental injury as a potential mechanism of TCE-induced adverse birth outcomes. In this study, we investigated if co-exposure with the antioxidant N-acetylcysteine (NAC) attenuates TCE exposure effects on RNA expression. Timed-pregnant Wistar rats were exposed orally to 480 mg TCE/kg/day on gestation days 6-16. Exposure of 200 mg NAC/kg/day alone or as a pre/co-exposure with TCE occurred on gestation days 5-16 to stimulate antioxidant genes prior to TCE exposure. Tissue was collected on gestation day 16. In male and female placentae, we evaluated TCE- and/or NAC-induced changes to gene expression and pathway enrichment analyses using false discovery rate (FDR) and fold-change criteria. In female placentae, exposure to TCE caused significant differential expression 129 genes while the TCE+NAC altered 125 genes, compared with controls (FDR< 0.05 + fold-change >1). In contrast, in male placentae TCE exposure differentially expressed 9 genes and TCE+NAC differentially expressed 35 genes, compared with controls (FDR< 0.05 + fold-change >1). NAC alone did not significantly alter gene expression in either sex. Differentially expressed genes observed with TCE exposure were enriched in mitochondrial biogenesis and oxidative phosphorylation pathways in females whereas immune system pathways and endoplasmic reticulum stress pathways were differentially expressed in both sexes (FDR<0.05). TCE treatment was differentially enriched for genes regulated by the transcription factors ATF6 (both sexes) and ATF4 (males only), indicating a cellular condition triggered by misfolded proteins during endoplasmic reticulum stress. This study demonstrates novel genes and pathways involved in TCE-induced placental injury and showed antioxidant co-treatment largely did not attenuate TCE exposure effects.

Apoptotic responses stimulated by the trichloroethylene metabolite S-(1,2-dichlorovinyl)-L-cysteine depend on cell differentiation state in BeWo human trophoblast cells.

During pregnancy, the placental villous cytotrophoblasts differentiate via cell fusion and multinucleation to create syncytiotrophoblasts, a cell type at the maternal-fetal interface. Apoptosis of syncytiotrophoblasts is associated with adverse pregnancy outcomes. The human trophoblast BeWo cell line has been used as an in vitro model for this differentiation process, also known as syncytialization. In the current study, we exposed unsyncytialized BeWo cells, BeWo cells undergoing syncytialization, and syncytialized BeWo cells to S-(1,2-dichlorovinyl)-L-cysteine (DCVC), a metabolite of the industrial chemical trichloroethylene (TCE). DCVC exposure at 50 µM for 48 h decreased cell viability, increased cytotoxicity, increased caspase 3/7 activity, and increased nuclear condensation or fragmentation in BeWo cells regardless of their differentiation status. Investigating mechanisms of apoptosis, DCVC increased H2O2 abundance and decreased PRDX2 mRNA in all three BeWo cell models. DCVC decreased tumor necrosis factor-receptor 1 (TNF-R1) concentration in media and decreased NFKB1 and PRDX1 mRNA expression in syncytialized BeWo cells only. DCVC decreased BCL2 mRNA expression in syncytializing BeWo cells and in syncytialized BeWo cells only. Decreased LGALS3 mRNA was seen in unsyncytialized BeWo cells only. Together, these data suggest roles for oxidative stress and pro-inflammatory mechanisms underlying apoptosis in BeWo cells with differences depending on differentiation state.

The trichloroethylene metabolite S-(1,2-dichlorovinyl)-l-cysteine inhibits lipopolysaccharide-induced inflammation transcriptomic pathways and cytokine secretion in a macrophage cell model.

