Primary Trophoblast Cells Research Articles

Revisiting Steroidogenic Pathways in the Human Placenta and Primary Human Trophoblast Cells.

Steroid hormones play a crucial role in supporting a successful pregnancy and ensuring proper fetal development. The placenta is one of the principal tissues in steroid production and metabolism, expressing a vast range of steroidogenic enzymes. Nevertheless, a comprehensive characterization of steroidogenic pathways in the human placenta and potential developmental changes occurring during gestation are poorly understood. Furthermore, the specific contribution of trophoblast cells in steroid release is largely unknown. Thus, this study aimed to (i) identify gestational age-dependent changes in the gene expression of key steroidogenic enzymes and (ii) explore the role of trophoblast cells in steroid biosynthesis and metabolism. Quantitative and Droplet Digital PCR analysis of 12 selected enzymes was carried out in the first trimester (n = 13) and term (n = 20) human placentas. Primary trophoblast cells (n = 5) isolated from human term placentas and choriocarcinoma-derived cell lines (BeWo, BeWo b30 clone, and JEG-3) were further screened for gene expression of enzymes involved in placental synthesis/metabolism of steroids. Finally, de novo steroid synthesis by primary human trophoblasts was evaluated, highlighting the functional activity of steroidogenic enzymes in these cells. Collectively, we provide insights into the expression patterns of steroidogenic enzymes as a function of gestational age and delineate the cellular origin of steroidogenesis in the human placenta.

The BMP2 Signaling Axis Promotes Invasive Differentiation of Human Trophoblasts.

Embryo implantation and trophoblast invasion are principal limiting factors of pregnancy establishment. Aberrant embryo development or improper trophoblast differentiation and invasion may lead to various unfavorable pregnancy-related outcomes, including early pregnancy loss (EPL). Our clinical data show that the serum BMP2 levels were significantly increased during the first trimester of pregnancy and that the serum and BMP2 expression levels were lower in women with EPL than in women with normal early pregnancies. Moreover, we observed that BMP2 was expressed in oocytes and trophoblast cells of cleaved embryos and blastocysts prior to implantation in both humans and mice. Exogenous BMP2 promoted embryonic development by enhancing blastocyst formation and hatching in mice. LncRNA NR026833.1 was upregulated by BMP2 and promoted SNAIL expression by competitively binding to miR-502-5p. SNAIL induced MMP2 expression and promoted cell invasion in primary extravillous trophoblast cells. BMP2 promotes the invasive differentiation of mouse trophoblast stem cells by downregulating the expression of TS cell marker and upregulating the expression of trophoblast giant cell marker and labyrinthine/spongiotrophoblast marker. Our findings provide significant insights into the regulatory roles of BMP2 in the development of the placenta, which may give us a framework to explore new therapeutic strategies to pregnancy-related complications.

1008 Autophagy and ZIKV viral replication co-occur in human and primate placentae

Evidence suggests that ZIKV induces autophagy via LC3-dependent mechanisms in trophoblasts, and inhibition of autophagy restricts placental infection in mice and cytotrophoblasts in vitro. Thus, anti-autophagal drugs (i.e, hydroxychloroquine) could inhibit vertical transmission. To generate preclinical data, we assessed primary cell culture and placentae from human congenital infections, and an experimental non-human primate (Callithrix jacchus) model for associated autophagal activity. Primary trophoblast cells were isolated from placenta (n=10) and infected in vitro with ZIKV. Autophagy-associated gene expression (ULK-1, BECN1, ATG5, ATG7, ATG12, ATG16L1, MAP1LC3A, MAP1LC3B, p62) was determined by TaqMan qPCR for changes with ZIKV. In in vivo validation experiments, autophagy genes LC3B and p62 were probed using in situ hybridization (ISH) in the placentae of Congenital Zika Syndrome (n=3) and ZIKV-infected primate placenta and fetal tissue (n=1). Infected and uninfected villi were compared for mean density and colocalization of autophagal markers. Studies of primary human trophoblasts revealed key autophagy genes ATG5 and p62 were decreased ∼2 fold in ZIKV-infected trophoblasts (p=0.04 and p=0.03). Histologic examination by ISH confirmed ZIKV replication in primate congenital infection cases (Fig 1A). Immunohistological identification of autophagal proteins LC3B and p62 demonstrates co-localization (Fig 1B-C). Protein expression was assessed via densitometry and revealed ∼0.8 fold decrease in p62 in villi with active ZIKV replication (p=0.007), confirming autophagal flux (Fig 2). We demonstrate for the first time that the association of autophagy at the maternal fetal barrier is observed following ZIKV in vivo using three methodologies. Downregulation of key autophagy genes is consistent with findings in Dengue virus, where autophagy is initially induced then downregulated in late stage infection. These findings collectively suggest antiautophagal drugs may be effective in mediating vertical transmission of ZIKV and potentially other novel RNA viruses.View Large Image Figure ViewerDownload Hi-res image Download (PPT)

