Human First-trimester Trophoblast Research Articles

Galectin-9 regulates HTR8/SVneo function via JNK signaling.

To obtain a successful pregnancy, trophoblasts must provide a physical barrier, suppress maternal reactivity, produce immunosuppressive hormones locally, and enhance the production of blocking factors that are able to bind to several antigenic sites. Inadequate placental perfusion has been closely associated with several pregnancy-associated diseases. Galectin-9 (Gal-9) has a wide variety of regulatory functions in innate and adaptive immunity during infection, tumor growth, and organ transplantation. We utilized immortalized human first-trimester extravillous trophoblast cells (HTR8/SVneo) for our functional study and examined the effects of Gal-9 on apoptosis, cytokine production and angiogenesis of HTR8/SVneo cells. Gal-9 inhibited the apoptosis and IFN-γ and IL-17A production, promoted IL-4 production, and coordinated the crosstalk between HTR8/SVneo cells and human umbilical vein endothelial cells via its interaction with Tim-3. Blockade of JNK signaling inhibited Gal-9 activities in HTR8/SVneo cells. In addition, we detected a correlation between low levels of Gal-9 and spontaneous abortion. So Gal-9 could inhibit the apoptosis and proinflammatory cytokine expression, and promote the angiogenesis and IL-4 production in HTR8/SVneo cells via Tim-3 in a JNK dependent manner to help the maintenance of normal pregnancy. These findings possibly identify Gal-9 as a key regulator of trophoblast cells and suggest its potential as a biomarker and target for the treatment of recurrent pregnancy loss.

Galectin-7 Impairs Placentation and Causes Preeclampsia Features in Mice.

Preeclampsia is a serious pregnancy-induced disorder unique to humans. The etiology of preeclampsia is poorly understood; however, poor placental formation is thought causal. Galectin-7 is produced by trophoblast and is elevated in first-trimester serum of women who subsequently develop preeclampsia. We hypothesized that elevated placental galectin-7 may be causative of preeclampsia. Here, we demonstrated increased galectin-7 production in chorionic villous samples from women who subsequently develop preterm preeclampsia compared with uncomplicated pregnancies. In vitro, galectin-7 impaired human first-trimester trophoblast outgrowth, increased placental production of the antiangiogenic sFlt-1 splice variant, sFlt-1-e15a, and reduced placental production and secretion of ADAM12 (a disintegrin and metalloproteinase12) and angiotensinogen. In vivo, galectin-7 administration (E8-E12) to pregnant mice caused elevated systolic blood pressure, albuminuria, impaired placentation (reduced labyrinth vascular branching, impaired decidual spiral artery remodeling, and a proinflammatory placental state demonstrated by elevated IL1β, IL6 and reduced IL10), and dysregulated expression of renin-angiotensin system components in the placenta, decidua, and kidney, including angiotensinogen, prorenin, and the angiotensin II type 1 receptor. Collectively, this study demonstrates that elevated galectin-7 during placental formation contributes to abnormal placentation and suggests that it leads to the development of preeclampsia via altering placental production of sFlt-1 and renin-angiotensin system components. Targeting galectin-7 may be a new treatment option for preeclampsia.

Peli1 signaling blockade attenuates congenital zika syndrome.

Zika virus (ZIKV) infects pregnant women and causes devastating congenital zika syndrome (CZS). How the virus is vertically transmitted to the fetus and induces neuronal loss remains unclear. We previously reported that Pellino (Peli)1, an E3 ubiquitin ligase, promotes p38MAPK activation in microglia and induction of lethal encephalitis by facilitating the replication of West Nile virus (WNV), a closely related flavivirus. Here, we found that Peli1 expression was induced on ZIKV-infected human monocytic cells, peripheral blood mononuclear cells, human first-trimester placental trophoblasts, and neural stem cell (hNSC)s. Peli1 mediates ZIKV cell attachment, entry and viral translation and its expression is confined to the endoplasmic reticulum. Moreover, Peli1 mediated inflammatory cytokine and chemokine responses and induced cell death in placental trophoblasts and hNSCs. ZIKV-infected pregnant mice lacking Peli1 signaling had reduced placental inflammation and tissue damage, which resulted in attenuated congenital abnormalities. Smaducin-6, a membrane-tethered Smad6-derived peptide, blocked Peli1-mediated NF-κB activation but did not have direct effects on ZIKV infection. Smaducin-6 reduced inflammatory responses and cell death in placental trophoblasts and hNSCs, and diminished placental inflammation and damage, leading to attenuated congenital malformations in mice. Collectively, our results reveal a novel role of Peli1 in flavivirus pathogenesis and suggest that Peli1 promotes ZIKV vertical transmission and neuronal loss by mediating inflammatory cytokine responses and induction of cell death. Our results also identify Smaducin-6 as a potential therapeutic candidate for treatment of CZS.

