Arsenic-Associated Oxidative Stress, Inflammation, and Immune Disruption in Human Placenta and Cord Blood

BACKGROUND: Arsenic (As) exposure during pregnancy induces oxidative stress and increases the risk of fetal loss and low birth weight. OBJECTIVES: In this study we aimed to elucidate the effects of As exposure on immune markers in the placenta and cord blood, and the involvement of oxidative stress. METHODS: Pregnant women were enrolled around gestational week (GW) 8 in our longitudinal, population-based, mother-child cohort in Matlab, an area in rural Bangladesh with large variations in As concentrations in well water. Women (n = 130) delivering at local clinics were included in the present study. We collected maternal urine twice during pregnancy (GW8 and GW30) for measurements of As, and placenta and cord blood at delivery for assessment of immune and inflammatory markers. Placental markers were measured by immunohistochemistry, and cord blood cytokines by multiplex cytokine assay. RESULTS: In multivariable adjusted models, maternal urinary As (U-As) exposure both at GW8 and at GW30 was significantly positively associated with placental markers of 8-oxoguanine (8-oxoG) and interleukin-1β (IL-1β); U-As at GW8, with tumor necrosis factor-α (TNFα) and interferon-γ (IFNγ); and U-As at GW30, with leptin; U-As at GW8 was inversely associated with CD3+ T cells in the placenta. Cord blood cytokines (IL-1β, IL-8, IFNγ, TNFα) showed a U-shaped association with U-As at GW30. Placental 8-oxoG was significantly positively associated with placental proinflammatory cytokines. Multivariable adjusted analyses suggested that enhanced placental cytokine expression (TNFα and IFNγ) was primarily influenced by oxidative stress, whereas leptin expression appeared to be mostly mediated by As, and IL-1β appeared to be influenced by both oxidative stress and As. CONCLUSION: As exposure during pregnancy appeared to enhance placental inflammatory responses (in part by increasing oxidative stress), reduce placental T cells, and alter cord blood cytokines. These findings suggest that effects of As on immune function may contribute to impaired fetal and infant health

Hypoxia induced HIF-1/HIF-2 activity alters trophoblast transcriptional regulation across gestation

Background: Delivery of oxygen to placental and fetal tissues changes across gestation with a significant increase once maternal blood flow into the placenta is established in late first trimester. Prior to 10 weeks of gestation, fetal and placental development takes place in a physiologically hypoxic environment of 1-2% oxygen tension. Subsequently, the oxygen concentration rises to a level considered normoxic (5-8%) for other tissues following dislocation of extravillous trophoblast cells that have invaded into and initially occluded the maternal spiral arterioles where they remodel the maternal decidual vasculature. Hypoxia inducible factors (HIFs) are transcription factors which respond to changes in oxygen tension and are active in a low oxygen environment. Therefore, oxygen, hypoxia and HIFs play a crucial role in placentation and inadequate trophoblast invasion which when impaired has been associated with pregnancy pathologies such as preeclampsia (PE). The aims of this study were to examine (1) the gene expression profile of first trimester trophoblast cells cultured in different oxygen concentrations and (2) the differential expression of hypoxia responsive genes in the placenta of early and late first trimester, term uncomplicated and term complicated pregnancy. Methods: HTR8/SVneo trophoblast cells were cultured in 1% (hypoxia), 5% (normoxia) and 20% oxygen (standard culture conditions) for 6 hours. Localisation of HIF-1α and HIF-2α protein in these cells was performed using immunofluorescence. Microarray analyses were undertaken to determine the differential gene expression profile of HTR8/SVneo cells in response to oxygen. Differential expression of selected genes (IGFBP3, IGFBP5, MMP1, VEGFA, P4HA1, P4HA2 and ANGPTL4) was validated by qPCR in independent samples. HIF-1α and HIF-2α protein were localised by immunohistochemistry in early (6-8 weeks of gestation) and late (10-12 weeks of gestation) first trimester, and term, placenta tissue samples. Expression of selected genes in placenta samples of early (n=11) and late (n=7) first trimester, term uncomplicated (n=10), term complicated by gestational hypertension (n=8) and term complicated by PE (n=12) pregnancy was also quantified by qPCR. Results: 290 genes were differentially expressed in HTR8/SVneo cells treated with 1% compared to 5% oxygen. HIF-1α was identified as the top upstream regulator of 41 genes using Ingenuity Pathway Analysis. qPCR validation showed that the expression of IGFBP3 (P<0.0001), VEGFA (P<0.0001), P4HA1 (P=0.0023), P4HA2 (P=0.0009) and ANGPTL4 (P=0.0001) are significantly increased in 1% oxygen compared to 5% oxygen. These genes were predicted to contain HIF-1 transcription factor binding sites. Immunofluorescence showed that HIF-1α is highly expressed in the nucleus of 1% oxygen treated trophoblasts compared to 5% oxygen whereas expression of HIF-2α remained unchanged by oxygen treatment. HIF-1α and HIF-2α were localised to the nucleus and cytoplasm of syncytiotrophoblast, cytotrophoblast and villous stroma of early and late first trimester placenta with an increased expression in the nucleus in early first trimester. Subtle but significantly lower expression of HIF-1α (-4% P<0.013) and higher expression of HIF-2α (+57% P<0.001) was observed in late first trimester placenta compared to early first trimester placenta. qPCR showed that the expression of IGFBP3 (P<0.0001), IGFBP5 (P<0.0001), VEGFA (P=0.0195) and ANGPTL4 (P<0.0001) was significantly increased in term control compared to first trimester placenta. No difference was observed in the expression of any genes between term control vs complicated pregnancy. Conclusion: HIF translocation to the nucleus was confirmed in trophoblasts under low oxygen tension. Genome wide assessment of trophoblast cells in vitro identified a large number of differentially expressed genes in different oxygen atmospheres. Of the seven selected genes of interest, IGFBP3, VEGFA and ANGPTL4 are known to be involved in trophoblast function and P4HA1 and P4HA2 have known roles in cellular proliferation and migration. Together our findings provide additional molecular evidence that expression of HIFs and hypoxia responsive genes is altered with the change of oxygen tension which may mediate known altered trophoblast activity in low oxygen tension.Thesis (MPhil) -- University of Adelaide, Adelaide Medical School, 201