Vasohibin 1, a clinically relevant biomarker, contributes to pre-eclampsia.

Pre-eclampsia is a leading health threat for pregnant women which is characterised by hypertension and proteinuria. The detailed mechanism is elusive and no effective therapy is available. Predictive biomarkers are needed for accurate diagnosis. Vasohibin-1 (VASH1) is an intrinsic inhibitor of angiogenesis induced by angiogenic factors in endothelial cells. This study aimed to evaluate the role of VASH1 as a useful biomarker for pre-eclampsia. VASH1 level was examined by ELISA and immunoblotting assay in the serum and placental samples from healthy pregnant women and pre-eclampsia patients. Cellular assay was performed to assess cell migration and invasion with different levels of VASH1. The level of VASH1 was measured under different oxygen conditions by qPCR. VASH1 was highly expressed in the serum and placenta of pre-eclampsia patients. Overexpression of VASH1 led to attenuated cell migration and invasion ability and reduced levels of matrix metallopeptidase 2 and 9. VASH1 was significantly induced in primary human trophoblast cells and placental explants under hypoxic condition in hypoxia-inducible factor 1 alpha-dependent manner. Our study suggested that VASH1 could be used as a potentially novel biomarker for pre-eclampsia and its level may positively correlate with the disease status.

Role of IL-36 Cytokines in the Regulation of Angiogenesis Potential of Trophoblast Cells.

IL-36 cytokines (the agonists IL-36α, IL-36β, IL-36γ, and the antagonist IL-36Ra) are expressed in the mouse uterus and associated with maternal immune response during pregnancy. Here, we characterize the expression of IL-36 members in human primary trophoblast cells (PTC) and trophoblastic cell lines (HTR-8/SVneo and JEG-3) and upon treatment with bacterial and viral components. Effects of recombinant IL-36 on the migration capacity of trophoblastic cells, their ability to interact with endothelial cells and the induction of angiogenic factors and miRNAs (angiomiRNAs) were examined. Constitutive protein expression of IL-36 (α, β, and γ) and their receptor (IL-36R) was found in all cell types. In PTC, transcripts for all IL-36 subtypes were found, whereas in trophoblastic cell lines only for IL36G and IL36RN. A synthetic analog of double-stranded RNA (poly I:C) and lipopolysaccharide (LPS) induced the expression of IL-36 members in a cell-specific and time-dependent manner. In HTR-8/SVneo cells, IL-36 cytokines increased cell migration and their capacity to interact with endothelial cells. VEGFA and PGF mRNA and protein, as well as the angiomiRNAs miR-146a-3p and miR-141-5p were upregulated as IL-36 response in PTC and HTR-8/SVneo cells. In conclusion, IL-36 cytokines are modulated by microbial components and regulate trophoblast migration and interaction with endothelial cells. Therefore, a fundamental role of these cytokines in the placentation process and in response to infections may be expected.

Inhibition of mechanistic target of rapamycin signaling decreases levels of O-GlcNAc transferase and increases serotonin release in the human placenta.

