The granulocyte colony-stimulating factor (G-CSF) upregulates metalloproteinase-2 and VEGF through PI3K/Akt and Erk1/2 activation in human trophoblast Swan 71 cells

IntroductionAlthough the expression of the granulocyte colony-stimulating factor (G-CSF) and its receptor (G-CSFR) in placental tissues suggests that the cytokine could play a role in placental development, the relevance of G-CSF:G-CSFR interaction in trophoblast cells remains to be studied. Thus, the possible functional role of G-CSF was examined in a human trophoblast cell line (Swan 71 cells).Methods and resultsThe expression of G-CSFR was detected by immunocytochemistry and Western blot assays. G-CSF treatment exerted neither a proliferative nor a protective effect on H₂O₂-mediated cell death in trophoblast cells. Gelatin zymography of supernatants collected from G-CSF-treated cells showed an increment of metalloproteinase-2 (MMP-2) activity. We also found higher MMP-2 and VEGF expression levels in conditioned medium from cells exposed to G-CSF. In addition, it was demonstrated that G-CSF induced the activation of PI3K/Akt and Erk1/2 pathways, which in turn activated NF-kB. By using selective pharmacological inhibitors, it was showed that these pathways are mediating the biological effects produced by G-CSF in Swan 71 cells.Discussion and conclusionWe have demonstrated for the first time that G-CSF increases MMP-2 activity and VEGF secretion in Swan 71 cells through activation of PI3K/Akt and Erk signaling pathways, both contributing to the translocation of NF-kB to the nucleus. These data suggest that G-CSF is involved in the regulation of trophoblast function, and should be considered as a locally produced cytokine probably contributing to embryo implantation and the development of a functional placenta.     

Pentoxifylline inhibits lipopolysaccharide-induced inflammatory mediators in human second trimester placenta explants

BackgroundIntrauterine infection and inflammation during pregnancy, which leads to up-regulation of inflammatory cytokines and prostaglandin synthesis, has been implicated in the pathogenesis of preterm delivery and other pregnancy complications. Effective preventive and therapeutic strategies to reduce these outcomes are lacking to date. Pentoxifylline (PTX) is a non-specific phosphodiesterase inhibitor which raises intracellular cyclic adenosine monophosphate and decreases production of pro-inflammatory mediators while enhancing anti-inflammatory cytokines. We hypothesized that pentoxifylline will decrease lipopolysaccharide (LPS)-induced pro-inflammatory cytokines production in human placental explants.MethodsPlacental explants derived from normal second trimester human placentas were treated with PTX, stimulated with LPS and cultured at 37 °C in 5% CO₂. Conditioned media were assayed for pro- and anti-inflammatory mediators with multiplex immunoassays or ELISA, and explant tissues for mRNA with real time PCR. Means of PTX-treated and untreated samples were compared using paired t tests and Wilcoxon-signed rank tests.ResultsPTX preferentially inhibited placental expression and production of LPS-induced pro-inflammatory cytokines including TNF-α (25461 vs. 1908 pg/ml, p < 0.001), IL-1β (2921 vs. 1067 pg/ml, p < 0.001) and IFN-γ (2190 vs 427 pg/ml, p < 0.001) with relative preservation of anti-inflammatory mediators. The suppressive effects on LPS-induced placental inflammation were independent of the timing of PTX administration in relation to LPS-induced stimulation.ConclusionOur study suggests that PTX attenuates the LPS-induced pro-inflammatory milieu in human placental explants. We speculate that PTX may have utility as a candidate anti-inflammatory agent for prophylaxis and/or treatment of human placental inflammation.     

