Cathy Vaillancourt

Prenatal stress and brain development.

Prenatal stress (PS) has been linked to abnormal cognitive, behavioral and psychosocial outcomes in both animals and humans. Animal studies have clearly demonstrated PS effects on the offsprings brain, however, while it has been speculated that PS most likely affects the brains of exposed human fetuses as well, no study has to date examined this possibility prospectively using an independent stressor (i.e., a stressful event that the pregnant woman has no control over, such as a natural disaster). The aim of this review is to summarize the existing animal literature by focusing on specific brain regions that have been shown to be affected by PS both macroscopically and microscopically. These regions include the hippocampus, amygdala, corpus callosum, anterior commissure, cerebral cortex, cerebellum and hypothalamus. We first discuss the mechanisms by which the effects of PS might occur. In particular, we show that maternal and fetal hypothalamic-pituitary-adrenal (HPA) axes, and the placenta, are the most likely candidates for these mechanisms. We see that, although animal studies have obvious advantages over human studies, the integration of findings in animals and the transfer of these findings to human populations remains a complex issue. Finally, we show how it is possible to circumvent these challenges by studying the effects of PS on brain development directly in humans, by taking advantage of natural or man-made disasters and assessing the impact and consequences of such stressful events on pregnant women and their offspring prospectively.

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.

Melatonin: The smart killer: The human trophoblast as a model

Melatonin has both the ability to induce intrinsic apoptosis in tumor cells while it inhibits it in non-tumor cells. Melatonin kills tumor cells through induction of reactive oxygen species generation and activation of pro-apoptotic pathways. In contrast, melatonin promotes the survival of non-tumor cells due to its antioxidant properties and the inhibition of pro-apoptotic pathways. In primary human villous trophoblast, a known pseudo-tumorigenic tissue, melatonin promotes the survival through inhibition of the Bax/Bcl-2 pathway while in BeWo choriocarcinoma cell line melatonin induces permeabilization of the mitochondrial membrane leading to cellular death. These findings suggest that the trophoblast is a good model to study the differential effects of melatonin on the intrinsic apoptosis pathway. This review describes the differential effects of melatonin on the intrinsic apoptosis pathway in tumor and non-tumor cells and presents the trophoblast as a novel model system in which to study these effects of melatonin.

Placental melatonin production and melatonin receptor expression are altered in preeclampsia: new insights into the role of this hormone in pregnancy

Abstract:  The melatonin system in preeclamptic pregnancies has been largely overlooked, especially in the placenta. We have previously documented melatonin production and expression of its receptors in normal human placentas. In addition, we and others have shown a beneficial role of melatonin in placental and fetal functions. In line with this, decreased maternal blood levels of melatonin are found in preeclamptic compared with normotensive pregnancies. However, melatonin production and expression of its receptors in preeclamptic compared with normotensive pregnancy placentas has never been examined. This study compares (i) melatonin‐synthesizing enzyme expression and activity, (ii) melatonin and serotonin, melatonin’s immediate precursor, levels and (iii) expression of MT1 and MT2 melatonin receptors in placentas from preeclamptic and normotensive pregnancies. Protein and mRNA expression of aralkylamine N‐acetyltransferase (AANAT) and hydroxyindole O‐methyltransferase (HIOMT), the melatonin‐synthesizing enzymes, as well as MT1 and MT2 receptors were determined by RT‐qPCR and Western blot, respectively. The activities of melatonin‐synthesizing enzymes were assessed by radiometric assays while melatonin levels were determined by LC‐MS/MS. There is a significant inhibition of AANAT, melatonin’s rate‐limiting enzyme, expression and activity in preeclamptic placentas, correlating with decreased melatonin levels. Likewise, MT1 and MT2 expression is significantly reduced in preeclamptic compared with normotensive pregnancy placentas. We propose that reduced maternal plasma melatonin levels may be an early diagnostic tool to identify pregnancies complicated by preeclampsia. This study indicates a clinical utility of melatonin as a potential treatment for preeclampsia in women where reduced maternal plasma levels have been identified.

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.

First steps for integrating sex and gender considerations into basic experimental biomedical research

In recent decades there has been an increasing recognition of the need to account for sex and gender in biology and medicine, in order to develop a more comprehensive understanding of biological phenomena and to address gaps in medical knowledge that have arisen due to a generally masculine bias in research. We have noted that as basic experimental biomedical researchers, we face unique challenges to the incorporation of sex and gender in our work, and that these have remained largely unarticulated, misunderstood, and unaddressed in the literature. Here, we describe some of the specific challenges to the incorporation of sex and gender considerations in research involving cell cultures and laboratory animals. In our view, the main‐streaming of sex and gender considerations in basic biomedical research depends on an approach that will allow scientists to address these issues in ways that do not undermine our ability to pursue our fundamental scientific interests. To that end, we suggest a number of strategies that allow basic experimental researchers to feasibly and meaningfully take sex and gender into account in their work.—Ritz, S.A., Antle, D. M., Côté, J., Deroy, K., Fraleigh, N., Messing, K., Parent, L., St‐Pierre, J., Vaillancourt, C., Mergler, D. First steps for integrating sex and gender considerations into basic experimental biomedical research. FASEB J. 28, 4–13 (2014). www.fasebj.org

Quantitative PCR pitfalls : the case of the human placenta.

