Joey St-Pierre

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.

Effects of prenatal maternal stress on serotonin and fetal development.

Fetuses are exposed to many environmental perturbations that can influence their development. These factors can be easily identifiable such as drugs, chronic diseases or prenatal maternal stress. Recently, it has been demonstrated that the serotonin synthetized by the placenta was crucial for fetal brain development. Moreover, many studies show the involvement of serotonin system alteration in psychiatric disease during childhood and adulthood. This review summarizes existing studies showing that prenatal maternal stress, which induces alteration of serotonin systems (placenta and fetal brain) during a critical window of early development, could lead to alteration of fetal development and increase risks of psychiatric diseases later in life. This phenomenon, termed fetal programming, could be moderated by the sex of the fetus. This review highlights the need to better understand the modification of the maternal, placental and fetal serotonin systems induced by prenatal maternal stress in order to find early biomarkers of psychiatric disorders.

Inhibition of placental 11beta-hydroxysteroid dehydrogenase type 2 by lead

Lead interferes with cortisol blood concentration, increases the risk of obstetrical complications, and could alter fetal development. The placenta controls maternal cortisol transfer to the fetus by the activity of the type 2 11β-hydroxysteroid dehydrogenase (11β-HSD2), which converts cortisol into inactive cortisone. This study determines the effect of lead on the expression and activity of the placental 11β-HSD2 in human trophoblast-like BeWo cells. Cells were treated with increasing concentration (0-1000nM) of PbCl2 for 24h. 11β-HSD2 protein expression was reduced by 45% at 1000nM of PbCl2 compared to untreated cells, while the activity was significantly reduced by PbCl2 at 10, 100 and 1000nM. This study shows the direct inhibitory action of lead on placental 11β-HSD2 activity and suggests that this heavy metal reduces the efficiency of the placental protection against the adverse effects of high cortisol level during fetal development.

A simple method to assess group difference in RT-qPCR reference gene selection using GeNorm: The case of the placental sex

Normalization with proper reference genes is a crucial step in obtaining accurate mRNA expression levels in RT-qPCR experiments. GeNorm and NormFinder are two commonly used software packages that help in selecting the best reference genes, based on their expression stability. However, GeNorm does not take into account a group variable, such as sample sex, in its calculation. We demonstrate a simple calculation step to assess the variability of such parameters by multiplying the GeNorm M value with the difference of Cq values between groups. To test this, we used 28 reference gene candidates, to analyze 20 placental samples (10 of each sex), and by using HPRT1 (lower Cq values in male placentas (Pu2009=u20090.017)), as a target gene. Our calculation demonstrates that the RPL30 – GAPDH reference gene combination is the better option to assess small placental sex differences in mRNA level, versus the selection obtained from GeNorm or NormFinder. The HPRT1 normalized mRNA expression level is different between placental sexes, using RPL30 and GAPDH as reference genes (Pu2009=u20090.01), but not when using genes suggested by GeNorm or NormFinder. These results indicate that the proposed calculation is appropriate to assess small variations in mRNA expression between 2 groups.

Prenatal Maternal Stress Alters Placental Endocrine Functions in Sex Dependent Manner

INTRODUCTION: n Prenatal Maternal stress (PNMS) is associated with nreduced type 2 11 nβ n- hydroxysteroid deshydrogenase (11 nβ n-HSD2) and type n1 glucose transporter (GLUT1). Cortisol exerts its action by binding to nglucocorticoid receptor alpha (GR- nα n) that acts as a transcription factor. nThe enzyme 11 nβ n-HSD2 protects the fetus from adverse cortisol levels nfrom the mother by converting cortisol to inactive cortisone. This study naims to determine if the placenta mediates the effects of disaster-related nPNMS (i.e., 2011 Queensland Flooding, Australia) on placental endocrine nfunction. We hypothesize that: (i) Increased PNMS will be associated nwith lower placental 11 nβ n-HSD2 gene expression which will be moderated nby fetal sex; and (ii) Increased PNMS will be associated with a lower nplacental index (fetal weight to placental weight) which will be moderated nby placental 11 nβ n-HSD2, GLUT-1 and/or GR- nα n gene expression. METHODS: n We assessed the women’s level of objective hardship (i.e., nevents they experienced) and subjective distress (i.e., their psychological nreaction to the flooding) shortly after the flooding. Placental villi n(trophoblastic tissues) from 96 placentas were processed and samples nflash frozen immediately after delivery. Gene expression was evaluated by nRT-qPCR. Regression and moderation were used for statistical analyses. RESULTS: n Results indicate that a higher level of subjective distress is nassociated with greater 11 nβ n-HSD2 gene expression in male fetuses and nlower 11 nβ n-HSD2 gene expression in female fetuses ( n∆ nR n2 n=0.074). Results nalso indicate that high levels of objective hardship coupled with high levels nof 11 nβ n-HSD2 gene expression (R n2 n=0.092), low levels of GLUT-1 gene nexpression (R n2 n=0,126), or low levels of GR- nα n gene expression (R n2 n=0.117) nis associated with higher placental index. CONCLUSIONS: n These results suggest that disaster-related PNMS ninfluences placental gene expression in a sex dependent manner. These nchanges in placental gene/protein expression demonstrate the different nsurvival strategies of the feto-placental unit in case of PNMS depending non fetal sex.INTRODUCTION: n Depression during pregnancy occurs in about n20% of women, of which 13% take antidepressants. SSRIs are the most ncommonly prescribed antidepressants for pregnant women, although ntheir effects on placental function have never been studied. A successful npregnancy depends on healthy placental development and function. The nextravillous trophoblast cells (evTBs), which invade the uterine wall, nare crucial for embryo implantation and the adaptation of maternal nspiral arteries. Poor invasion/migration of evTBs can cause important npregnancy complications such as preeclampsia and possibly maternal and nfetal mortality. The aim of this study was to determine whether SSRIs ncommonly used during pregnancy affect migratory and invasive properties nof JEG3 cells, used as a model of the evTBs. METHODS: n JEG3 cells were treated with increasing concentrations n(0.03-10 μM) of fluoxetine, norfluoxetine or sertraline. Cell nproliferation was monitored in real-time using a cell impedance-based nxCELLigence n system. JEG3 cell-cycle distribution was analyzed by flow cytometry. Migration was determined using a scratch test. Activities nof metalloproteinases MMP-2 and MMP-9 (markers of invasion) were ndetermined by gelatin zymography. RESULTS: n Fluoxetine and sertraline significantly decreased JEG3 ncell proliferation at 10 μM by 93% and 98%, respectively ([Figure n1]-fluoxetine), compared to control, whereas norfluoxetine had no effect. nFluoxetine decreased the number of cells in the G2-M at 1 and 10 μM, and nthe number of cells in G0-G1 at 10 μM. None of the SSRIs affected JEG3 nmigration ([Figure 2]-fluoxetine) or the activities of MMP-2 and MMP-9. Legend: nFigure 1: Effect of fluoxetine on extravillous trophoblast-like JEG3 cell nproliferation determined by real-time impedance monitoring. Statistically nsignificantly different from DMSO-treated cells. nFigure 2: Effect of fluoxetine on extravillous trophoblast-like JEG3 cell nmigration determined by scratch test. CONCLUSIONS: n This study suggests that the SSRIs fluoxetine, nnorfluoxetine and sertraline do not alter extravillous trophoblast viability nor migration at therapeutic levels. Our observations will be verified using nprimary cultures of evTBs and will include additional SSRIs.