Mouse prolyl oligopeptidase plays a role in trophoblast stem cell differentiation into trophoblast giant cell and spongiotrophoblast

IntroductionProlyl oligopeptidase (prolyl endopeptidase, Prep), a multifunctional protease hydrolyzing -Pro-X- peptide bonds, is highly expressed in the mouse placenta, but the function during development is not known. We explored the possibility of Prep's involvement in placental differentiation.MethodsWe cultured trophoblast stem cells (TSCs) derived from the E6.5 mouse embryo and investigated the detailed expression pattern of Prep during their differentiation. Prep-specific inhibitors were added to the TSC culture, and the effect on the differentiation was assessed by microscopic observation and the expression of marker gene for each placental cell.ResultsDuring TSC differentiation for 6 days, Prep was constantly detected at mRNA, protein, and activity levels, and the protein was found mainly in the cytoplasm. The addition of 30 μM and 10 μM SUAM-14746, a Prep-specific inhibitor, effectively inhibited the differentiation into spongiotrophoblasts (SpTs) and trophoblast giant cells (TGCs), while the TSC viability was not affected. 5 μM SUAM-14746 impaired the differentiation into SpTs, and 1 μM SUAM-14746 exhibited no effects. Another Prep-specific inhibitor, KYP-2047, did not affect the differentiation. We confirmed efficient inhibition of Prep enzymatic activity in TSCs by both inhibitors.ConclusionThe dose-dependent effect of SUAM-14746 on TSCs suggests that Prep plays an important role in the differentiation into SpTs and TGCs in the mouse placenta.     

Endometrial androgen signaling and decidualization regulate trophoblast expansion and invasion in co-culture: A time-lapse study

IntroductionTo elucidate whether trophoblast expansion and invasion are modulated by androgen signaling in an in vitro co-culture model system with decidualizing endometrial stromal cells (ESCs).MethodsWe employed an in vitro co-culture model of early embryo implantation, consisting of human ESCs (EtsT499 cells) and spheroids generated by extravillous trophoblast (EVT) derived HTR8/Svneo. The ESCs were decidualized with 8-bromo-cAMP (8-br-cAMP) in the presence or absence of dihydrotestosterone (DHT) at various concentrations for 5 days before co-culture with EVT spheroids. Trophoblast expansion was monitored by fluorescent time-lapse imaging microscopy. ESCs motility was visualized by using CellTracker™ Orange CMRA fluorescent probe. Apoptosis of ESCs was detected by CellEvent™ Caspase-3/7^® green detection reagent. Invasion assays were performed to quantify EVT invasion through a chemotaxis cell membrane.ResultsExpansion of EVT spheroids was significantly enhanced by decidualized compared to undifferentiated ESCs. This process was further stimulated if ESCs were first decidualized in the presence of DHT. In contrast to decidualized ESCs, undifferentiated cells actively migrated away from expanding EVT spheroids. Invasiveness of EVT toward decidualized ESCs was significantly attenuated in comparison to undifferentiated ESCs. DHT had no effect on EVT invasion. However, an inhibitor of intercellular gap junction communication significantly enhanced EVT invasion towards decidualized ESCs.ConclusionsThese results indicate distinct roles for androgen signaling and gap junction formation in decidual cells in regulating trophoblast expansion and invasion.     

Increased placental phospholipase A2 gene expression and free F2-isoprostane levels in response to oxidative stress in preeclampsia

Vasoactive eicosanoids such as thromboxane (TX) A₂ and F₂-isoprostanes (F₂-isoPs) were shown to be increased in the preeclamptic placenta. Only one of the 64 possible isomers of F₂-isoPs derived from the oxidation of arachidonic acid was investigated in the placenta so far. F₂-isoPs are released from membrane phospholipids by phospholipases A₂ (PLA₂) and were shown to act on the TXA₂ receptor (TBXA2R). However, the PLA₂ deregulated in preeclampsia (PE) remains to be determined. In this study, we analyzed the concentrations of six isomers of F₂-isoPs; 8-iso-PGF_2α, 8-iso-15(R)-PGF_2α, 15(R)-PGF_2α, iPF_2α-IV, iPF_2α-VI, 5-iPF_2α-VI and the concentrations of the stable metabolites of TXA₂, TXB_2, by high performance liquid chromatography coupled with tandem mass spectrometry in placentas of PE (n = 17) and normotensive (n = 15) pregnancies according to the biopsy site: peri-insertion or periphery. In the same biopsies, relative mRNA expression of PLA2G2A, PLA2G4A, PLA2G5, PLA2G7, the PLA2 receptor (PLA2R1), the TXA₂ synthase and TBXAR2 were measured by quantitative RT-PCR. We observed similar concentrations of total F₂-isoP isomers between groups whereas higher concentrations (>40%) of free F₂-isoP were observed for all isomers (p ≤ 0.033) in PE than normotensive controls. As expected, we also observed higher placental concentrations of TXB₂ in PE (p = 0.005). Interestingly, we concomitantly found higher mRNA expression of secretory PLA2G2A (p = 0.010), PLA2G5 (p = 0.038) and TBXA2R (p = 0.023) in PE than normotensive placentas. In sum, deregulated PLA₂ could potentially be implicated in freeing F₂-isoP which could participate in local hypertension observed in the PE placenta through the TX pathway.     