Changes in placental function, in particular down-regulation of placental O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT) in response to maternal stress and increased placental secretion of serotonin into the fetal circulation following maternal infection, have been mechanistically linked to adverse neurodevelopment in mice. We hypothesized that mechanistic target of rapamycin (mTOR) signaling is a key regulator of trophoblast serotonin synthesis and OGT protein expression and that serotonin is secreted by the human placenta into the fetal circulation. Placental homogenates (n=46) from elective terminations at 8-22 weeks of gestation and from healthy-term women were sexed and the protein levels of OGT and enzymes involved in serotonin synthesis was determined. Primary human trophoblast (PHT) cells were isolated from normal term placenta (n=27), cultured and transfected (n=8) with siRNA targeting a scramble sequence (control), raptor (inhibits mTOR Complex 1 (mTORC1)), or rictor (inhibits mTOR Complex 2 (mTORC2)). Subsequently, conditioned media and PHT cell lysates were collected. Free serotonin concentration was measured using ELISA in cell culture media and in platelet-depleted normal term umbilical vein and artery plasma (n=38). Both mTORC1 and mTORC2 inhibition down-regulated OGT levels in PHT cells. The level of serotonin synthesis enzyme tryptophan hydroxylase (TPH-1) was higher in early gestation female placentas and at term serotonin concentration was three-fold higher in the umbilical vein than in the umbilical artery. Inhibition of mTORC2, but not mTORC1, increased cultured PHT cell serotonin secretion. Our data are consistent with the model that mTOR signaling is a key regulator of trophoblast serotonin synthesis and OGT protein expression.

MTORC1 Transcriptional Regulation of Ribosome Subunits, Protein Synthesis, and Molecular Transport in Primary Human Trophoblast Cells.

Mechanistic Target of Rapamycin Complex 1 (mTORC1) serves as positive regulator of placental nutrient transport and mitochondrial respiration. The role of mTORC1 signaling in modulating other placental functions is largely unexplored. We used gene array following silencing of raptor to identify genes regulated by mTORC1 in primary human trophoblast (PHT) cells. Seven hundred and thirty-nine genes were differentially expressed; 487 genes were down-regulated and 252 up-regulated. Bioinformatic analyses demonstrated that inhibition of mTORC1 resulted in decreased expression of genes encoding ribosomal proteins in the 60S and 40S ribosome subunits. Furthermore, down-regulated genes were functionally enriched in genes involved in eIF2, sirtuin and mTOR signaling, mitochondrial function, and glutamine and zinc transport. Stress response genes were enriched among up-regulated genes following mTORC1 inhibition. The protein expression of ribosomal proteins RPL26 (RPL26) and Ribosomal Protein S10 (RPS10) was decreased and positively correlated to mTORC1 signaling and System A amino acid transport in human placentas collected from pregnancies complicated by intrauterine growth restriction (IUGR). In conclusion, mTORC1 signaling regulates the expression of trophoblast genes involved in ribosome and protein synthesis, mitochondrial function, lipid metabolism, nutrient transport, and angiogenesis, representing novel links between mTOR signaling and multiple placental functions critical for normal fetal growth and development.

Activin A increases human trophoblast invasion by upregulating integrin β1 through ALK4.

Activin A promotes human trophoblast invasion during the first trimester of pregnancy and is associated with preeclampsia and pregnancy-induced hypertension (PE/PIH) in naturally conceived pregnancies. However, whether integrin β1 mediates activin A-increased trophoblast invasion remains unknown and the evidence is limited regarding the predictive value of activin A for PE/PIH in women receiving in vitro fertilization (IVF) treatment. Here, we studied the role and underlying molecular mechanisms of integrin β1 in activin A-promoted invasion in immortalized (HTR8/SVneo) and primary human extravillous trophoblast (EVT) cells. A nest case-control study was designed to investigate the predictive/diagnostic value of activin A in IVF pregnancies. Results showed that integrin β1 expression increased after activin A treatment and knockdown of integrin β1 significantly decreased both basal and activin A-increased HTR8/SVneo cell invasion. SB431542 (TGF-β type I receptors inhibitor) abolished activin A-induced SMAD2/SMAD3 phosphorylation and integrin β1 overexpression. Activin A-upregulated integrin β1 expression was attenuated after the depletion of ALK4 or SMAD4 in both HTR8/SVneo and primary EVT cells. Furthermore, we found similar first-trimester activin A levels in IVF patients with or without subsequent PE/PIH. These results reveal that integrin β1 mediates activin A-promoted trophoblast invasion through ALK4-activated SMAD2/3-SMAD4 pathway, and the predictive/diagnostic value of first-trimester maternal serum activin A for hypertensive disorders of pregnancy might be different in IVF population.