Studies have shown that the trichloroethylene metabolite S-(1,2-dichlorovinyl)-l-cysteine (DCVC) inhibits cytokine secretion in pathogen stimulated fetal membrane tissue but little is known about the mechanism for these effects, including which cell types or transcriptomic pathways are impacted. Macrophages play a critical role in fetal membrane immune responses during infection. We tested the hypothesis that DCVC inhibits lipopolysaccharide (LPS) stimulated inflammation pathways in macrophage-like THP-1 cells. We treated THP-1 cells for 24 h then treated with 1, 5, or 10 µM DCVC for 24 h. After a 4 h incubation with lipopolysaccharide (LPS), we collected RNA and cell media. We performed transcriptomic analysis using RNA sequencing for 5 µM DCVC treatments and quantified cytokine release (IL-1ß, IL-6, and TNF-α) for 1, 5 and 10 µM DCVC treatments. RNA sequencing analysis revealed 1399 differentially expressed genes (FDR < 0.05 and log 2 fold change magnitude>2.5) in cells co-treated with DCVC and LPS compared to LPS alone. For example, TNF had a log2(fold-change) = -3.5 with the addition of DCVC. Pathways downregulated (adjusted p-value<0.05) in DCVC+LPS treatments versus LPS-only treatments included: "acute inflammatory response", "production of molecular mediator of immune response" and "phagocytosis". LPS increased IL-1ß, IL-6, and TNF-α levels in culture media (p < 0.001), but this was inhibited by co-treatment with DCVC (p < 0.001 for LPS vs. LPS + DCVC treatments). Our results demonstrate that DCVC suppresses inflammatory responses in macrophages.

Trichloroethylene modifies energy metabolites in the amniotic fluid of Wistar rats.

Exposure to trichloroethylene (TCE), an industrial solvent, is associated with several adverse pregnancy outcomes in humans and decreased fetal weight in rats. However, effects of TCE on energy metabolites in amniotic fluid, which have associations with pregnancy outcomes, has not been published previously. In the current exploratory study, timed-pregnant Wistar rats were exposed to 480 mg TCE/kg/day via vanilla wafer or to vehicle (wafer) alone from gestational day (GD) 6-16. Amniotic fluid collected on GD 16 was analyzed for metabolites important in energy metabolism using short chain fatty acid and tricarboxylic acid plus platforms (N = 4 samples/sex/treatment). TCE decreased concentrations of the following metabolites in amniotic fluid for both fetal sexes: 6-phosphogluconate, guanosine diphosphate, adenosine diphosphate, adenosine triphosphate, and flavin adenine dinucleotide. TCE decreased fructose 1,6-bisphosphate and guanosine triphosphate concentrations in amniotic fluid of male but not female fetuses. Moreover, TCE decreased uridine diphosphate-D-glucuronate concentrations, and increased arginine and phosphocreatine concentrations, in amniotic fluid of female fetuses only. No metabolites were increased in amniotic fluid of male fetuses. Pathway analysis suggested that TCE altered folate biosynthesis and pentose phosphate pathway in both sexes. Using metabolite ratios to investigate changes within specific pathways, some ratio alterations, including those in arginine metabolism and phenylalanine metabolism, were detected in females only. Ratio analysis also suggested enzymes, including gluconokinase, as potential TCE targets. Together, results from this exploratory study suggest that TCE differentially modified energy metabolites in amniotic fluid based on sex. These findings may inform future studies of TCE reproductive toxicity.

Toxicity assessments of selected trichloroethylene and perchloroethylene metabolites in three in vitro human placental models.

Residential and occupational exposures to the industrial solvents perchloroethylene (PERC) and trichloroethylene (TCE) present public health concerns. In humans, maternal PERC and TCE exposures can be associated with adverse birth outcomes. Because PERC and TCE are biotransformed to toxic metabolites and placental dysfunction can contribute to adverse birth outcomes, the present study compared the toxicity of key PERC and TCE metabolites in three in vitro human placenta models. We measured cell viability and caspase 3 + 7 activity in the HTR-8/SVneo and BeWo cell lines, and caspase 3 + 7 activity in first trimester villous explant cultures. Cultures were exposed for 24 h to 5-100 µM S-(1,2-dichlorovinyl)-L-cysteine (DCVC) and S-(1,2,2-trichlorovinyl)-L-cysteine (TCVC), or 5-200 µM trichloroacetate (TCA) and dichloroacetate (DCA). DCVC significantly reduced cell viability and increased caspase 3 + 7 activity in HTR-8/SVneo cells at a lower concentration (20 µM) compared with concentrations toxic to BeWo cells and villous explants. Similarly, TCVC reduced cell viability and increased caspase 3 + 7 activity in HTR-8/SVneo cells but not in BeWo cells. TCA and DCA had only negligible effects on HTR-8/SVneo or BeWo cells. This study advances understanding of potential risks of PERC and TCE exposure during pregnancy by identifying metabolites toxic in placental cells and tissues.