YAP mediates human decidualization of the uterine endometrial stromal cells

IntroductionThe decidualization of uterine endometrial stromal cells (ESCs) is critical for the successful establishment and maintenance of pregnancy and involves extensive cell proliferation and differentiation. A newly established signaling pathway, the Hippo/Yes-associated protein (YAP) pathway, plays a critical role in these proliferation processes. Our previous study demonstrated that YAP is expressed in human ESCs. However, its role in decidualization remains unclear. The objective of the present study was to explore the role of YAP in the decidualization of human ESCs.MethodsThe expression of YAP was first investigated in the endometrium of non-pregnant women and in the decidua of pregnant women. The role of YAP was investigated by transfecting ESCs with mRNA silencing constructs and observing the negative effects of this action upon decidualization induced in vitro.ResultsOur results revealed that the expression of YAP was higher in decidual cells from early pregnant decidua compared with ESCs from non-pregnant endometrium. The expression levels of YAP and TEA domain 1 (TEAD1) were both increased in ESCs during in vitro decidualization and the knockdown of YAP in ESCs caused negative effects upon decidualization in vitro.DiscussionOur study suggests that YAP is upregulated in human decidual cells compared with ESCs and influences the decidualization of ESCs.     

Association of first-trimester angiogenic factors with placental histological findings in late-onset preeclampsia

ObjectiveTo explore in women with late-onset preeclampsia (PE) the association between maternal levels of angiogenic/antiangiogenic factors in the first trimester of pregnancy and histological findings attributable to placental underperfusion (PUP).MethodsA nested case-control cohort study was conducted in 73 women with pregnancies complicated by late-onset PE (>34 weeks at delivery) matched with controls. First trimester uterine artery Doppler (UtA); maternal levels of placental growth factor (PlGF) and soluble fms-like tyrosine kinase-1 (sFlt-1) were retrieved. Placentas were histologically evaluated using a hierarchical and standardized classification system. One-way ANOVA with linear polynomial contrast or linear-by-linear association test was performed to test the hypothesis of a linear association across study groups (controls, PE without PUP and PE with PUP).ResultsIn 54 (74%) placentas, 89 placental histological findings qualifying for PUP were found. Across study groups, significant values were observed in maternal levels of decreased PlGF (MoM values: 1.53, 1.41 and 1.37; p < 0.001), increased sFlt-1 (MoM values: 3.11, 3.11 and 3.22; p = 0.002), increased sFlt-1/PlGF ratio (MoM values: 2.3, 2.3 and 2.44; p < 0.001), abnormal UtA Doppler (MoM values: 1, 1.26 and 1.32; p < 0.001), and worse perinatal outcomes in terms of gestational age at delivery, cesarean section for not reassuring fetal status, birth weight and neonatal acidosis.DiscussionIn late-onset PE an imbalance of circulating angiogenic and anti-angiogenic factors already present at 8–10 weeks of pregnancy was associated with histological findings reflecting placental insufficiency. An early first trimester screening by angiogenic factors might help to identify patients with placental involvement among late-onset PE cases.ConclusionIn late-onset preeclampsia, first-trimester uterine Doppler and circulating levels of angiogenic/antiangiogenic factors are associated with placental underperfusion.     

