First-trimester Trophoblast Research Articles

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.

MicroRNA-218-5p Promotes Endovascular Trophoblast Differentiation and Spiral Artery Remodeling.

Preeclampsia (PE) is the leading cause of maternal and neonatal morbidity and mortality. Defects in trophoblast invasion, differentiation of endovascular extravillous trophoblasts (enEVTs), and spiral artery remodeling are key factors in PE development. There are no markers clinically available to predict PE, leaving expedited delivery as the only effective therapy. Dysregulation of miRNA in clinical tissues and maternal circulation have opened a new avenue for biomarker discovery. In this study, we investigated the role of miR-218-5p in PE development. miR-218-5p was highly expressed in EVTs and significantly downregulated in PE placentas. Using first-trimester trophoblast cell lines and human placental explants, we found that miR-218-5p overexpression promoted, whereas anti-miR-218-5p suppressed, trophoblast invasion, EVT outgrowth, and enEVT differentiation. Furthermore, miR-218-5p accelerated spiral artery remodeling in a decidua-placenta co-culture. The effect of miR-218-5p was mediated by the suppression of transforming growth factor (TGF)-β2 signaling. Silencing of TGFB2 mimicked, whereas treatment with TGF-β2 partially reversed, the effects of miR-218-5p. Taken together, these findings demonstrate that miR-218-5p promotes trophoblast invasion and enEVT differentiation through a novel miR-218-5p-TGF-β2 pathway. This study elucidates the role of an miRNA in enEVT differentiation and spiral artery remodeling and suggests that downregulation of miR-218-5p contributes to PE development.

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.

Glucocorticoid signaling regulates cell invasion and migration in the human first-trimester trophoblast cell line Sw.71.

The development of the placenta and its functions are sensitive to infection and stress, which can activate the hypothalamic-pituitary-adrenal axis. Adrenally produced glucocorticoids are the body's primary mediators of the inflammatory and stress response. Although the glucocorticoid receptor (GR) is expressed in all human villous trophoblast tissue, the effect of glucocorticoids on placentation is not well understood. Using microarray analysis, we identified the glucocorticoid-regulated transcriptional profile in the immortalized first-trimester extravillous trophoblast cell line Swan.71 (Sw.71). The synthetic glucocorticoid dexamethasone significantly regulated 3829 genes, including genes associated with cell movement, growth, and survival. SERPINE1, an inhibitor of trophoblast invasion, was induced by glucocorticoids in Sw.71 cells and is associated with the pathogenesis of preeclampsia. Glucocorticoid treatment induced recruitment of activated polymerase II and GR to the SERPINE1 promoter, suggesting a mechanism for transcriptional regulation. Functionally, glucocorticoid treatment inhibited cell proliferation, migration, and invasion. These findings suggest that glucocorticoids regulate extravillous trophoblast functions by altering the gene expression profile, which may contribute to the pathogenesis of reproductive disorders such as preeclampsia and IUGR.

Placentation-related processes in a human first-trimester extravillous trophoblast cell line (HTR-8/SVneo cells) are affected by several xenobiotics

Our aim was to investigate the effect of some xenobiotics on placentation-related processes in an extravillous trophoblastic cell line (HTR-8/SVneo cells). Amphetamine, MDMA, theophylline, and fluoxetine, but not nicotine, cocaine, and caffeine, had a negative effect on cell proliferation rates, culture growth, viability, or migratory capacity. These compounds have a detrimental effect in placentation-related processes of HTR-8/SVneo cells.

Zika, dengue and yellow fever viruses induce differential anti-viral immune responses in human monocytic and first trimester trophoblast cells