A Data Mining Approach Reveals Chemicals Detected at Higher Levels in Non-Hispanic Black Women Target Preterm Birth Genes and Pathways.

Preterm birth occurs disproportionately in the USA non-Hispanic Black population. Black women also face disproportionate exposure to certain environmental chemicals. The goal of this study was to use publicly available toxicogenomic data to identify chemical exposures that may contribute to preterm birth disparities. We tested 19 chemicals observed at higher levels in the blood or urine of non-Hispanic Black women compared to non-Hispanic White women. We obtained chemical-gene interactions from the Comparative Toxicogenomics Database and a list of genes involved in preterm birth from the Preterm Birth Database. We tested chemicals for enrichment with preterm birth genes using chi-squared tests. We then conducted pathway enrichment analysis for the preterm birth genes using DAVID software and identified chemical impacts on genes involved in these pathways. Genes annotated to all 19 chemicals were enriched with preterm birth genes (FDR-adjusted p value < 0.05). Preterm birth enriched chemicals that were detected at the highest levels in non-Hispanic Black women included methyl mercury, methylparaben, propylparaben, diethyl phthalate, dichlorodiphenyldichloroethylene, and bisphenol S. The preterm birth genes were enriched for pathways including "inflammatory response" (FDR-adjusted p value = 3 × 10-19), "aging" (FDR-adjusted p value = 4 × 10-8) and "response to estradiol" (FDR-adjusted p value = 2 × 10-4). Chemicals enriched with preterm birth genes impacted genes in all three pathways. This study adds to the body of knowledge suggesting that exposures to environmental chemicals contribute to racial disparities in preterm birth and that multiple chemicals drive these effects. These chemicals affect genes involved in biological processes relevant to preterm birth such as inflammation, aging, and estradiol pathways.

Maternal blood metal concentrations and whole blood DNA methylation during pregnancy in the Early Autism Risk Longitudinal Investigation (EARLI).

The maternal epigenome may be responsive to prenatal metals exposures. We tested whether metals are associated with concurrent differential maternal whole blood DNA methylation. In the Early Autism Risk Longitudinal Investigation cohort, we measured first or second trimester maternal blood metals concentrations (cadmium, lead, mercury, manganese, and selenium) using inductively coupled plasma mass spectrometry. DNA methylation in maternal whole blood was measured on the Illumina 450 K array. A subset sample of 97 women had both measures available for analysis, all of whom did not report smoking during pregnancy. Linear regression was used to test for site-specific associations between individual metals and DNA methylation, adjusting for cell type composition and confounding variables. Discovery gene ontology analysis was conducted on the top 1,000 sites associated with each metal. We observed hypermethylation at 11 DNA methylation sites associated with lead (FDR False Discovery Rate q-value <0.1), near the genes CYP24A1, ASCL2, FAT1, SNX31, NKX6-2, LRC4C, BMP7, HOXC11, PCDH7, ZSCAN18, and VIPR2. Lead-associated sites were enriched (FDR q-value <0.1) for the pathways cell adhesion, nervous system development, and calcium ion binding. Manganese was associated with hypermethylation at four DNA methylation sites (FDR q-value <0.1), one of which was near the gene ARID2. Manganese-associated sites were enriched for cellular metabolism pathways (FDR q-value<0.1). Effect estimates for DNA methylation sites associated (p < 0.05) with cadmium, lead, and manganese were highly correlated (Pearson ρ > 0.86). DNA methylation sites associated with lead and manganese may be potential biomarkers of exposure or implicate downstream gene pathways.