Reverse-transcription quantitative polymerase chain reaction (RT-qPCR) is a rapid and high throughput gene expression quantification technology. In order to obtain accurate results, several key experimental design and standardization steps must be rigorously followed as previously described in the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines. This study investigates the effect of reference gene normalization and the impact of RNA degradation on gene expression of 8-oxoguanine DNA glycosylase in human placenta from pregnancies complicated by preeclampsia and gestational diabetes mellitus and their gestation-matched controls. The data presented here show how RNA quality and appropriate reference gene selection is not only important to obtain accurate and reproducible RT-qPCR data but how different and even opposite results can be reported if the key steps outlined in the MIQE guidelines are not followed. The procedures and associated results presented in this study provide the first practical application of the MIQE guidelines to placental analysis in normal and pathological pregnancies.

Birth Insult Alters Dopamine-Mediated Behavior in a Precocial Species, the Guinea Pig: Implications for Schizophrenia

Schizophrenia is associated with increased birth complications, suggesting that birth complications might alter CNS dopaminergic activity later in life. In rats, Caesarean section (C-section) birth can produce long term changes in dopaminergic biochemistry and behavior. However rat brain is somewhat immature compared to human brain at birth. The current study tested if mild birth complications also alter dopamine-mediated function in a species with a more mature CNS at birth, the guinea pig. As adults, guinea pigs born by C-section showed increased amphetamine-induced locomotion and disruption of prepulse inhibition (PPI) of acoustic startle, compared to vaginally born controls. Guinea pigs born by C-section with 1 min of added global anoxia showed reduced amphetamine-induced locomotion and disrupted PPI, while a C-section plus 2 min anoxia group showed no change in amphetamine-induced locomotion but increased amphetamine-induced startle. No group differences in effects of amphetamine or apomorphine on PPI were observed. Taken with previous findings, these results indicate that mild birth complications can cause long term changes in dopamine-mediated behavior in both guinea pig and rat, two species spanning the level of human brain maturity at birth.

Melatonin: the watchdog of villous trophoblast homeostasis against hypoxia/reoxygenation-induced oxidative stress and apoptosis.

Human placenta produces melatonin and expresses its receptors. We propose that melatonin, an antioxidant, protects the human placenta against hypoxia/reoxygenation (H/R)-induced damage. Primary term villous cytotrophoblasts were cultured under normoxia (8% O2) with or without 1mM melatonin for 72h to induce differentiation into the syncytiotrophoblast. The cells were then cultured for an additional 22h under normoxia or subjected to hypoxia (0.5% O2) for 4h followed by 18h reoxygenation (8% O2) with or without melatonin. H/R induced oxidative stress, which activated the Bax/Bcl-2 mitochondrial apoptosis pathway and the downstream fragmentation of DNA. Villous trophoblast treatment with melatonin reversed all the negative effects induced by H/R to normoxic levels. This study shows that melatonin protects the villous trophoblast against H/R-induced oxidative stress and apoptosis and suggests a potential preventive and therapeutic use of this indolamine in pregnancy complications characterized by syncytiotrophoblast survival alteration.

Low environmental contamination by lead in pregnant women: effect on calcium transfer in human placental syncytiotrophoblasts.

There is an extensive literature on the neurotoxic effects of lead (Pb) on the developing fetus; however, little is known about the mechanisms of action at low levels. Heavy metals are known to affect calcium (Ca2 + ) homeostasis through perturbation of Ca2 + channels and pumps and interference with protein kinase C (PKC) and Ca2 + binding protein (CaBP). During pregnancy, placental Ca2 + exchange is one of the most important mechanisms for fetal survival. This ion is an essential element for healthy fetal growth and development. The aim of the present study was to determine the influence of low maternal blood Pb levels on Ca2 + levels in serum and placenta and placental Ca2 + transfer. Blood samples (maternal and cord) and placental tissue were obtained at birth from 30 women residing in southwest Quebec. Total 2+and Pb levels were measured in maternal and umbilical cord samples and placental tissue at term. The placentas were taken for trophoblast cell isolation and Ca2 + incorporation kinetic experiments. Data showed that Ca2 + in maternal blood did not influence Ca2 + uptake by syncytiotrophoblast. However, although maternal and cord blood Pb levels were low, maternal blood Pb concentration was significantly linked to a decrease in Ca2 + uptake by syncytiotrophoblast. This suggests that exposure to very levels of Pb significantly modifies Ca2 + transfer in syncytiotrophoblasts.