Abstract Zika virus (ZIKV) is a mosquito-borne flavivirus associated with severe neonatal birth defects, but the causative mechanism is incompletely understood. ZIKV shares sequence homology and early clinical manifestations with yellow fever virus (YFV) and dengue virus (DENV) and are all transmitted in urban cycles by the same species of mosquitoes. However, YFV and DENV have been rarely reported to cause congenital diseases. Here, we compared infection with a contemporary ZIKV strain (FSS13025) to YFV17D and DENV-4 in human monocytic cells (THP-1) and first-trimester trophoblasts (HTR-8). Our results suggest that all three viruses have similar tropisms for both cells. Nevertheless, ZIKV induced strong type 1 IFN and inflammatory cytokine and chemokine production in monocytes and peripheral blood mononuclear cells. Furthermore, ZIKV infection in trophoblasts induced lower IFN and higher inflammatory immune responses. Placental inflammation is known to contribute to the risk of brain damage in preterm newborns. Inhibition of toll-like receptor (TLR)3 and TLR8 each abrogated the inflammatory cytokine responses in ZIKV-infected trophoblasts. Our findings identify a potential link between maternal immune activation and ZIKV-induced congenital diseases, and a potential therapeutic strategy that targets TLR-mediated inflammatory responses in the placenta.

TROPHOBLAST INVASION AND UTERIOIMPLANTATION GROWTH IN EARLY RAT GESTATION REQUISITE ADRENOMEDULLIN

Objective: Adrenomedullin (ADM) is a currently exposed hypotensive peptide that is articulated in a variety of cell and tissue types. The existence of ADM has been immunohistochemically demonstrated in pathologic uterioimplantation regions, but no systematic study of ADM expression in early pregnancy in uterioimplantation region has been reported.Methods: Rats on the gestational day 2 were implanted subcutaneously with osmotic (Alzet) minipumps delivering 125 and 250 μg rat/day/of AM22–52 and were killed on the gestational day 9. We have ascertained the hypothesis that ADM, a multiregulatory ubiquitous peptide hormone, works as a trophoblast pro-invasion factor.Results: We confirmed ADM significance in in vitro assay using ACH-3P cell line, which is first-trimester trophoblast cell line.Conclusion: Our data support the conception that ADM is involved in the human implantation process through regulating trophoblast proliferation and differentiation.

Antiphospholipid Antibodies Inhibit Trophoblast Toll-Like Receptor and Inflammasome Negative Regulators.

Women with antiphospholipid antibodies (aPL) are at risk for pregnancy complications associated with poor placentation and placental inflammation. Although these antibodies are heterogeneous, some anti-β2 -glycoprotein I (anti-β2 GPI) antibodies can activate Toll-like receptor 4 (TLR-4) and NLRP3 in human first-trimester trophoblasts. The objective of this study was to determine the role of negative regulators of TLR and inflammasome function in aPL-induced trophoblast inflammation. Human trophoblasts were not treated or were treated with anti-β2 GPI aPL or control IgG in the presence or absence of the common TAM (TYRO3, AXL, and Mer tyrosine kinase [MERTK]) receptor ligand growth arrest-specific protein 6 (GAS6) or the autophagy-inducer rapamycin. The expression and function of the TAM receptor pathway and autophagy were measured by quantitative reverse transcription-polymerase chain reaction (qRT-PCR), Western blotting, and enzyme-linked immunosorbent assay (ELISA). Antiphospholipid antibody-induced trophoblast inflammation was measured by qRT-PCR, activity assays, and ELISA. Anti-β2 GPI aPL inhibited trophoblast TAM receptor function by reducing cellular expression of the receptor tyrosine kinases AXL and MERTK and the ligand GAS6. The addition of GAS6 blocked the effects of aPL on the TLR-4-mediated interleukin-8 (IL-8) response. However, the NLRP3 inflammasome-mediated IL-1β response was not affected by GAS6, suggesting that another regulatory pathway was involved. Indeed, anti-β2 GPI aPL inhibited basal trophoblast autophagy, and reversing this with rapamycin inhibited aPL-induced inflammasome function and IL-1β secretion. Basal TAM receptor function and autophagy may serve to inhibit trophoblast TLR and inflammasome function, respectively. Impairment of TAM receptor signaling and autophagy by anti-β2 GPI aPL may allow subsequent TLR and inflammasome activity, leading to a robust inflammatory response.