93-OR: Low Maternal Insulin Sensitivity Associates with DNA-Related Functions in Human First-Trimester Trophoblast

In first trimester (FT) of pregnancy the human placenta is rapidly growing making it sensitive to environmental influence. We hypothesized that the altered intrauterine milieu associated with maternal obesity modifies placental proteome and function. We used maternal BMI and leptin as measures of obesity and adiposity, characterized the glucose-insulin axis (glucose, C-peptide, insulin sensitivity (ISHOMA)) and quantified fatty acids (GC) in maternal fasted serum (n=123-323). Among those, only higher C-peptide levels and lower ISHOMA were associated with obesity. Subsequent untargeted proteomics (LC-MS/MS, qTOF Bruker maXis ETD II) in FT placentas (weeks 5-6 p.m.) from lean (n=11) and obese (n=11) women and Principal Component Analysis clustered the samples in two groups. Only ISHOMA differed between both clusters (ISHOMA cluster 1: median 0.95, IQR 0.69-1.17; ISHOMA cluster 2: median 0.57, IQR 0.20 - 1.00, P<0.05). Comparing proteomic profiles between ISHOMA groups (separated by median ISHOMA 0.61) identified 86 proteins enriched in the low ISHOMA cluster (P<0.05, FDR 0.05). Biological processes (KEGG pathway analysis) enriched in this group were related to DNA replication and cell cycle (FDR<0.001). Members of the MCM family, involved in DNA damage response and cell cycle arrest, associated with low ISHOMA. This suggested trophoblast growth related processes depend on ISHOMA. Semi-quantitative in-situ analysis found decreased trophoblast proliferation (Ki67, n=22, Δ -33.4%, p<0.05) and increased apoptosis (TUNEL assay, n=22, 5.6 fold, p<0.05) in the low ISHOMA group. These differences were related to an increased DNA damage (𝛄;;H2AX, n=12, 6.9 fold, p<0.001). We conclude that low ISHOMA associated with maternal obesity in FT of pregnancy induces placental proteome changes related to DNA metabolism. This might alter trophoblast, i.e., placental, growth early in pregnancy, which is known to associate with fetal growth and birth weight. Disclosure J. Bandres-Meriz: None. D. Hoch: None. S. Honeder: None. A. Majali-Martinez: None. E. Herrera: None. M. Schittmayer: None. R. Birner-Gruenberger: None. G. Desoye: None.

SIRT1 Alleviates LPS-Induced IL-1β Production by Suppressing NLRP3 Inflammasome Activation and ROS Production in Trophoblasts.

Emerging evidence indicates that aberrant maternal inflammation is associated with several pregnancy-related disorders such as preeclampsia, preterm birth, and intrauterine growth restriction. Sirtuin1 (SIRT1), a class III histone deacetylase, is involved in the regulation of various physiopathological processes including cellular inflammation and metabolism. However, the effect of SIRT1 on the placental proinflammatory environment remains to be elucidated. In this study, we investigated the effect of SIRT1 on lipopolysaccharide (LPS)-induced NLRP3 inflammasome activation and its underlying mechanisms in human first-trimester trophoblasts (Sw.71 and HTR-8/SVneo cells). Treatment with LPS elevated SIRT1 expression and induced NLRP3 inflammasome activation in mouse placental tissues and human trophoblasts. Knockdown of SIRT1 enhanced LPS-induced NLRP3 inflammasome activation, inflammatory signaling, and subsequent interleukin (IL)-1β secretion. Furthermore, knockdown of NLRP3 considerably attenuated the increase of IL-1β secretion in SIRT1-knockdown cells treated with LPS. Moreover, SIRT1 inhibited LPS-induced NLRP3 inflammasome activation by reducing oxidative stress. This study revealed a novel mechanism via which SIRT1 exerts anti-inflammatory effects, suggesting that SIRT1 is a potential therapeutic target for the prevention of inflammation-associated pregnancy-related complications.

