Julie Lafond

Thyroid hormones in pregnancy in relation to environmental exposure to organochlorine compounds and mercury.

Polychlorinated biphenyls (PCBs), chlorinated pesticides, and mercury are global environmental contaminants that can disrupt the endocrine system in animals and humans. However, there is little evidence that they can interfere with endocrine status in pregnant women and neonates at low levels of exposure. The aim of this study was to examine thyroid hormone levels during pregnancy and in cord blood in relation to blood concentrations of organochlorine compounds (OCs) and Hg in healthy women recruited during pregnancy. We found a significant negative correlation between maternal total triiodothyronine levels and three non-coplanar congeners (PCB-138, PCB-153, and PCB-180), three pesticides (p,p′-DDE, cis-nanochlor, and hexachlorobenzene), and inorganic Hg independently, without any other changes in thyroid status. No significant relationships were observed between OCs and cord serum thyroid hormones. Cord serum free thyroxin was negatively correlated with inorganic Hg. These results suggest that at even low levels of exposure, persistent environmental contaminants can interfere with thyroid status during pregnancy.

Combining biochemical and ultrasonographic markers in predicting preeclampsia : a systematic review

BACKGROUND Early identification of pregnant women at risk for preeclampsia is a priority to implement preventive measures. Some biochemical and ultrasonographic parameters have shown promising predictive performance, but so far there is no clinically validated screening procedure. CONTENT Using a series of keywords, we reviewed electronic databases (Medline, Embase, all records to May 2009) reporting the performance of biological and ultrasonographic markers to predict preeclampsia, both single markers and combinations of markers. We analyzed the data according to gestational age and risk levels of the studied populations. We evaluated the methodological quality of included publications using QUADAS (quality assessment of diagnostic accuracy studies). We identified 37 relevant studies that assessed 71 different combinations of biochemical and ultrasonographic markers. Most studies were performed during the second trimester on small-scale high-risk populations with few cases of preeclampsia. Combinations of markers generally led to an increase in sensitivity and/or specificity compared with single markers. In low-risk populations, combinations including placental protein 13 (PP13), pregnancy-associated plasma protein A (PAPP-A), a disintegrin and metalloprotease-12 (ADAM12), activin A, or inhibin A measured in first or early second trimester and uterine artery Doppler in second trimester appear promising (sensitivity 60%-80%, specificity >80%). In high-risk populations, the combination of PP13 and pulsatility index in first trimester showed 90% sensitivity and 90% specificity in a single study limited to severe preeclampsia. SUMMARY Combinations of biochemical and ultrasonographic markers improved the performance of early prediction of preeclampsia. From a perspective of integrative medicine, large population-based studies evaluating algorithms combining multiple markers are needed, if screening approaches are to be eventually implemented.

ERK1/2 and p38 regulate trophoblasts differentiation in human term placenta

Mitogen‐activated protein kinases (MAPKs) control many cellular events from complex programmes, such as embryogenesis, cell differentiation and proliferation, and cell death, to short‐term changes required for homeostasis and acute hormonal responses. However, little is known about expression and activation of classical MAPKs, extracellular signal‐regulated kinase1/2 (ERK1/2) and p38 in human placenta. Therefore, we examined the expression of ERK1/2 and p38 in trophoblasts from human term placenta, and their implication in differentiation. In vitro, freshly isolated cytotrophoblast cells, cultivated in 10% fetal bovine serum (FBS), spontaneously aggregate and fuse to form multinucleated cells that phenotypically resemble mature syncytiotrophoblasts, that concomitantly produce human chorionic gonadotropin (hCG) and human placental lactogen (hPL). This study shows that the level of ERK1/2 and p38 decreases with increasing days of culture, to reach an undetectable level after 5 days of culture. Moreover, pretreatment of cells with an ERK1/2‐specific inhibitor (PD98059) and/or a p38‐specific inhibitor (SB203580) suppressed trophoblast differentiation. Our results also demonstrate that the p38 pathway is highly solicited as compared to the ERK1/2 pathway in the differentiation process. Furthermore, ERK1/2 and p38 are rapidly activated upon addition of FBS, but the activation of p38 is delayed compared to that of ERK1/2. In summary, this study showed that ERK1/2 and p38 pathways are essential to mediate initiation of trophoblast differentiation.

Syncytin-2 plays an important role in the fusion of human trophoblast cells.