Trophoblast-derived CXCL16 induces M2 macrophage polarization that in turn inactivates NK cells at the maternal\u2013fetal interface

Decidual macrophages (dMΦ) are distinct from the conventional macrophages present in other tissues and express M2 macrophage markers, but the molecular mechanisms of formation and the roles of M2 MΦ during pregnancy have not been completely elucidated. The crosstalk between decidual natural killer cells (dNK) and dMΦ plays an important role in the maintenance of maternal–fetal immune tolerance. Here, CXCL16 derived from first-trimester trophoblast cells induces the polarization of human M2 macrophages. The M2 MΦ polarized by CXCL16 exhibit decreased interleukin-15 production, which facilitates the inactivation of NK cells. The cytotoxicity of NK cells is attenuated by the CXCL16-polarized M2 MΦ. The data shown in the present study provide evidence to support the hypothesis that CXCL16 secreted by trophoblast cells is a key molecule involved in decidual M2 MΦ polarization, which in turn regulates the killing ability of NK cells, thereby contributing to the homeostatic and immune-tolerant milieu required for successful fetal development.

Soluble Fms-Like Tyrosine Kinase-1 Alters Cellular Metabolism and Mitochondrial Bioenergetics in Preeclampsia.

Preeclampsia is a maternal hypertensive disorder that affects up to 1 out of 12 pregnancies worldwide. It is characterized by proteinuria, endothelial dysfunction, and elevated levels of the soluble form of the vascular endothelial growth factor receptor-1 (VEGFR-1, known as sFlt-1). sFlt-1 effects are mediated in part by decreasing VEGF signaling. The direct effects of sFlt-1 on cellular metabolism and bioenergetics in preeclampsia, have not been established. The goal of this study was to evaluate whether sFlt-1 causes mitochondrial dysfunction leading to disruption of normal functioning in endothelial and placental cells in preeclampsia. Endothelial cells (ECs) and first-trimester trophoblast (HTR-8/SVneo) were treated with serum from preeclamptic women rich in sFlt-1 or with the recombinant protein. sFlt-1, dose-dependently inhibited ECs respiration and acidification rates indicating a metabolic phenotype switch enhancing glycolytic flux. HTR-8/SVneo displayed a strong basal glycolytic metabolism, remaining less sensitive to sFlt-1-induced mitochondrial impairment. Moreover, results obtained in ECs exposed to serum from preeclamptic subjects demonstrated that increased sFlt-1 leads to metabolic perturbations accountable for mitochondrial dysfunction observed in preeclampsia. sFlt-1 exacerbated mitochondrial reactive oxygen species (ROS) formation and mitochondrial membrane potential dissipation in ECs and trophoblasts exposed to serum from preeclamptic women. Forcing oxidative metabolism by culturing cells in galactose media, further sensitized cells to sFlt-1. This approach let us establish that sFlt-1 targets mitochondrial function in ECs. Effects of sFlt-1 on HTR-8/SVneo cells metabolism were amplified in galactose, demonstrating that sFlt-1 only target cells that rely mainly on oxidative metabolism. Together, our results establish the early metabolic perturbations induced by sFlt-1 and the resulting endothelial and mitochondrial dysfunction in preeclampsia.

Modulatory effects of trophoblast-secreted CXCL12 on the migration and invasion of human first-trimester decidual epithelial cells are mediated by CXCR4 rather than CXCR7

BackgroundMaternal-fetal crosstalk during embryo implantation is complex and regulated by local signaling molecules. Chemokines and their receptors are critical signaling components required for implantation and the process of pregnancy. This study aimed to explore whether human first-trimester trophoblast cells (TCs) were capable of modulating the migration and invasion of human first-trimester decidual epithelial cells (DECs) via CXCL12/CXCR4/CXCR7 signaling.MethodThe expression of CXCR4 and CXCR7 in DECs was examined by immunohistochemistry, immunocytochemistry, immunofluorescence, flow cytometry, real-time polymerase chain reactions and western blotting. The effects of recombinant human CXCL12 (rhCXCL12) and TC-conditioned medium (TC-CM) on DEC viability in vitro were explored using a viability assay. The modulatory effects of rhCXCL12 and TC/DEC co-cultures on DEC migration and invasion were examined with migration/invasion assays.ResultCXCR4 and CXCR7 were co-expressed in human first-trimester DECs. Human rhCXCL12 and TC-CM had no effects on DEC viability in vitro (P > 0.05). Both exogenous CXCL12 and co-culture with TCs significantly increased the migration and invasion of DECs (P < 0.05). Neutralizing antibodies against CXCR4 (P < 0.05) or CXCL12 (P < 0.05), but not CXCR7 (P > 0.05), significantly blocked the enhanced migration and invasion of DECs induced by exogenous CXCL12 or TC co-culture.ConclusionsCXCR4 and CXCR7 were co-expressed in human first-trimester DECs. TC-derived CXCL12 promoted the migration and invasion of DECs via CXCR4, but not CXCR7, in a paracrine manner during early pregnancy.