H19 regulates angiogenic capacity of extravillous trophoblasts by H19/miR-106a-5p/VEGFA axis.

To investigate the role and underlying mechanism of H19 in regulating angiogenic capacity of extravillous trophoblasts. Gain and loss of function experiments were performed using a human first-trimester extravillous trophoblast (EVT) cell line, HTR-8/SVneo cells. H19 was overexpressed or knocked down in HTR-8 cells by transfecting plasmid harboring whole-length H19 sequence (pH19) or siRNA specially targeting H19, respectively (siH19). Cell migration and tube-formation assay were assessed in the indicated groups. Gene expression was detected by RT-qPCR, Western blot, and ELISA assay. Overexpression of H19 in EVT cells increased cell migration and tube formation, while downregulation of H19 in EVT cells decreased cell migration and tube formation. Furthermore, we found that H19 played its role by VEGFA. In addition, we demonstrated the H19/miR-106a-5p/VEGFA regulatory axis in EVT. Experiments of the clinical specimen showed that H19 was very abundantly expressed in human first-trimester trophoblasts, and we found that the expression of H19 and VEGFA were significantly downregulated in the villous tissues from idiopathic recurrent miscarriage (RM) patients; moreover, the expression of H19 and VEGFA was positively correlated. H19/miR-106a-5p/VEGFA axis plays a role in regulating the angiogenic capacity of EVT, which might contribute to idiopathic RM.

Sestrin2 alleviates palmitate-induced endoplasmic reticulum stress, apoptosis, and defective invasion of human trophoblast cells.

Maternal obesity induces elevated saturated fatty acid palmitate levels in the blood and causes pregnancy complications such as gestational diabetes, preeclampsia, fetal growth abnormalities, and stillbirth. Sestrin2, a highly conserved stress-inducible protein, is involved in the cellular responses of various stress conditions and homeostatic regulation. However, the effects of Sestrin2 on trophoblast cells have not yet been investigated. Here, we investigated the role of Sestrin2 in palmitate-induced lipotoxicity and its underlying mechanisms in human first-trimester trophoblast cells (Sw.71). Mouse placental tissues were obtained from low-fat diet-fed mice (n=14) and high-fat diet-fed mice (n=14) at gestation day 17.5. Sw.71 cells were treated with palmitate or bovine serum albumin as vehicle controls. The role of Sestrin2 in palmitate-induced lipotoxicity was examined by immunocytochemistry, immunoblot analysis, quantitative real-time PCR, and invasion assay. Expression of placental Sestrin2 was elevated in high-fat diet-fed dams compared to that of low-fat diet-fed dams. Prolonged treatment of Sw.71 cells with palmitate-induced endoplasmic reticulum (ER) stress-dependent expressions of Sestrin2 protein and mRNA, and the treatment also triggered apoptosis. Knockdown of Sestrin2 increased palmitate-mediated ER stress, inflammatory signaling, and apoptosis. Furthermore, Sestrin2 suppressed impaired trophoblast invasion caused by palmitate and attenuated palmitate-induced ER stress and inflammation via AMPK/mTORC1 pathways. Our study provides the relationship between Sestrin2, AMPK/mTORC1 pathway, and trophoblast function, suggesting that Sestrin2 may be a novel potential therapeutic target for the prevention of pregnancy complications.

Resveratrol induces SIRT1-Dependent autophagy to prevent H2O2-Induced oxidative stress and apoptosis in HTR8/SVneo cells

IntroductionPre-eclampsia (PE) is a serious complication of pregnancy, and the likely pathogenic basis of early onset PE are placental dysfunction and increased oxidative stress. Resveratrol (RES) is a potent antioxidant which has shown beneficial effects in many diseases. The aim of this study was to investigate the protective effects of RES against oxidative stress-induced damage in trophoblasts, and elucidate the potential mechanisms. MethodsWe established an in vitro model of oxidative stress by exposing the human first-trimester extravillous trophoblast cell line HTR8/SVneo to H2O2. The level of oxidative stress was reflected by ROS, MDA and SOD. The viability of cells was determined by the MTS assay. Apoptosis was detected using Annexin V-FITC staining and flow cytometry. Levels of SIRT1(sirtuin 1) and autophagy-related proteins (LC3, Beclin-1, p62) were detected by western blot. Autophagosomes were observed by transmission electron microscopy (TEM). ResultsPre-treatment with RES significantly ameliorated H2O2-induced cytotoxicity, morphological damage, oxidative stress and apoptosis. Mechanistically, RES restored the levels of SIRT1 and autophagy-related proteins including LC3-II, Beclin-1 and p62 that were dysregulated by H2O2. Blocking autophagy by 3-methyladenine (3-MA) completely abolished the protective effects of RES, as did knocking down SIRT1. ConclusionRES may protect human trophoblasts against H2O2-induced oxidative stress by activating SIRT1-dependent autophagy, and therefore has therapeutic potential in PE.