Gestational Hormone Concentrations Are Associated With Timing of Delivery in a Fetal Sex-Dependent Manner.

Background: Early delivery remains a significant public health problem that has long-lasting impacts on mother and child. Understanding biological mechanisms underlying timing of labor, including endocrine disruption, can inform prevention efforts. Methods: Gestational hormones were measured among 976 women in PROTECT, a longitudinal birth cohort in Puerto Rico. We evaluated associations between hormone concentrations at 18 and 26 weeks gestation and gestational age at birth, while assessing effect modification by fetal sex. Exploratory analyses assessed binary outcomes of overall preterm birth (PTB, <37 weeks gestation) and the spontaneous PTB subtype, defined as preterm premature rupture of membranes, spontaneous preterm labor, or both. Multivariable logistic and linear regressions were fit using visit-specific hormone concentrations, and fetal sex-specific effects were estimated using interaction terms. Main outcome models were adjusted for maternal age, education, marital status, alcohol consumption, environmental tobacco smoke exposure, and pre-pregnancy body mass index (BMI). Exploratory models adjusted for maternal age and education. Results: We observed reduced gestational age at birth with higher circulating CRH (ß: -2.73 days, 95% CI: -4.97, -0.42), progesterone (ß: -4.90 days, 95% CI: -7.07, -2.73), and fT4 concentrations (ß: -2.73 days, 95% CI: -4.76, -0.70) at 18 weeks specifically among male fetuses. Greater odds of overall and spontaneous PTB were observed among males with higher CRH, estriol, progesterone, total triiodothyronine (T3), and free thyroxine (fT4) concentrations. Greater odds of PTB among females was observed with higher testosterone concentrations. Conclusions: Various associations between hormones and timing of delivery were modified by fetal sex and timing of hormone measurement. Future studies are needed to understand differential mechanisms involved with timing of labor between fetal sexes.

Transcriptional profiling of the response to the trichloroethylene metabolite S-(1,2-dichlorovinyl)-L-cysteine revealed activation of the eIF2α/ATF4 integrated stress response in two in vitro placental models.

Trichloroethylene (TCE) is an industrial solvent and widespread environmental contaminant. Although TCE exposure is prevalent, epidemiological studies of TCE exposure associations with adverse birth outcomes are inconclusive. Prior studies show that the TCE metabolite S-(1,2-dichlorovinyl)-L-cysteine (DCVC) exhibits toxicity in a placental cell line. In the current study, genome-wide gene expression and gene set enrichment analyses were used to identify novel genes and pathway alterations in the HTR-8/SVneo human trophoblast cell line and human placental villous explants treated with DCVC at concentrations relevant to human exposures. In the cells, concentration- and time-dependent effects were observed, as evidenced by the magnitude of altered gene expression after treatment with 20 µM DCVC versus 10 µM, and 12-h versus 6-h of treatment. Comparing the two models for the transcriptional response to 12-h 20 µM DCVC treatment, no differentially expressed genes reached significance in villous explants, whereas 301 differentially expressed genes were detected in HTR-8/SVneo cells compared with non-treated controls (FDR < 0.05 + LogFC > 0.35 [FC > 1.3]). GSEA revealed five upregulated enriched pathways in common between explants and cells (FDR < 0.05). Moreover, all 12-h DCVC treatment groups from both models contained upregulated pathways enriched for genes regulated by the ATF4 transcription factor. The overrepresentation of ATF4 regulation of differentially expressed genes indicated activation of the integrated stress response (ISR), a condition triggered by multiple stress stimuli, including the unfolded protein response. DCVC-induced ISR activation was confirmed by elevated eIF2α phosphorylation, ATF4 protein concentrations, and decreased global protein synthesis in HTR-8/SVneo cells. This study identifies a mechanism of DCVC-induced cytotoxicity by revealing the involvement of a specific stress signaling pathway.