Syncytialization and prolonged exposure to palmitate impacts BeWo respiration.

Placental villous trophoblast mitochondrial respiratory function is critical for a successful pregnancy and environmental influences such as maternal obesity have been associated with respiratory impairment at term. More recently, a gestational high fat diet independent of maternal body composition, has been highlighted as a potential independent regulator of placental mitochondrial metabolism. The current study aimed to characterize the direct impact of a prolonged and isolated exposure to the dietary fatty acids Palmitate (PA) and Oleate (OA) upon placental cell mitochondrial respiratory function. BeWo cytotrophoblast (CT) and syncytiotrophoblast (SCT) cells were treated for 72 h with 100 µM PA, OA or PA+OA (P/O). Live-cell metabolic function was analyzed via the Seahorse XF Mito and Glycolysis Stress tests. Immunoblots and spectrophotometric activity assays were utilized to examine the protein expression and function of electron transport chain (ETC) complexes and key mitochondrial regulatory enzymes. Syncytialization of BeWo cells resulted reduced respiratory activity in conjunction with altered complex I and II activity and decreased pyruvate dehydrogenase (PDH) protein expression and activity. PA and P/O treatments were associated with increased basal and maximal respiratory activities in BeWo CT cells without alterations in protein expression or activity of individual ETC complexes and mitochondrial substrate regulators. The metabolic suppression in BeWo SCTs was consistent with that previously observed in primary human trophoblast cell cultures, while the observed increases in respiratory activity in PA-treated BeWo CTs may be indicative of an early timepoint of specific dietary saturated fat-mediated placental cell mitochondrial dysfunction.

Dynamics of Tryptophan Metabolic Pathways in Human Placenta and Placental-Derived Cells: Effect of Gestation Age and Trophoblast Differentiation.

L-Tryptophan is an essential amino acid and a precursor of several physiologically active metabolites. In the placenta, the serotonin and kynurenine metabolic pathways of tryptophan metabolism have been identified, giving rise to various molecules of neuroactive or immunoprotective properties, such as serotonin, melatonin, kynurenine, kynurenic acid, or quinolinic acid. Current literature suggests that optimal levels of these molecules in the fetoplacental unit are crucial for proper placenta functions, fetal development and programming. Placenta is a unique endocrine organ that, being equipped with a battery of biotransformation enzymes and transporters, precisely orchestrates homeostasis of tryptophan metabolic pathways. However, because pregnancy is a dynamic process and placental/fetal needs are continuously changing throughout gestation, placenta must adapt to these changes and ensure proper communication in the feto-placental unit. Therefore, in this study we investigated alterations of placental tryptophan metabolic pathways throughout gestation. Quantitative polymerase chain reaction (PCR) analysis of 21 selected genes was carried out in first trimester (n = 13) and term (n = 32) placentas. Heatmap analysis with hierarchical clustering revealed differential gene expression of serotonin and kynurenine pathways across gestation. Subsequently, digital droplet PCR, Western blot, and functional analyses of the rate-limiting enzymes suggest preferential serotonin synthesis early in pregnancy with a switch to kynurenine production toward term. Correspondingly, increased function and/or protein expression of serotonin degrading enzyme and transporters at term indicates efficient placental uptake and metabolic degradation of serotonin. Lastly, gene expression analysis in choriocarcinoma-derived cell lines (BeWo, BeWo b30, JEG-3) revealed dissimilar expression patterns and divergent effect of syncytialization compared to primary trophoblast cells isolated from human term placentas; these findings show that the commonly used in vitro placental models are not suitable to study placental handling of tryptophan. Altogether, our data provide the first comprehensive evidence of changes in placental homeostasis of tryptophan and its metabolites as a function of gestational age, which is critical for proper placental function and fetal development.

Acetylcholine ameliorated TNF-α-induced primary trophoblast malfunction via muscarinic receptors†.