1063: Endometrial and myometrial cell CXCL12 expression in response to mechanical injury: implications for placenta accreta.

In view of increasing incidence of placenta accreta spectrum disorders, there is an urge to better understand the molecular mechanisms of abnormal trophoblast invasion. CXCL12 and its receptor, CXCR4, have been implicated in many physiologic and pathologic processes including cell migration and trafficking, tissue healing in the setting of mechanical, ischemic and inflammatory damage and cancer metastasis. CXCL12 functions as a chemokine, attracting cells expressing CXCR4. Prior studies demonstrate that CXCL12 is produced by endometrium while CXCR4 is present on trophoblast cells. In this study, we sought to assess if tissue injury would affect the expression of CXCL12 in endometrial and myometrial cells as well as CXCR4 expression in trophoblast cells using an in vitro model. Cultured primary human endometrial stromal and myometrial cells were subjected to a scratch injury. Cells were collected 4 hours after injury and CXCL12 mRNA levels in injured and non-injured cells were assessed using RT-qPCR. CXCR4 mRNA levels were assessed in 2 human first-trimester trophoblast cell lines and in primary cytotrophoblast from term placentas with RT-qPCR. Baseline levels of CXCL12 mRNA were higher in myometrial than endometrial stromal cell (2-ΔCt; 0.076 vs 0.011 respectively, p< 0.01). Scratch injury resulted in significant increase in CXCL12 mRNA levels in endometrial stromal (2-ΔCt; 0.048 from 0.011, p=0.02) but not in myometrial cells (2-ΔCt; 0.076 vs 0.069, p >0.05). CXCR4 mRNA was expressed at high levels in cytotrophoblast from term placentas, but not in the established trophoblast cell lines. Injury results in enhanced expression of CXCL12 in cultured endometrial stromal cells. CXCL12 secretion will attract the CXCR4 expressing trophoblasts. These results suggest that activation of this axis as part of the normal healing process is likely a factor modulating the increased invasiveness of the trophoblast in the setting of uterine injury. Modulation of CXCL12 expression or use of CXCR4 antagonists may prevent excessive placental invasion associated with placenta accreta.

Human placental trophoblast cells contribute to maternal\u2013fetal tolerance through expressing IL-35 and mediating iTR35 conversion

During pregnancy, trophoblast cells sustain the maternal–fetal tolerance via expressing and secreting various chemokines and cytokines. Our previous study revealed the expression of interleukin-35 (IL-35) in human first-trimester trophoblasts. Here we show that IL-35 is expressed in both human first-trimester primary trophoblast cells and a trophoblast cell line. Trophoblast cells inhibit the proliferation of human naive conventional T cells (Tconv cells) and convert suppressed Tconv cells into iTR35 in an IL-35-dependent manner. Mechanistically, trophoblast cell derived IL-35 mediates its function through phosphorylation of STAT1 and STAT3. In vivo studies confirm that mice with immunologically spontaneous abortion have lower levels of IL-35 and iTR35 cells at the maternal–fetal interface, and neutralizing anti-IL-35 mAb enhances abortion rates. Meanwhile, exogenous IL-35 induces iTR35 and prevents immunological abortion. Our findings thus suggest that trophoblast cells have a critical function in preserving maternal–fetal tolerance via secreting IL-35 during pregnancy.

Effect of placental growth factor in models of experimental pre-eclampsia and trophoblast invasion.