Human endogenous retrovirus (HERV)-encoded Syncytin-1 has been suggested to play a major role in trophoblast cell fusion and thereby placenta development. However, recent studies have strongly suggested that other HERV envelope proteins could also be implicated in this process. Based on this premise, herein we compared the expression and functional implication of Syncytin-1 with the more recently described Syncytin-2 protein in various trophoblast cells. Real-time reverse transcription PCR and Western blot analyses in differentiating primary trophoblast cells first indicated a direct correlation between mRNA and protein levels of Syncytin-2 and cell fusion, while an inverse correlation for Syncytin-1 was noted. Similar reverse transcription PCR experiments and promoter studies showed that cell fusion-inducing agents in the trophoblastic BeWo cell line increased the expression of Syncytin-1 but, more importantly, augmented Syncytin-2 expression. Confocal microscopy experiments further revealed that in BeWo cells and in freshly isolated primary human trophoblast cells, Syncytin-1 was present as a cytoplasmic punctuated structure in proximity to regions of cell-to-cell contact. On the other hand, Syncytin-2 presented an inducible signal, which mainly localized to the cytoplasmic membrane. Experiments with siRNA (small interfering RNA)-transfected BeWo and primary human trophoblast cells demonstrated an important diminution in the number of cell fusion events upon repression of Syncytin-2 expression, whereas transfection experiments with Syncytin-1-specific siRNA resulted in a more modest effect. Overall, these results highlight the importance of Syncytin-2 in BeWo and primary human trophoblast cell fusion.

Modulation of Fatty Acid Transport and Metabolism by Maternal Obesity in the Human Full-Term Placenta

ABSTRACT Knowledge of the consequences of maternal obesity in human placental fatty acids (FA) transport and metabolism is limited. Animal studies suggest that placental uptake of maternal FA is altered by maternal overnutrition. We hypothesized that high maternal body mass index (BMI) affects human placental FA transport by modifying expression of key transporters. Full-term placentas were obtained by vaginal delivery from normal weight (BMI, 18.5–24.9 kg/m2) and obese (BMI > 30 kg/m2) women. Blood samples were collected from the mother at each trimester and from cord blood at delivery. mRNA and protein expression levels were evaluated with real-time RT-PCR and Western blotting. Lipoprotein lipase (LPL) activity was evaluated using enzyme fluorescence. In vitro linoleic acid transport was studied with isolated trophoblasts. Our results demonstrated that maternal obesity is associated with increased placental weight, decreased gestational age, decreased maternal high-density lipoprotein (HDL) levels during the first and third trimesters, increased maternal triglyceride levels during the second and third trimesters, and increased maternal T3 levels during all trimesters, and decreased maternal cholesterol (CHOL) and low-density lipoprotein (LDL) levels during the third trimester; and increased newborn CHOL, LDL, apolipoprotein B100, and T3 levels. Increases in placental CD36 mRNA and protein expression levels, decreased SLC27A4 and FABP1 mRNA and protein and FABP3 protein expression, and increased LPL activity and decreased villus cytotrophoblast linoleic acid transport were also observed. No changes were seen in expression of PPARA, PPARD, or PPARG mRNA and protein. Overall this study demonstrated that maternal obesity impacts placental FA uptake without affecting fetal growth. These changes, however, could modify the fetus metabolism and its predisposition to develop diseases later in life.

Human placental trophoblasts synthesize melatonin and express its receptors.

Abstract:  Although the role of melatonin on fetal development has been the subject of a number of studies, little is known about the function of melatonin in the placenta. We previously showed that melatonin receptors are expressed and are functional in JEG‐3 and BeWo cell lines, both in vitro models of human trophoblast. Local synthesis of melatonin in placenta has been proposed, but the human placenta’s ability to synthesize melatonin de novo has never been studied. The purpose of this study was to investigate the expression [reverse transcription‐polymerase chain reaction (RT‐PCR) and western blot analysis] and activity (radiometric assay) of melatonin synthesizing enzymes, and characterize the expression of the melatoninergic receptors in human term villous trophoblast. The results show that arylalkylamine N‐acetyltransferase and hydroxyindole O‐methyltransferase melatonin synthesizing enzymes are expressed and active in villous trophoblast as well as in JEG‐3 and BeWo placental choriocarcinoma cells. In addition, immunohistochemical analysis demonstrated the presence of MT1, MT2, and retinoid‐related orphan nuclear receptor α melatonin receptor proteins in both villous cytotrophoblast and syncytiotrophoblast (STB) as well as in endothelial cells surrounding the fetal capillaries and in the villous mesenchymal core. RT‐PCR and western blot analysis in primary cultures of human term trophoblast confirmed the expression of all three melatonin receptors in villous cytotrophoblast and STB cells. This study demonstrates for the first time a local synthesis of melatonin and expression of its receptors in human trophoblasts and strongly suggests a paracrine, autocrine, and/or intracrine role for this indolamine in placental function and development as well as in protection from oxidative stress.

Syncytin proteins incorporated in placenta exosomes are important for cell uptake and show variation in abundance in serum exosomes from patients with preeclampsia.