Oxidative stress and apoptosis of trophoblasts are involved in preeclampsia (PE). Numerous studies have shown that acetylcholine (ACh), the principal vagal neurotransmitter, plays a crucial role in attenuating oxidative stress, inflammation, and apoptosis in a variety of human diseases. However, the role of ACh in PE management remains unclear. Here, we aimed to determine the effects of ACh on TNF-α-treated human primary trophoblast cells. Western blotting, CCK-8, DHE, TUNEL immunofluorescence staining, transwell assays, and wound-healing assays were performed to evaluate the role of ACh in vitro. We found that both TNF-α expression and the apoptotic index were higher in placentas from preeclamptic women than in normal placentas. TNF-α enhanced oxidative stress and increased the number of TUNEL-positive nuclei, Bax/Bcl-2 ratio, and the cleaved caspase-3/caspase-3 ratio while decreasing cell viability in primary human trophoblast cells. TNF-α promoted cell migration and invasion. PDTC, a selective NF-κB inhibitor, significantly blunted TNF-α-induced effects. ACh treatment attenuated oxidative stress and apoptosis while further promoting migration and invasion of TNF-α-treated primary trophoblast cells. The effects of ACh could be reversed by the muscarinic receptor antagonist atropine. Overall, our findings indicate that ACh significantly ameliorates TNF-α-induced oxidative stress and apoptosis of human primary trophoblast cells via muscarinic receptors. This is the first time that the improvement of vagal activity served as a therapeutic strategy for PE-like trophoblasts, suggesting its potential value in clinical practice.

GNRH ANTAGONISTS INHIBIT TROPHOBLAST INVASION, PROLIFERATION AND OUTGROWTH IN PLACENTA FROM WOMEN WITH ECTOPIC PREGNANCY

Ectopic pregnancy (EP) is a leading cause of maternal morbidity and mortality. Besides surgical intervention, methotrexate (MTX) therapy is the only approved medical treatment for EP. In addition to its side effects, the rate of treatment failure could reach up to 30%. Gonadotropin-releasing hormone (GnRH) and its receptor are abundantly expressed in EP implantation sites. GnRH also has been shown to facilitate implantation by promoting trophoblast invasion. The objective of our study is to investigate the role of GnRH antagonists in inhibiting invasion, proliferation, and outgrowth in a de novo primary EP trophoblast cell model and a 3D placental explant model.

Aryl hydrocarbon receptor (AhR) agonist β-naphthoflavone regulated gene networks in human primary trophoblasts

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that is highly expressed in placenta. AhR belongs to a class of transcriptional regulators that control many developmental and physiological events (e.g. xenobiotic metabolism). Our study describes AhR regulated transcriptional responses in human primary trophoblast by using the AhR agonist, β-naphthoflavone (BNF). Human primary trophoblast cells were isolated from full term placenta after delivery. The trophoblasts were exposed to 25 μM of AhR agonist, BNF, for 72 hours. Gene expression profiling was conducted with Illumina HT-12 expression beadchips. Expression of selected genes was confirmed with RT-qPCR. Ingenuity pathway analysis (IPA) was used to predict functional pathways and upstream regulators of differentially expressed genes in order to identify regulatory networks associated with AhR. In response to BNF exposure, 64 genes were upregulated, and 257 genes were downregulated compared to control trophoblasts (±1.5-fold, p < 0.05). BNF regulated genes included placental hormones and genes implicated in immune- and inflammatory responses in addition to their well-known effects on xenobiotic metabolism, oxidative stress, antioxidant defense. In conclusion, these results show that BNF has wide-ranging effects on placental gene expression beyond xenobiotic metabolism e.g. disruption of inflammatory processes and hormones in the placenta.