Placental growth factor (PlGF) is decreased in early gestation of pregnant women who subsequently develop pre-eclampsia. In this study, pre-emptive treatment with PlGF to prevent pre-eclampsia was evaluated in an in vivo rodent model of experimental pre-eclampsia (EPE) induced by TNF-α and in an in vitro model of human first-trimester trophoblast invasion. Pregnant C57/BL6 mice were treated with recombinant mouse placental growth factor-2 (rmPlGF-2) 100μg/kg/day IP from gestational day (gd) 10. Animals had EPE induced by continuous TNF-α infusion on gd 13 and were subject to either continuous blood pressure monitoring by radiotelemetry throughout pregnancy or live placenta T2 -weighted magnetic resonance imaging (MRI) to demonstrate placental function on gd 17. There was no difference in BP (P>.99), proteinuria (P=.9) or T2 values on MRI (P=.9) between control and rmPlGF-2-treated animals. On gd 13, animals treated with rmPlGF-2 demonstrated increased placenta PlGF (P=.01) and Toll-like receptor-3 (P=.03) mRNA expression as compared with controls. Fluorescent-labelled human uterine microvascular endothelial cells and HTR8/SVNeo cells were co-cultured on Matrigel™ and treated with recombinant human PlGF (rhPlGF) (10ng/mL) and/or TNF-α (0.5ng/mL). Trophoblast integration into endothelial networks was reduced by added TNF-α (P=.006), as was rhPlGF concentration in conditioned media (P<.0001). Cell integration was not ameliorated by addition of rhPlGF (P>.9). Although TNF-α-induced EPE was not reversed with pre-emptive rmPlGF-2, a further trial of pre-emptive rhPlGF in vivo is required to determine whether the absence of effect of rhPlGF demonstrated in vitro precludes PlGF as a preventative therapy for pre-eclampsia.

Trophoblasts promote induction of a regulatory phenotype in B cells that can protect against detrimental T cell-mediated inflammation.

A successful outcome to pregnancy is critically dependent on the initiation of maternal immune tolerance before embryo implantation. Cells of embryonic origin that come in contact with the uterine microenvironment can exert influence over the phenotype and function of immune cells to facilitate robust implantation; however, what influence they may have on B cells remains unknown. In this study, we investigate the effect of human trophoblast cells on B-cell phenotype and the subsequent effect on peri-implantation events. We cultured purified human B cells with the first-trimester human trophoblast cell line Swan 71 to investigate trophoblast-B-cell interactions and utilized trophoblast spheroids in an in vitro implantation model of migration and invasion. Trophoblast-educated B cells or TE-B cells were found to consist of B cells in committed lineages such as plasmablasts and memory B cells, as well as increased proportions in subsets of CD24hi CD27+ regulatory B cells and CD19+ IL-10+ B cells. Conditioned media from the TE-B cells showed reduced production of pro-inflammatory cytokines that influenced the T-cell proliferation and cytokine production. Using trophoblast spheroids, we assessed the role of TE-B cells in trophoblast invasion and migration. Our results demonstrate a protective effect of TE-B-conditioned media against deleterious inflammation as evidenced by survival of the trophoblast spheroid in the presence of an immune assault and promotion of a migratory phenotype. We posit that trophoblast-mediated education of B cells leads to their acquisition of properties capable of modulating inflammation in the uterine environment during the peri-implantation period.

Regulation of RECK expression by TGF-β1 and oxygen in human first-trimester trophoblast cells

Matrix metalloproteinases (MMPs) are a cluster of enzymes that degrade the extracellular matrix (ECM) and some intracellular proteins; as such, they play an important role in tissue regeneration, infant growth, animal reproduction, and immunity. Most research into MMPs focuses mainly on their effects on the mammalian immune system. However, it is not clear how MMPs affect immune processes in crustaceans. Here, we cloned the open reading frame (ORF) of Eriocheir sinensis (Chinese mitten crab) MMP-14 (EsMMP-14) to explore the role of MMPs in crustacean innate immune responses. RT-PCR results showed that stimulation of crab with LPS and poly I:C upregulated expression of EsMMP-14 markedly. Besides, following the stimulation of 20-Hydroxyecdysone, the expression level of EsMMP-14 increased robustly, suggesting that EsMMP-14 involved in the molt process of E. sinensis. Hematoxylin and eosin staining of hepatopancreas and intestine revealed that knocking down EsMMP-14 maintained morphology following infection by Bacillus thuringiensis. Moreover, downregulated expression of EsMMP-14 increased the survival rate of infected E. sinensis. These results show that EsMMP-14 plays a role in innate immune responses of E. sinensis and fills a gap in our knowledge about the function of MMPs in crustaceans.