Oxygen concentration modulates cellular senescence and autophagy in human trophoblast cells.

We investigated the effect of oxygen concentrations on cellular senescence and autophagy and examined the role of autophagy in human trophoblast cells. Human first-trimester trophoblast cells (Sw.71) were incubated under 21%, 5%, or 1% O2 concentrations for 24hours. We examined the extent of senescence caused using senescence-associated β-galactosidase (SA-β-Gal) and senescence-associated secretory phenotype (SASP) as markers. Moreover, we examined the role of autophagy in causing cellular senescence using an autophagy inhibitor (3-methyladenine, 3MA). Physiological normoxia (5% O2 ) decreased SA-β-Gal-positive cells and SASP including interleukin-6 (IL-6) and IL-8 compared with cultured cells in 21% O2 . Pathophysiological hypoxia (1% O2 ) caused cytotoxicity, including extracellular release of ATP and lactate dehydrogenase, and decreased senescence phenotypes. 3MA-treated trophoblast cells significantly suppressed senescence markers (SA-β-Gal-positive cells and SASP secretion) in O2 -independent manner. We conclude that O2 concentration modulates cellular senescence phenotypes regulating autophagy in the human trophoblast cells. Moreover, inhibiting autophagy suppresses cellular senescence, suggesting that autophagy contributes to oxygen stress-induced cellular senescence.

Pathway of Maternal Serotonin to the Human Embryo and Fetus.

Serotonin [5-hydroxytryptamine (5-HT)] is essential to intrauterine development, but its source is debated. We used immunocytochemistry to gauge 5-HT, its biosynthetic enzyme tryptophan hydroxylase 1 (TPH1); an importer (serotonin transporter, 5-HTT/SERT/SLC6A); other transporters [P-glycoprotein 1 (P-gp/ABCB1), OCT3/SLC22A3, and gap junction connexin-43]; and the 5-HT degradative enzyme monoamine oxidase A (MAOA) in sections of placentas. In humans, 5-HT was faintly stained only in first-trimester trophoblasts, whereas TPH1 was not seen at any stage. SERT was expressed in syncytiotrophoblasts and, more strongly, in cytotrophoblasts. MAOA was prominent in syncytiotrophoblasts, OCT3 and gap junctions were stained in cytotrophoblasts, and P-gp was present at the apical surfaces of both epithelia. 5-HT added to cultured placental explants accumulated in the trophoblast epithelium and reached the villus core vessels. Trophoblast uptake was blocked by the SERT inhibitor escitalopram. Inhibition of gap junctions with heptanol prevented the accumulation of 5-HT in cytotrophoblasts, whereas blocking OCT3 with decynium-22 and P-gp with mitotane led to its accumulation in cytotrophoblasts. Reducing 5-HT destruction by inhibiting MAOA with clorgyline increased the accumulation of 5-HT throughout the villus. In the mouse fetus, intravascular platelets stained prominently for 5-HT at day 13.5, whereas the placenta and yolk sac endoderm were both negative. TPH1 was not detected, but SERT was prominent in these mouse tissues. We conclude that serotonin is conveyed from the maternal blood stream through syncytiotrophoblasts, cytotrophoblasts and the villus core to the fetus through a physiological pathway that involves at least SERT, gap junctions, P-gp, OCT3, and MAOA.