Cleavage of the soluble (pro)renin receptor (sATP6AP2) in the placenta

IntroductionThe (pro)renin receptor (ATP6AP2) is cleaved and released as soluble ATP6AP2 (sATP6AP2). The sATP6AP2 is detected in plasma and urine and is elevated in women with gestational diabetes and preeclampsia. The source and cleavage pathway of sATP6AP2 in pregnancy is unknown. The syncytiotrophoblast is the major placental secretory layer and is in direct contact with maternal blood. Both FURIN and Site 1 protease (MBTPS1) cleave sATP6AP2 in non-placental cells. We postulated that ATP6AP2 was cleaved by FURIN and/or MBTPS1 and that sATP6AP2 is secreted by the placental syncytiotrophoblast. MethodsTerm primary trophoblast cells were transfected with FURIN siRNA, negative control siRNA or vehicle. In a separate experiment, primary trophoblasts were treated with a pro-protein convertase inhibitor (DEC-RVKR-CMK), an MBTPS1 inhibitor (PF 429242) or vehicle. Trophoblasts were left to spontaneously syncytialise before cells and supernatants were collected and intracellular and extracellular sATP6AP2 levels analysed by immunoblot.Results: sATP6AP2 is secreted by placental trophoblasts. Levels of intra and extra-cellular sATP6AP2 decrease with syncytialisation (P = 0.01 and P = 0.02, respectively), as do FURIN mRNA (P = 0.0003) and protein (P = 0.0007). FURIN siRNA decreased FURIN mRNA and protein levels (both P < 0.0001). Neither FURIN siRNA or PF 429242 affected sATP6AP2 levels. DEC-RVKR-CMK significantly decreased extracellular sATP6AP2 protein levels (P = 0.02). DiscussionSoluble ATP6AP2 is secreted by placental trophoblasts and levels decrease with syncytialisation. DEC-RVKR-CMK, a broad inhibitor of pro-protein convertases reduced extracellular sATP6AP2 levels, but FURIN siRNA and MBTPS1 inhibition had no effect. Hence, a convertase other than FURIN or MBTPS1 is most likely responsible for placental sATP6AP2 secretion.

Establishment and characterization of a new human first trimester Trophoblast cell line, AL07

IntroductionThe limited cell number of primary trophoblasts and contamination of trophoblast cell lines promote us to develop a novel stable trophoblast cell line. Method of studyPrimary trophoblast cells were isolated from first-trimester placenta and telomerase-induced immortalization was used to immortalize these cells. Subsets of cells were then evaluated by flow cytometry using CK7, HLA-G, CD45 and CD14, specific markers for trophoblast cells, extra-villous trophoblast, pan leucocyte and monocyte/macrophage, respectively. Immunofluorescence staining and immunocytochemistry were used to detect CK7 expression in trophoblast cells. The level of secreted human Chorionic Gonadotropin (hCG) was measured by electrochemiluminescence (ECL). The Bio-Plex MAGPIX System was used to analyze the cytokines and chemokines produced by AL07 cell line. ResultsWe were able to isolate primary trophoblast cells from several first-trimester placentas. One clone, AL07 trophoblast cells, isolated from a week 7 placenta, was morphologically stable and positive for the expression of CK7 by immunofluorescence and immunocytochemistry staining. Characterization of AL07 cells reveled that they are CD45 or CD14 negative and had constitutive secretion of hCG and low HLA-G expression. Furthermore, clone AL07 secret high levels of several cytokines and chemokines, including IL-6, IL-8 and VEGF, and moderately secreted MCP-1 IP-10 and RANTES. DiscussionWe report the successful isolation, immortalization and characterization of AL07 cells, a novel cell clone isolated from first trimester human placenta. The clone is free of contamination of immune cells, and exhibits similar cytokine profile as other trophoblast cell lines. This new cytotrophoblast-like AL07 cell, can be a valuable tool for in-vitro trophoblast studies in the future.

Trophoblast lineage specific expression of the alternative splicing factor RBFOX2 suggests a role in placental development