LIN28B regulates androgen receptor in human trophoblast cells through Let‐7c

LIN28B is an RNA-binding protein necessary for maintaining pluripotency in stem cells and plays an important role in trophoblast cell differentiation. LIN28B action on target gene function often involves the Let-7 miRNA family. Previous work in cancer cells revealed that LIN28 through Let-7 miRNA regulates expression of androgen receptor (AR). Considering the similarities between cancer and trophoblast cells, we hypothesize that LIN28B also is necessary for the presence of AR in human trophoblast cells. The human first-trimester trophoblast cell line, ACH-3P was used to evaluate the regulation of AR by LIN28B, and a LIN28B knockdown cell line was constructed using lentiviral-based vectors. LIN28B knockdown in ACH-3P cells resulted in significantly decreased levels of AR and increased levels of Let-7 miRNAs. Moreover, treatment of ACH-3P cells with Let-7c mimic, but not Let-7e or Let-7f, resulted in a significant reduction in LIN28B and AR. Finally, forskolin-induced syncytialization and Let-7c treatment both resulted in increased expression of syncytiotrophoblast marker ERVW-1 and a significant decrease in AR in ACH-3P. These data reveal that LIN28B regulates AR levels in trophoblast cells likely through its inhibitory actions on let-7c, which may be necessary for trophoblast cell differentiation into the syncytiotrophoblast.

Is Atrial Natriuretic Peptide (ANP) and Natriuretic Peptide Receptor-A (NPR-A) Expression in Human Placenta and Decidua Normal?

BackgroundAtrial natriuretic peptide (ANP) is a cardiac hormone that regulates blood pressure and the salt-water balance in the blood. It acts through natriuretic peptide receptors (NPR), and the major biologically active ANP receptor is natriuretic peptide receptor-A (NPR-A). Aberrant forms of ANP and its receptors have been reported in patients with preeclampsia. However, whether aberrant forms of ANP or NPR-A are present in preeclamptic placenta, and what their role is in preeclampsia pathogenesis, has not yet been elucidated clearly. The aim of this study was to assess the expression of ANP and NPR-A in the placenta and decidua and its role in preeclampsia development.Material/MethodsThe expression of ANP and NPR-A in the first-trimester villous and decidua, full-term placenta, and preeclamptic placenta was determined using immunohistochemistry and Western blot analysis. The HTR8/SVneo cell line was used to investigate the role of NPR-A in proliferation, apoptosis, and invasion using Cell Counting Kit-8 analysis, flow cytometry analysis, and a Transwell invasion assay, respectively.ResultsANP and NPR-A were localized in the syncytiotrophoblasts, cytotrophoblasts, and trophoblast columns of human first-trimester villous trophoblast cells of decidua, and in the glandular epithelium and extravillous trophoblast cells of decidua. ANP-positive and NPR-A-positive cells in the decidual stroma were clustered around and infiltrated into the vascular wall of the spiral artery undergoing remodeling. NPR-A expression was significantly reduced in preeclamptic placentas, and NPR-A knockdown significantly impaired the invasion ability of HTR8/SVneo cells, although it had no effect on cell proliferation and apoptosis.ConclusionsANP and NPR-A are involved in human placental development. Decreased levels of NPR-A may contribute to the development of preeclampsia.

In Vitro Assays to Evaluate the Migration, Invasion, and Proliferation of Immortalized Human First-trimester Trophoblast Cell Lines.

Cell movement is a critical property of trophoblasts during placental development and early pregnancy. The significance of proper trophoblast migration and invasion is demonstrated by pregnancy disorders such as pre-eclampsia and intrauterine growth restriction, which are associated with inadequate trophoblast invasion of the maternal vasculature. Unfortunately, our understanding of the mechanisms by which the placenta develops from migrating trophoblasts is limited. In vitro analysis of cell migration via the scratch assay is a useful tool in identifying factors that regulate trophoblast migratory capacity. However, this assay alone does not define the cellular changes that can result in altered cell migration. This protocol describes three different in vitro assays that are used collectively to evaluate trophoblast cell movement: the scratch assay, the invasion assay, and the proliferation assay. The protocols described here may also be modified for use in other cell lines to quantify cell movement in response to stimuli. These methods allow investigators to identify individual factors that contribute to the cell movement and provide a thorough examination of potential mechanisms underlying apparent changes in cell migration.