Zika, dengue and yellow fever viruses induce differential anti-viral immune responses in human monocytic and first trimester trophoblast cells

Zika virus (ZIKV) is a mosquito-borne flavivirus associated with severe neonatal birth defects, but the causative mechanism is incompletely understood. ZIKV shares sequence homology and early clinical manifestations with yellow fever virus (YFV) and dengue virus (DENV) and are all transmitted in urban cycles by the same species of mosquitoes. However, YFV and DENV have been rarely reported to cause congenital diseases. Here, we compared infection with a contemporary ZIKV strain (FSS13025) to YFV17D and DENV-4 in human monocytic cells (THP-1) and first-trimester trophoblasts (HTR-8). Our results suggest that all three viruses have similar tropisms for both cells. Nevertheless, ZIKV induced strong type 1 IFN and inflammatory cytokine and chemokine production in monocytes and peripheral blood mononuclear cells. Furthermore, ZIKV infection in trophoblasts induced lower IFN and higher inflammatory immune responses. Placental inflammation is known to contribute to the risk of brain damage in preterm newborns. Inhibition of toll-like receptor (TLR)3 and TLR8 each abrogated the inflammatory cytokine responses in ZIKV-infected trophoblasts. Our findings identify a potential link between maternal immune activation and ZIKV-induced congenital diseases, and a potential therapeutic strategy that targets TLR-mediated inflammatory responses in the placenta.

Myelin Oligodendrocyte Glycoprotein-Independent Rubella Infection of Keratinocytes and Resistance of First-Trimester Trophoblast Cells to Rubella Virus In Vitro.

Rubella virus (RuV), which belongs to the family Togaviridae and genus Rubivirus, causes systemic infection in children and young adults and congenital rubella syndrome in developing fetuses if the infection occurs during pregnancy. The mechanisms of fetal infection by RuV are not completely understood. Myelin oligodendrocyte glycoprotein (MOG) is reported to be a cellular receptor for RuV; however, it is mainly expressed in the central nervous system. Therefore, it is thought that other receptors are also responsible for virus entry into susceptible cells. In this study, we found that first-trimester trophoblast cells were resistant to RuV. In addition, we showed that HaCaT cells (an immortalized keratinocyte cell line) that did not express MOG on their surface were infected with RuV. This finding is one of the first demonstrations of MOG-independent RuV infection of susceptible host cells and suggests that it is important to continue searching for alternative RuV receptors. In addition, this study reports the resistance of first-trimester trophoblast cells to RuV and suggests that utilizing an epithelial–mesenchymal transition approach to study the mechanisms of transplacental vertical RuV infection.

Nematicidal activity of grammicin produced by Xylaria grammica KCTC 13121BP against Meloidogyne incognita.

The endolichenic fungus Xylaria grammica KCTC 13121BP showed strong nematicidal activity against Meloidogyne incognita. This study aimed to identify the nematicidal metabolites and to evaluate the efficacy of the strain as a biocontrol agent under pot and field conditions. Bioassay-guided fractionation and instrumental analyses led to grammicin being identified as the nematicidal metabolite. Because patulin is a mycotoxic isomer of grammicin and is known to have strong antibacterial and cytotoxic activities, several biological activities of the two compounds were compared. Grammicin showed strong second-stage juvenile killing and egg-hatching inhibitory effects, with a 50% effective concentration at 72 h (EC50/72 h ) of 15.9 µg/mL and a 50% effective concentration at 14 days (EC50/14 days ) of 5.87 µg/mL, respectively, whereas patulin was virtually inactive in both respects. Patulin was strongly active toward various phytopathogenic bacteria in vitro, whereas grammicin was weakly so. Patulin at the concentration range of 0.1-10 µg/mL also showed dose-dependent cytotoxicity toward the human first-trimester trophoblast cell line SW.71, whereas grammicin was not toxic toward this cell line. In pot and field experiments, a wettable powder-type formulation and fermentation broth filtrate of X. grammica KCTC 13121BP effectively suppressed the development of root-knot nematode disease on tomato and melon plants. The results suggest that X. grammica and grammicin may have potential applications for control of root-knot nematode disease of various crops. © 2017 Society of Chemical Industry.

