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

Biochemical Screening for Fetal Trisomy 21: Pathophysiology of Maternal Serum Markers and Involvement of the Placenta

It is now well established that maternal serum markers are often abnormal in fetal trisomy 21. Their determination is recommended for prenatal screening and pregnancy follow-up. However, mechanisms leading to abnormal maternal serum levels of such markers are still debated. Our objective was to help clinicians and scientists unravel the pathophysiology of these markers via a review of the main studies published in this field, both in vivo and in vitro, focusing on the six most widely used markers (hCG, its free subunit hCGβ, PAPP-A, AFP, uE3, and inhibin A) as well as cell-free feto–placental DNA. Analysis of the literature shows that mechanisms underlying each marker’s regulation are multiple and not necessarily directly linked with the supernumerary chromosome 21. The crucial involvement of the placenta is also highlighted, which could be defective in one or several of its functions (turnover and apoptosis, endocrine production, and feto–maternal exchanges and transfer). These defects were neither constant nor specific for trisomy 21, and might be more or less pronounced, reflecting a high variability in placental immaturity and alteration. This explains why maternal serum markers can lack both specificity and sensitivity, and are thus restricted to screening

IDH1 immunohistochemistry reactivity and mosaic IDH1 or IDH2 somatic mutations in pediatric sporadic enchondroma and enchondromatosis

Mosaic somatic mutations in the isocitrate dehydrogenase 1/2 (IDH1/2) genes have been identified in most enchondromas by targeted mutation analysis. Next-generation sequencing (NGS), that may detect even low-level mosaic mutation rates, has not previously been applied to enchondromas. Immunohistochemistry using the H09 clone is routinely used as a surrogate for the common R132H IDH1 mutation in gliomas. We compared immunohistochemistry and NGS results in a series of 13 enchondromas from 8 pediatric patients. NGS identified a heterozygous IDH mutation in all enchondromas, showing identical mutation status in patients with multiple tumors assessed, thereby confirming somatic mosaicism. A majority of the tumors harbored an IDH1 mutation (p.R132H in 3 tumors; p.R132C in 4 tumors from 2 patients; p.R132L and p.R132G in one tumor each). A p.R172S IDH2 mutation was identified in 4 enchondromas, but not in the ependymoma from one patient with Ollier disease, who further displayed a heterozygous STK11 missense mutation. IDH mutation rates varied between 14% (indicative of mutations in 28% of the cells and of intratumoral mosaicism) and 45% (tumor content was close to 100%). Cytoplasmic H09 reactivity was observed as expected in tumors with an IDH1 p.R132H mutation; cross-reactivity was seen with the p.R132L variant. This first NGS study of pediatric enchondromas confirms that IDH mutations may occur in a mosaic fashion. STK11 gene mutations may provide insights in the development of multiple cartilaginous tumors in enchondromatosis, this tumor suppressor gene having been shown in animal models to regulate both chondrocyte maturation and growth plate organization during 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

Effects of pulse width variations in pallidal stimulation for primary generalized dystonia.

International audienceBACKGROUND: Various pulse widths (from 60-450 mus) have been used for bilateral pallidal stimulation in generalized dystonia but, to date, no comparison of this parameter's effects is available. OBJECTIVE: To provide an analysis of the differential effects of bilateral short, medium and long stimulus pulse width (PW) on clinical improvement in primary generalized dystonia. METHODS: The most effective therapeutic stimulation parameters were recorded in 22 patients using bilateral pallidal stimulation. Six months after surgery, the effects of bilateral pallidal short (60-90 micros), medium (120-150 micros) and long (450 micros) PWs were studied in 20 of those patients. The effect of the stimulation was assessed by reviewing videotaped sessions by an observer blinded to treatment status (Burke-Fahn-Marsden movement score). Patients were tested on separate days, in random order, for the stimulation conditions (acute effect with the stimulation condition lasting 10 hours). The same contact was used for each stimulation condition. All the electrodes were set at 130 Hz (monopolar stimulation) and the intensity was set individually 10% below the side effect threshold. RESULTS: Median PWs of 60 (short), 120 (medium) and 450 micros (long) were compared,with a mean intensity of 4.46, 3.45 and 2.47 V, respectively. This study failed to demonstrate any significant difference in the movement scale dystonia mean scores depending on PW. CONCLUSION: According to our findings, short duration stimulus PWs are as effective as longer ones during a 10 hour period of observation. Confirmation of this finding for chronic use could be of importance in saving stimulator energy. Moreover, the use of smaller stimulus pulse widths are said to reduce charge injection and increase the therapeutic window between therapeutic effects and side effects