In Vitro Assays to Evaluate the Migration, Invasion, and Proliferation of Immortalized Human First-trimester Trophoblast Cell Lines

In Vitro Assays to Evaluate the Migration, Invasion, and Proliferation of Immortalized Human First-trimester Trophoblast Cell Lines

Modulation of trophoblast function by concurrent hyperglycemia and antiphospholipid antibodies is in part TLR4‐dependent

While diabetes and APS are individually associated with increased risk of poor perinatal outcomes, in particular preeclampsia, recent studies have demonstrated an association between concurrent aPL and diabetes leading to an increased risk of pregnancy morbidity. Hyperglycemia and aPL have independently been shown to alter human trophoblast function by inducing a pro-inflammatory, anti-angiogenic, and antimigratory response. However, little is known about the effects of concurrent hyperglycemia and aPL on trophoblast function. A human first-trimester extravillous trophoblast cell line was exposed to glucose at 5mmol/L (normoglycemia) or 25mmol/L (hyperglycemia), all in the presence or absence of low-dose aPL or control IgG. For some experiments, the TLR4 antagonist, LPS-RS, was included. Cell culture supernatants were measured for inflammatory IL-1β and IL-8, and angiogenic PlGF, sFlt-1, and sEndoglin by ELISA. Inflammasome-associated uric acid was measured using a bioassay; caspase-1 was measured using an activity assay. Trophoblast migration was quantified using a two-chamber colorimetric assay. Compared to excess glucose alone, combination excess glucose and low-dose aPL (a) further augmented trophoblast inflammatory IL-1β, inflammasome-associated uric acid and caspase-1, and pro-angiogenic PlGF; (b) dampened trophoblast inflammatory IL-8, anti-angiogenic sEndoglin, and sFlt-1; and (c) further reduced trophoblast migration. Our findings indicate that while concurrent aPL and hyperglycemia are overall detrimental to trophoblast function, the presence of two simultaneous insults triggers some protective effects.

Arachidonic Acid Reverses Xanthohumol-Induced Insufficiency in a Human First-Trimester Extravillous Trophoblast Cell Line (HTR-8/SVneo Cells).

We previously described a negative effect of xanthohumol (XN) upon placentation-related processes. We aimed to better characterize this effect by investigating the effect of XN upon the uptake of arachidonic acid (ARA), a crucial nutrient during pregnancy, by the HTR-8/SVneo human first-trimester extravillous trophoblast cell line and its relationship with the negative effect of XN upon placentation-related processes. Uptake of 14C-ARA (100 nM) was time dependent and inhibited by short-term (26 minutes) or long-term (24 hours) exposure to XN. Xanthohumol (24 hours; 5 µM) behaved as an uncompetitive inhibitor of 14C-ARA uptake; the mammalian target of rapamycin, tyrosine kinases, and c-Jun N-terminal kinases intracellular pathways were involved in this effect; and it markedly reduced long-chain acyl-CoA synthetase 1 messenger RNA levels. Moreover, the effects of XN (24 hours; 5 µM) upon cell proliferation, culture growth, migration, viability, and apoptosis index were prevented by high extracellular ARA but not by the peroxisome proliferator-activated receptor-γ (PPAR-γ) agonist rosiglitazone. We thus conclude that ARA is an essential nutrient regulating cell viability, proliferation, culture growth, migration, and apoptosis of HTR-8/SVneo cells and that the deleterious effects of XN involve inhibition of ARA cellular uptake but appears to be independent of PPAR-γ activation.

Involvement of mTOR, JNK and PI3K in the negative effect of ethanol and metformin on the human first-trimester extravillous trophoblast HTR-8/SVneo cell line

Our aim was to investigate the effect of two xenobiotics to which pregnant woman may be exposed, the drug of abuse ethanol (EtOH) (and its metabolite acetaldehyde (ACA)) and the therapeutic agent metformin (METF), on placentation-related processes in an extravillous trophoblastic (EVTs) cell line (HTR-8/SVneo cells). EtOH, ACA and METF (24 h) significantly reduced cell proliferation rates, culture growth, viability and migratory capacity of HTR-8/SVneo cells. Moreover, both EtOH (100 μM) and METF (1 mM) increased the apoptosis index and inhibited 3H-deoxy-D-glucose (3H-DG) and 3H-folic acid (3H-FA) uptake. mTOR, JNK and PI3K intracellular signaling pathways were involved in the effect of EtOH upon 3H-FA uptake and in the effect of METF upon cell viability, and mTOR and JNK in the effect of EtOH upon cell viability and 3H-DG uptake.We show that EtOH and METF have a detrimental effect in placentation-related processes of HTR-8/SVneo cells. Moreover, mTOR, JNK and PI3K appear to mediate some of these negative effects.