Age and Sex-Related Changes in Human First-Trimester Placenta Transcriptome and Insights into Adaptative Responses to Increased Oxygen

Physiological oxygen tension rises dramatically in the placenta between 8 and 14 weeks of gestation. Abnormalities in this period can lead to gestational diseases, whose underlying mechanisms remain unclear. We explored the changes at mRNA level by comparing the transcriptomes of human placentas at 8–10 gestational weeks and 12–14 gestational weeks. A total of 20 samples were collected and divided equally into four groups based on sex and age. Cytotrophoblasts were isolated and sequenced using RNAseq. Key genes were identified using two different methods: DESeq2 and weighted gene co-expression network analysis (WGCNA). We also constructed a local database of known targets of hypoxia-inducible factor (HIF) subunits, alpha and beta, to investigate expression patterns likely linked with changes in oxygen. Patterns of gene enrichment in and among the four groups were analyzed based on annotations of gene ontology (GO) and KEGG pathways. We characterized the similarities and differences between the enrichment patterns revealed by the two methods and the two conditions (age and sex), as well as those associated with HIF targets. Our results provide a broad perspective of the processes that are active in cytotrophoblasts during the rise in physiological oxygen, which should benefit efforts to discover possible drug-targeted genes or pathways in the human placenta

Formaldehyde Crosses the Human Placenta and Affects Human Trophoblast Differentiation and Hormonal Functions

International audienceThe chorionic villus of the human placenta is the source of specific endocrine functions and nutrient exchanges. These activities are ensured by the syncytiotrophobast (ST), which bathes in maternal blood. The ST arises and regenerates throughout pregnancy by fusion of underlying cytotrophoblasts (CT). Any anomaly of ST formation or regeneration can affect pregnancy outcome and fetal growth. Because of its direct interaction with maternal blood, the ST is sensitive to drugs, pollutants and xenohormones. Ex vivo assays of perfused cotyledon show that formaldehyde, a common pollutant present in furniture, paint and plastics, can accumulate in the human placenta and cross to the fetal compartment. By means of RT-qPCR, immunoblot and immunocytochemistry experiments, we demonstrate in vitro that formaldehyde exerts endocrine toxicity on human trophoblasts, including a decrease in the production of protein hormones of pregnancy. In addition, formaldehyde exposure triggered human trophoblast fusion by upregulating syncitin-1 receptor expression (ASC-type amino-acid transporter 2: ASCT2). Moreover, we show that formaldehyde-exposed trophoblasts present an altered redox status associated with oxidative stress, and an increase in ASCT2 expression intended to compensate for this stress. Finally, we demonstrate that the adverse effects of formaldehyde on trophoblast differentiation and fusion are reversed by N-acetyl-L-cysteine (Nac), an antioxidant

Assessment of dually labelled PEGylated liposomes transplacental passage and placental penetration using a combination of two ex-vivo human models: the dually perfused placenta and the suspended villous explants

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In Utero Exposure to Citalopram Mitigates Maternal Stress Effects on Fetal Brain Development.

Human epidemiological and animal-model studies suggest that separate exposure to stress or serotonin-selective reuptake inhibitor (SSRI) antidepressants during pregnancy increases risks for neurodevelopmental disorders in offspring. Yet, little is known about the combined effects of maternal stress and SSRIs with regard to brain development in utero. We found that the placenta is highly permeable to the commonly prescribed SSRI (±)-citalopram (CIT) in humans and mice, allowing rapid exposure of the fetal brain to this drug. We investigated the effects of maternal chronic unpredictable stress in mice with or without maternal oral administration of CIT from embryonic day (E)8 to E17. We assessed fetal brain development using magnetic resonance imaging and quantified changes in serotonergic, thalamocortical, and cortical development. In utero exposure to maternal stress did not affect overall fetal brain growth. However, serotonin tissue content in the fetal forebrain was increased in association with maternal stress; this increase was reversed by maternal CIT. In utero exposure to stress increased the numbers of deep-layer neurons in specific cortical regions, whereas CIT increased overall cell numbers without changing the proportions of layer-specific neurons to offset the effects of stress on deep-layer cortical development. These findings suggest that stress and SSRI exposure in utero differentially impact serotonin-dependent fetal neurodevelopment such that CIT reverses key effects of maternal gestational stress on offspring brain development

N-acetyl-L-cysteine (Nac) reverses the effects of formaldehyde on human trophoblast differentiation and fusion.

<p>(A) Immunocytofluorescence of desmoplakin (magenta) on human trophoblasts at 24 h of culture with or without formaldehyde, Nac, H₂O₂, or a combination of formaldehyde and Nac or H₂O₂ and Nac. Nuclei were counterstained with DAPI (cyan). Syncytia (ST) boundaries are indicated by dashed lines. Scale bar: 15 μm. (left panel). (B) Effect of formaldehyde, Nac and H₂O₂ alone or in combination on cell fusion after 24 h of culture, represented as remaining mononuclear cells (upper panel) and fusion index histograms (lower panel). (C) Levels of hCG secreted in the culture medium at 24 h in the same cultures. (D) The mRNA expression of OXSR1, GPx-3, GSR in trophoblasts exposed for 24 h to formaldehyde and/or Nac. The mRNA data are expressed as the level of each marker normalized to beta actin mRNA expression (ACTB). (E) Immunoblots of OXSR1, GPx-3, GSR and actin in the same conditions. (F) Analysis of previous immunoblots normalized to actin (a.u. = arbitrary units). (G) GPx activity measured in the same trophoblasts and conditions after 24 h of exposure. (H) The mRNA expression of ASCT2 normalized to beta actin mRNA expression (ACTB) in trophoblasts exposed for 24 h to formaldehyde and/or Nac. (I) Immunoblot analysis of ASCT2 and actin levels in the same conditions (left panel). Levels of the proteins listed above were assessed by densitometric scanning of immunoblots and normalized to actin levels in the same blots (histograms; a.u. for arbitrary units). Results are expressed as the mean ± SEM of 3 independent experiments (* p < 0.05, ** p < 0.01, *** p < 0.001).</p

Effect of formaldehyde on human trophoblasts.