N-Acetyl-L-cysteine and aminooxyacetic acid differentially modulate trichloroethylene reproductive toxicity via metabolism in Wistar rats.

Exposure to the industrial solvent trichloroethylene (TCE) has been associated with adverse pregnancy outcomes in humans and decreased fetal weight in rats. TCE kidney toxicity can occur through formation of reactive metabolites via its glutathione (GSH) conjugation metabolic pathway, largely unstudied in the context of pregnancy. To investigate the contribution of the GSH conjugation pathway and oxidative stress to TCE toxicity during pregnancy, we exposed rats orally to 480 mg TCE/kg/day from gestational day (GD) 6 to GD 16 with and without N-acetyl-L-cysteine (NAC) at 200 mg/kg/day or aminooxyacetic acid (AOAA) at 20 mg/kg/day as pre/co-treatments from GD 5-16. NAC is a reactive oxygen species scavenger that modifies the GSH conjugation pathway, and AOAA is an inhibitor of cysteine conjugate ß-lyase (CCBL) in the GSH conjugation pathway. TCE decreased fetal weight, and this was prevented by AOAA but not NAC pre/co-treatment to TCE. Although AOAA inhibited CCBL activity in maternal kidney, it did not inhibit CCBL activity in maternal liver and placenta, suggesting that AOAA prevention of TCE-induced decreased fetal weight was due to CCBL activity inhibition in the kidneys but not liver or placenta. Unexpectedly, NAC pre/co-treatment with TCE, relative to TCE treatment alone, altered placental morphology consistent with delayed developmental phenotype. Immunohistochemical staining revealed that the decidua basale, relative to basal and labyrinth zones, expressed the highest abundance of CCBL1, flavin-containing monooxygenase 3, and cleaved caspase-3. Together, the findings show the differential effects of NAC and AOAA on TCE-induced pregnancy outcomes are likely attributable to TCE metabolism modulation.

Comparison of rat fetal sex determination using placental gDNA and mRNA via qRT-PCR.

A growing need exists to consider fetal sex as a biological variable and accurately assess sex-specific effects. Among the multiple methods used to determine fetal sex, quantitative real-time polymerase chain reaction (qRT-PCR) of Sry (sex-determining region Y) with genomic DNA (gDNA) is commonly used in addition to use of methodologies such as transcriptomics and detection of Barr body. However, Sry messenger RNA (mRNA), a product of Sry gDNA, has not been previously assessed for sex determination. Using placental samples from timed-pregnant Wistar rats at gestational day (GD) 16, this study assessed the compatibility of Sry detection using gDNA versus mRNA to determine fetal sex. Samples used in this current study come from a larger study that investigated trichloroethylene (TCE) reproductive toxicity and potential modulation by N-acetyl-L-cysteine (NAC) and aminooxyacetic acid (AOAA). In 90 out of 91 samples, the sex classification determined by gDNA matched the sex classification determined by mRNA analyzing Sry (Sry/B2m) values. For both gDNA and mRNA, statistically significant differences in Sry/B2m values between males and females were observed with samples considered in totality and when samples were separated by treatment groups (all comparisons were p<0.01 or below, and all but two comparisons were p<0.001 or below). Finally, the validity of using Sry Cq values to determine fetal sex and the B2m reference gene were also discussed. Together, this study suggests that determination of fetal sex in Wistar rats can be accomplished using Sry measurements in gDNA or mRNA with highly compatible results.

Toxicant Disruption of Immune Defenses: Potential Implications for Fetal Membranes and Pregnancy.