IntroductionRBFOX2, an RNA-binding protein, controls tissue-specific alternative splicing of exons in diverse processes of development. The progenitor cytotrophoblast of the human placenta differentiates into either the syncytiotrophoblast, formed via cell fusion, or the invasive extravillous trophoblast lineage. The placenta affords a singular system where a role for RBFOX2 in both cell invasion and cell fusion may be studied.We investigated a role for RBFOX2 in trophoblast cell differentiation, as a foundation for investigations of RBFOX2 in embryo implantation and placental development. MethodsImmunohistochemistry of RBFOX2 was performed on placental tissue sections from three trimesters of pregnancy and from pathological pregnancies. Primary trophoblast cell culture and immunofluorescence were employed to determine RBFOX2 expression upon cell fusion. Knockdown of RBFOX2 expression was performed with βhCG and syncytin-1 as molecular indicators of fusion. ResultsIn both normal and pathological placentas, RBFOX2 expression was confined to the cytotrophoblast and the extravillous trophoblast, but absent from the syncytiotrophoblast. Additionally, we showed that primary trophoblasts that spontaneously fused in cell culture downregulated RBFOX2 expression. In functional experiments, knockdown expression of RBFOX2 significantly upregulated βhCG, while the upregulation of syncytin-1 did not reach statistical significance. DiscussionRBFOX2, by conferring mRNA diversity, may act as a regulator switch in trophoblast differentiation to either the fusion or invasive pathways. By studying alternative splicing we further our understanding of placental development, yielding possible insights into preeclampsia, where expression of antiangiogenic isoforms produced through alternative splicing play a critical role in disease development and severity.

Uptake of Cerium Dioxide Nanoparticles and Impact on Viability, Differentiation and Functions of Primary Trophoblast Cells from Human Placenta.

The human placenta is at the interface between maternal and fetal circulations, and is crucial for fetal development. The nanoparticles of cerium dioxide (CeO2 NPs) from air pollution are an unevaluated risk during pregnancy. Assessing the consequences of placenta exposure to CeO2 NPs could contribute to a better understanding of NPs’ effect on the development and functions of the placenta and pregnancy outcome. We used primary villous cytotrophoblasts purified from term human placenta, with a wide range of CeO2 NPs concentrations (0.1–101 μg/cm2) and exposure time (24–72 h), to assess trophoblast uptake, toxicity and impact on trophoblast differentiation and endocrine function. We have shown the capacity of both cytotrophoblasts and syncytiotrophoblasts to internalize CeO2 NPs. CeO2 NPs affected trophoblast metabolic activity in a dose and time dependency, induced caspase activation and a LDH release in the absence of oxidative stress. CeO2 NPs decreased the fusion capacity of cytotrophoblasts to form a syncytiotrophoblast and disturbed secretion of the pregnancy hormones hCG, hPL, PlGF, P4 and E2, in accordance with NPs concentration. This is the first study on the impact of CeO2 NPs using human primary trophoblasts that decrypts their toxicity and impact on placental formation and functions.

1972-P: Adipose Tissue–Derived Exosomal MicroRNA Regulates Placental Glucose Uptake in Gestational Diabetes Mellitus Pregnancies

Adipose tissue exosomal miRNAs (exomiRs) serve as a signalling molecule that is involved in conveying intercellular message signalling. In this study, we aimed to profile the adipose tissue exomiRs in patients with normal glucose tolerant (NGT) and gestational diabetes mellitus (GDM). Omentum were obtained from BMI-matched NGT (n=10) and GDM (n=10) women at delivery (&amp;gt;37 weeks gestation). Isolated exosomes were characterised based on size distribution and protein enrichment. Total exosomal RNA was isolated by miRNeasy (Qiagen) according to manufacturer’s instructions. Illumina TrueSeq Small RNA kit was used to construct a small RNA library. Finally, the resulting sequencing FASTQ files were analysed using miRDeep2 to identify both known and novel miRNAs. A selected exomiR mimic was overexpressed in the human primary trophoblast (PHT) cells and its effect on glucose uptake was evaluated using 2-NBDG. We identified a total of 1209 miRNAs with 18 miRNAs were significantly differentially expressed in GDM exosomes. Comparatively, 12 miRNAs were upregulated while 6 were downregulated in the exosomes from GDM pregnancy compared to normal pregnancy. Consequently, has-miR-515-5p was selected to be investigated in further functional studies, following validation of its expression using real-time PCR. The QRT-PCR analysis of placental tissue showed a significant increase in the expression of GDM placenta compared to NGT placenta. In PHT cells, the overexpression of miR-515-5p significantly stimulated the glucose uptake (p&amp;lt;0.0001) compared to control group. The proteomics analysis of miR-515-5p overexpressed PHT cells showed differential expression of proteins that are associated with glycolysis. These findings suggest adipose tissue exosomal miR515-5p mediated placental glucose uptake could contribute to an excessive placental nutrient transfer GDM, which could instigate consequences, such as fetal overgrowth. Disclosure N. Jayabalan: None. D. Guanzon: None. A. Lai: None. D. McIntyre: Other Relationship; Self; Novo Nordisk A/S. M. Lappas: None. C. Salomon: None. Funding Diabetes Australia; Fondo Nacional de Desarrollo Científico y Tecnológico (1170809)