<p>(A) Immunohistofluorescence of cytokeratin 7 (CK7, magenta) in human placental biopsies; nuclei were counterstained with TOPRO-3 (cyan). Scale bar: 10 μm. (B) Histograms represent the viability (%) of untreated cells (control) and cells treated with formaldehyde (10, 50, 100 μM and 1 mM), as quantified with the Trypan blue exclusion assay. (C) Effect of formaldehyde exposure on human trophoblast apoptosis determined by immunocytofluorescence of cleaved cytokeratin 18 (cCK18, yellow) after 24 h and 72 h of culture; nuclei were counterstained with DAPI (cyan). Scale bar: 15 μm (left panel). Histograms represent viability (%) quantified by cCK18 immunostaining of untreated cells (control) and cells treated with 100 μM formaldehyde, after 24 h or 72 h of culture (right panel). (D) Immunocytofluorescence of desmoplakin (magenta) on human trophoblasts at 24 h and 72 h of untreated or formaldehyde-exposed (100 μM) culture; nuclei were counterstained with DAPI (cyan). Syncytia (ST) boundaries are indicated with dashed lines. Scale bar: 15 μm. (left panel). Effect of formaldehyde on cell fusion after 24 or 72 h of culture, represented as remaining mononuclear cells (middle left panel) and fusion index histograms (middle right panel). (E) Intracellular cAMP in control and formaldehyde-treated cells. Results are expressed as the mean ± SEM of 3 independent experiments (** p < 0.01, *** p < 0.001).</p

Pharmacological characteristics of antipyrine and formaldehyde placental transfer during a 90-min period.

<p>The concentrations (%) of compounds added to the maternal compartment are indicated, along with the concentrations found in the fetal compartment after 90 min (FTR: fetal transfer rate). The clearance index is represented, as well as the percentage of compounds retained in the human cotyledon after 90 min of perfusion.</p

Effect of formaldehyde exposure on syncytins and syncytin receptors in human trophoblasts.

<p>(A) mRNA expression of syncytins and their receptors (syncytin-1, ASCT2, syncytin-2 and MFSD2) in trophoblasts with or without (control) formaldehyde exposure for 24 h. The mRNA data are expressed as the level of each mRNA marker normalized by cyclophilin A mRNA expression (PPIA). (B) Immunoblot analysis of syncytin-1, ASCT2, syncytin-2, MFSD2 and actin levels in cells with or without formaldehyde exposure during trophoblast differentiation (upper panel). Levels of the protein listed above were assessed by densitometric scanning of immunoblots and normalized to actin levels in the same blots (histograms; a.u. for arbitrary units). Results are expressed as the mean ± SEM of 3 independent experiments (** p < 0.01, *** p < 0.001).</p

Characterization of transplacental formaldehyde transfer.

<p>(A) Fetal transfer rate of antipyrine (solid dark triangles) and formaldehyde (solid dark squares) and (B) clearance <i>versus</i> time, during 90 min of perfusion (mean ± SEM of 3 independent human perfused cotyledons).</p

First evidence that emerging pinnatoxin-G, a contaminant of shellfish, reaches the brain and crosses the placental barrier

International audienceMassive proliferation of some toxic marine dinoflagellates is responsible for the occurrence of harmful algal blooms and the contamination of fish and shellfish worldwide. Pinnatoxins (PnTx) (A-H) comprise an emerging phycotoxin family belonging to the cyclic imine toxin group. Interest has been focused on these lipophilic, fast-acting and highly potent toxins because they are widely found in contaminated shellfish, and can represent a risk for seafood consumers. PnTx display a potent antagonist effect on nicotinic acetylcholine receptors (nAChR), and in this study we assessed in vivo the ability of PnTx-G to cross physiological barriers to reach its molecular target. Radiolabeled [3H]-PnTx-G synthesized with good radiochemical purity and yield retained the high affinity of the natural toxin. Oral gavage or intravenous administration to adult rats and digital autoradiographic analyses revealed the biodistribution and toxicokinetics of [3H]-PnTx-G, which is rapidly cleared from blood, and accumulates in the liver and small intestine. The labeling of peripheral and brain adult/embryo rat tissues highlights its ability to cross the intestinal, blood-brain and placental barriers. High-resolution 3D-imaging and in vitro competition studies on rat embryo sections revealed the specificity of [3H]-PnTx-G binding and its selectivity for muscle and neuronal nAChR subtypes (such as α7 subtype). The use of a human perfused cotyledon model and mass spectrometry analyses disclosed that PnTx-G crosses the human placental barrier. The increasing worldwide occurrence of both the dinoflagellate Vulcanodinium rugosum and PnTx-contaminated shellfish, due to climate warming, raises concerns about the potential adverse impact that exposure to pinnatoxins may have for human health