Exosomes are extracellular vesicles that mediate intercellular communication and are involved in several biological processes. The objective of our study was to determine whether endogenous retrovirus group WE, member l (ERVWE1)/syncytin‐1 and endogenous retrovirus group FRD, member 1 (ERVFRDE1)/ syncytin‐2, encoded by human endogenous retrovirus (HERV) envelope (env) genes, are present at the surface of exosomes produced by placenta‐derived villous cytotrophoblasts and whether they play a role in cellular uptake of exosomes. In addition, we sought to determine whether these proteins are present in various abundances in serum‐derived exosomes from normal pregnant women vs. women with preeclampsia (PE). Isolated exosomes were analyzed for their content by Western blot, a bead‐associated flow cytometry approach, and a syncytin‐2 ELISA. Binding and uptake were tested through confocal and electron microscopy using the BeWo choriocarcinoma cell line. Quality control of exosome preparations consisted of detection of exosomal and nonexosomal markers. Exosome–cell interactions were compared between cells incubated in the presence of control exosomes, syncytin‐1 or syncytin‐2‐deprived exosomes, or exosomes solely bearing the uncleaved forms of these HERV env proteins. From our data, we conclude that villous cytotrophoblast exosomes are positive for both env proteins and are rapidly taken up by BeWo cells in a syncytin‐1‐ and syncytin‐2‐dependent manner and that syncytin‐2 is reduced in serum‐derived exosomes from women with PE when compared to exosomes from normal pregnant women.—Vargas, A., Zhou, S., Éthier‐Chiasson, M., Flipo, D., Lafond, J., Gilbert, C., Barbeau, B. Syncytin proteins incorporated in placenta exosomes are important for cell uptake and show variation in abundance in serum exosomes from patients with preeclampsia. FASEB J. 28, 3703–3719 (2014). www.fasebj.org

Calcium uptake and calcium transporter expression by trophoblast cells from human term placenta

Placental transfer of maternal calcium (Ca(2+)) is a crucial step for fetal development although the biochemical mechanisms responsible for this process are largely unknown. This process is carried out in vivo by the placental syncytiotrophoblast layer. The aim of this study was to define the membrane gates responsible for the syncytiotrophoblast Ca(2+) entry, the first step in transplacental transfer. We have investigated the basal Ca(2+) uptake by primary culture of human term placenta syncytiotrophoblast. Kinetic studies revealed an active extracellular Ca(2+) uptake by cultured human syncytiotrophoblast. We demonstrated by Northern blot the presence of transcript for calcium transporter type 1 (CaT1) in cultured human syncytiotrophoblast and CaT1 expression was further confirmed by reverse transcription polymerase chain reaction (RT-PCR). In addition, the expression of calcium transporter type 2 (CaT2) was revealed by RT-PCR in cultured human syncytiotrophoblast. It has been reported that the activity of this family of Ca(2+) channels is voltage-independent, and is not sensitive to L-type Ca(2+) channels agonist and antagonist. Interestingly, modulation of membrane potential by extracellular high potassium concentration and valinomycin had no effect on the basal Ca(2+) uptake of human syncytiotrophoblast. Moreover, the addition of L-type Ca(2+) channel modulators (Bay K 8644 and nitrendipine) to the incubation medium had also no effect on the basal Ca(2+) uptake, suggesting that the process is mainly voltage-independent and does not involved L-type Ca(2+) channels. On the other hand, we observed that two known blockers of CaT-mediated Ca(2+) transport, namely extracellular magnesium (Mg(2+)) and ruthenium red, dose-dependently inhibited Ca(2+) uptake by cultured human syncytiotrophoblast. Therefore, our results suggest that basal Ca(2+) uptake of human syncytiotrophoblast may be assured by CaT1 and CaT2.

Reduced Expression of Both Syncytin 1 and Syncytin 2 Correlates With Severity of Preeclampsia

Human endogenous retroviruses (HERVs) represent up to 8% of the human genome and express several of its genes in the placenta. Studies have demonstrated that HERV envelope proteins syncytins 1 and 2 play a crucial role in trophoblast fusion and placenta development. Here, we compared the levels of placental expression of syncytins with the severity of preeclampsia (PE) symptoms. Confocal microscopy experiments indicated a pronounced deficiency in cellular fusion in trophoblast cells from patients with PE when compared to controls. As determined by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) and Western blot analyses, syncytin mRNA and protein levels were decreased in PE placentas versus controls. Interestingly, syncytin 2 levels were more importantly impaired than syncytin 1. Our results further highlighted the existence of a correlation between the extent of the decrease in the expression levels of both fusogenic proteins and the degree of severity of PE symptoms. These HERV proteins could thereby be used as potential markers for the early diagnosis of PE.

Stability of reference proteins in human placenta: General protein stains are the benchmark

The stability of reference proteins in semi-quantitative Western blot experiments in normal and diseased placenta has never been studied. This study aims to determine the stability of five reference proteins and two general protein stains in placentas from preeclampsia, gestational diabetes mellitus and matched control pregnancies. The stability of the reference proteins was analysed using indicators of inter-group (P value) and intra-group (coefficient of variation) stability. The effect of different normalization strategies was determined by normalizing serotonin transporter (SERT) expression against the different reference protein markers. Results show significant expression variability of β-actin, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), hypoxanthine phosphoribosyltransferase 1 (HPRT1), peptidylprolyl isomerase A (PPIA) and α-tubulin, and that amido black staining is the most stable reference protein marker. Furthermore, results show that SERT expression significantly differs according to the reference protein markers used for its normalization. The present study demonstrated the importance of using stable reference protein markers and normalization strategy in order to get correct results in semi-quantitative Western blot experiments in placental tissues.