In addition to providing a physical compartment for gestation, the fetal membranes (FM) are an active immunological barrier that provides defense against pathogenic microorganisms that ascend the gravid reproductive tract. Pathogenic infection of the gestational tissues (FM and placenta) is a leading known cause of preterm birth (PTB). Some environmental toxicants decrease the capacity for organisms to mount an immune defense against pathogens. For example, the immunosuppressive effects of the widespread environmental contaminant trichloroethylene (TCE) are documented for lung infection with Streptococcus zooepidemicus. Group B Streptococcus (GBS; Streptococcus agalactiae) is a bacterial pathogen that is frequently found in the female reproductive tract and can colonize the FM in pregnant women. Work in our laboratory has demonstrated that a bioactive TCE metabolite, S-(1, 2-dichlorovinyl)-L-cysteine (DCVC), potently inhibits innate immune responses to GBS in human FM in culture. Despite these provocative findings, little is known about how DCVC and other toxicants modify the risk for pathogenic infection of FM. Infection of the gestational tissues (FM and placenta) is a leading known cause of PTB, therefore toxicant compromise of FM ability to fight off infectious microorganisms could significantly contribute to PTB risk. This Perspective provides the current status of understanding of toxicant-pathogen interactions in FM, highlighting knowledge gaps, challenges, and opportunities for research that can advance protections for maternal and fetal health.

Tert-Butyl Hydroperoxide Stimulated Apoptosis Independent of Prostaglandin E2 and IL-6 in the HTR-8/SVneo Human Placental Cell Line.

Significant gaps exist in our knowledge of how cellular redox status, sometimes referred to as oxidative stress, impacts placental trophoblasts. The present study used tert-butyl hydroperoxide (TBHP) as a known generator of reactive oxygen species (ROS) in the extravillous trophoblast cell line HTR-8/SVneo to examine the role of cellular redox disruption of prostaglandin E2 (PGE2) and the cytokine IL-6 in cell death. Cells were exposed to 0, 12.5, 25, or 50 µM TBHP for 4, 8, and 24 h to ascertain effects on cell viability, caspase 3/7 activity, PGE2 release, PTGS2 mRNA expression, and IL-6 release. Experiments with inhibitors included the cyclooxygenase inhibitor indomethacin, mitogen-activated protein kinase inhibitors (PD169316, U0126, or SP600125), or treatments to counter expected consequences of TBHP-stimulated generation of ROS (deferoxamine [DFO], butylated hydroxyanisole [BHA], and N,N'-diphenyl-1,4-phenylenediamine [DPPD]) using 24-h exposure to 50 µM TBHP. Cell viability, measured by ATP content, decreased 24% relative to controls with a 24-h exposure to 50 µM TBHP, but not at lower TBHP concentrations nor at earlier time points. Exposure to 50 µM TBHP increased caspase 3/7 activity, an indicator of apoptosis, after 8 and 24 h. Antioxidant treatment markedly reduced TBHP-stimulated caspase 3/7 activity, PGE2 release, and IL-6 release. TBHP-stimulated IL-6 release was blocked by PD169316 but unaltered by indomethacin. These data suggest that TBHP-stimulated IL-6 release and caspase 3/7 activation were independent of PGE2 yet were interrupted by treatments with known antioxidant properties, providing new insight into relationships between PGE2, IL-6, and apoptosis under conditions of chemically induced cellular oxidation.

Brominated diphenyl ether-47 differentially regulates cellular migration and invasion in a human first trimester trophoblast cell line.

Polybrominated diphenyl ethers (PBDEs) are flame retardant compounds detected in human placenta and linked to adverse pregnancy outcomes. Impaired trophoblast migration and invasion during early pregnancy have been implicated as potential mechanisms of pregnancy disorders. The present study investigated the effect of BDE-47, a prevalent PBDE congener, on cell migration, invasion, and matrix metalloproteinase (MMP) expression in a human first trimester extravillous trophoblast cell line, HTR-8/SVneo. BDE-47 stimulated cell migration in HTR-SV/neo cells while decreasing invasion of cells into Matrigel. In addition, BDE-47 led to differential expression of MMP-1, -2, -3, and -9 at protein and mRNA levels. In summary, BDE-47 differentially regulated cellular migration and invasion with divergent changes in MMP expression in trophoblasts. Because proper regulation of trophoblast migration and invasion is critical for placental development and function, further research is warranted to determine if exposure to PBDEs disrupts trophoblast functions with increased risk for adverse pregnancy outcomes.