Effect of regulating peroxisome proliferator-activated receptor γ on soluble endoglin expression in first-trimester trophoblasts

Objective To investigate the effect of regulating peroxisome proliferator-activated receptor γ (PPARγ) on soluble endoglin (sEng) expression in first-trimester trophoblasts via an in vitro study. Methods Chorionic villus were collected from 20 samples of first-trimester artificial abortion in Peking University First Hospital from July 1st to 31st, 2016. Primary culture of trophoblast cells was performed. Trophoblast cells from each sample were divided into three groups, which were PPARγ antagonist group, PPARγ antagonist and PPARγ agonist group, and control group. Supernatant sEng level was detected in each group by enzyme linked immunosorbent assay (ELISA). Paired-sample t test was used for statistical analysis. Results Compared with the control group, trophoblast cells in the PPARγ antagonist group grew slower and were reduced in number. No significant difference in growth or morphology of trophoblast cells was observed between the PPARγ antagonist and PPARγ agonist group and the control group. Supernatant sEng level was elevated in the PPARγ antagonist group, but was not significantly changed in the PPARγ antagonist and PPARγ agonist group as compared with that in the control group [(124.1±23.8) vs (94.0±12.7) pg/ml, t=4.31, P<0.05; (87.1±10.6) vs (94.0±12.7) pg/ml, t=1.62, P=0.12). Conclusions Suppression of PPARγ promotes sEng expression in trophoblast cells and that can be reversed by PPARγ agonist. Key words: Pregnancy trimester, first; Trophoblasts; PPARγ; Antigens, CD; Receptors, cell surface; Pre-eclampsia

Trichloroethylene metabolite S-(1,2-dichlorovinyl)-L-cysteine induces reactive oxygen species-mediated apoptosis via both the intrinsic and extrinsic apoptotic pathways in first-trimester human extravillous trophoblasts

Trichloroethylene metabolite S-(1,2-dichlorovinyl)-L-cysteine induces reactive oxygen species-mediated apoptosis via both the intrinsic and extrinsic apoptotic pathways in first-trimester human extravillous trophoblasts

Cortisol inhibits CSF2 and CSF3 via DNA methylation and inhibits invasion in first-trimester trophoblast cells.

Heightened maternal stress affects trophoblast function and increases risk for adverse pregnancy outcomes. Studies were performed using the first-trimester trophoblast cell line, Sw.71. Cytokines were quantified using qPCR and ELISA. Epigenetic regulation of cytokines was characterized by inhibiting histone deacetylation (1μmol/L suberoylanilide hydroxamic acid [SAHA]) or methylation (5μmol/L 5-azacytidine), or with chromatin immunoprecipitation (ChIP) with a pan-acetyl histone-3 antibody. Invasion assays used Matrigel chambers. Cortisol inhibited expression of CSF2 (GM-CSF) and CSF3 (G-CSF) in trophoblast cells. Cortisol-associated inhibition was dependent on DNA methylation and was not affected by acetylation. There was also a modest decrease in trophoblast invasion, not dependent on loss of CSFs. In first-trimester trophoblast cells, the physiological glucocorticoid, cortisol, inhibited two cytokines with roles in placental development and decreased trophoblast invasion. Cortisol-associated changes in trophoblast function could increase the risk for immune-mediated abortion or other adverse pregnancy outcomes.

Decreased expression of WNT2 in villi of unexplained recurrent spontaneous abortion patients may cause trophoblast cell dysfunction via downregulated Wnt/β-catenin signaling pathway.

WNT2 has been reported to be important for placental development, especially for the proper vascularization of the placenta. However, its precise role in first-trimester trophoblast cells is still unknown. WNT2 expression in the villous tissues of unexplained recurrent spontaneous abortion (URSA) patients was compared with that of healthy women by Western blot. The function of WNT2 in HTR-8/SVneo trophoblast cells was evaluated by altering the cellular WNT2 level through overexpression and shRNA knockdown. The molecular mechanism of the effect of WNT2 on trophoblast cells was investigated. The association of WNT2 with the Wnt/β-catenin signaling pathway was studied through Western blot and immunofluorescence. Results showed that WNT2 protein expression was significantly decreased in villi of the URSA group compared with the control group. In vitro studies showed that WNT2 could promote human trophoblast cell proliferation and migration through activating the Wnt/β-catenin signaling pathway. Moreover, upon the knockdown of WNT2, trophoblast cell proliferation and migration were significantly suppressed. In conclusion, our study indicated that WNT2 plays an important role in trophoblast function. WNT2 insufficiency might cause impaired trophoblast cell proliferation and migration via downregulation of Wnt/β-catenin signaling pathway.