The (pro)renin receptor (ATP6AP2) does not play a role in syncytialisation of term human primary trophoblast cells

IntroductionIn the placenta, the (pro)renin receptor (ATP6AP2) is localised to the syncytiotrophoblast. ATP6AP2 can activate the placental renin-angiotensin system (RAS), producing Angiotensin II (Ang II) which, acting via the angiotensin II type 1 receptor (AGTR1), is important for placental development and function. ATP6AP2 can also independently stimulate intracellular signalling pathways known to regulate trophoblast syncytialisation. We proposed that ATP6AP2 plays a role in trophoblast syncytialisation. MethodsPrimary trophoblast cells were isolated from human placentae and transfected with an ATP6AP2 siRNA, a negative control siRNA or vehicle and allowed to spontaneously syncytialise. Syncytialisation was determined by secretion of human chorionic gonadotrophin (hCG) and by decreased CDH1 (E-cadherin) levels. Expression of RAS mRNAs and proteins were measured by qPCR and immunoblotting, respectively. ResultsPrimary trophoblast cells spontaneously syncytialised in culture. Syncytialisation did not affect ATP6AP2 mRNA or protein levels. However, the expression of REN, AGT and AGTR1 mRNAs were increased (P = 0.02, P = 0.01 and P = 0.03, respectively). ATP6AP2 siRNA had no effect on syncytialisation. DiscussionIn primary trophoblasts, syncytialisation was associated with increased expression of the RAS. hCG was increased during syncytialisation and is known to stimulate REN and possibly AGT, however further experiments are needed to confirm that this was the mechanism via which the RAS was activated. Therefore, syncytialisation of primary trophoblasts may involve hCG-induced RAS activation and downstream activation of signalling pathways and growth factors, which can be stimulated via the interaction of Ang II with AGTR1. Nevertheless, it appears that the (pro)renin receptor is not involved.

L-carnitine increases cell proliferation and amino acid transporter expression via the activation of insulin-like growth factor I signaling pathway in rat trophoblast cells.

Early embryo implantation and development is primarily determined by the homeostasis between cellular apoptosis and proliferation as well as placental nutrient transporters. Recent studies showed that L‐carnitine enhances female reproductive performance. However, the potential function of L‐carnitine on placenta is largely unknown. In our study, primary rat trophoblast cells were separated and cultured for 12 hr in medium containing various concentrations of L‐carnitine (0, 1, 10, and 50 mM). Placenta trophoblast cells treated with 50 mM L‐carnitine increased the proportion of cells in S phase of the cell cycle (p < .05). In addition, live cell percentage was increased when treated with either 10 mM or 50 mM L‐carnitine, which was accompanied with decreased necrotic cells, late apoptotic cells, and early apoptotic cells (p < .05). Compared with the control treatment, the mRNA expression of insulin‐like growth factor I (IGF‐1) and insulin‐like growth factor I receptor (IGF‐1R) was higher in rat placenta trophoblasts treated with either 10 mM or 50 mM L‐carnitine (p < .05). Similarly, sodium‐dependent neutral amino acid transporter (SNAT)‐1 and SNAT2 were up‐regulated in both mRNA and protein levels when trophoblast cells were treated with 50 mM L‐carnitine (p < .05). Inhibiting downstream targets (Akt or ERK signaling pathways) of IGF‐1 signaling pathway partially blocked the effect the L‐carnitine‐induced increase in protein abundances of SNAT1 and SNAT2. Collectively, our data showed protective role of L‐carnitine on placenta trophoblast cells through the involvement of IGF‐1 signaling pathway.