Effects of sphingosine-1-phosphate on gene expression of two cell mouse embryos induced by C2-Ceramide

ObjectiveAs to understand the sphingosine-1-phosphate (S1P) on gene expression of the pre-implantation mouse embryo induced by C₂-Ceramide (CED).Materials and methodsGlobal gene-expression profiling of two cell mouse embryo was performed using Affymetrix Gene Chip® Mouse Genome 430 2.0 Array.ResultsFifty-five genes were identified with significant expression changes that are functionally involved in the two cell embryo after treated by S1P and CED. Of these genes, 30 were up-regulated and 25 were down-regulated. Genes Atm, Cdkn1b and Fgrfr2 are identified to be involved in the protective role of S1P against the apoptotic signals induced by CED.ConclusionsThis study provides a map of genes in the pre-implantation two cell mouse embryo. Further investigation based on these data will provide a better understanding of the effects of S1P on the pre-implantation embryos in other mammalian species, especially human.     

Protective proteins and telomere length in placentas from patients with pre-eclampsia in the last trimester of gestation

IntroductionVisfatin/nicotinamide phosphoribosyltransferase (Nampt), an enzyme involved in energy metabolism and sirtuins, SIRT1 and SIRT3, which are NAD-dependent deacetylases, are critical for cellular function. All three either regulate or are regulated by intracellular NAD+ levels and therefore available cellular energy, important for placental cell survival and successful pregnancy. This study investigates whether these protective proteins are involved in the placental pathophysiology of pre-eclampsia (PE) and if they are associated with 8-oxo-deoxyguanosine (8OHdG), a marker of oxidative damage or with placental telomere length.MethodsMaternal blood and placental samples were collected from 31 patients with PE and 30 controls between 31 and 40 weeks gestation. Quantitative immunohistochemistry was performed on placental specimens for visfatin/Nampt, SIRT1, SIRT3, and nuclear 8OHdG. Plasma visfatin was measured by ELISA and telomere length by Southern blot analysis of telomere restriction fragments.ResultsVisfatin/Nampt and SIRT1 in syncytiotrophoblast decreased in PE compared to controls (p < 0.0001, p = 0.004 respectively). SIRT3 decreased in PE most significantly at preterm (p = 0.002). 8OHdG was only significantly lower in preterm controls compared to term controls (p = 0.01) and correlated with SIRT1 in all samples (r = 0.27). Telomere length was not different in PE and controls.DiscussionDecreased visfatin/Nampt, SIRT1 and SIRT3 in syncytiotrophoblast in PE suggests a lack of placental reserve in metabolic energy efficiency, increased inflammation, and lower resistance to environmental stressors. However, there was little effect on nuclear function, or evidence of genomic DNA damage, which would lead to cellular senescence and death.     

ICAM-1 expression on immune cells in chronic villitis

IntroductionICAM-1 expression on the villous syncytiotrophoblast (ST) is believed to participate in migration of maternal cells into the inflamed villi regardless of villitis etiology. However, its expression on immune cells in chronic villitis (CV) has yet to be analyzed. ICAM-1 induces cell–cell adhesion allowing intercellular communication, T cell-mediated defense mechanism, and inflammatory response.Material and methods21 cases of CV (all without an identifiable etiologic agent) and 3 control placentas were analyzed using ICAM-1, and for immune cells CD45, CD3 and CD68. These cells were subdivided according to their location in inflamed villi: a) within the inflamed villi and b) outside forming perivillous aggregates.ResultsLarge amounts of CD45, CD3 and CD68 were found within the inflamed villi and forming perivillous aggregates attached to areas of trophoblastic loss. Inflamed villi usually showed ICAM-1+ ST. The majority of immune cells surrounding areas of trophoblastic rupture presented marked expression of ICAM-1. In contrast, a small number of immune cells within the inflamed villi exhibited ICAM-1 expression. Only some (<5%) inflamed villi without trophoblastic rupture and with ICAM-1+ ST presented adherence of immune cells.DiscussionIn inflamed villi of chronic villitis, the level of ICAM-1 expression on immune cells depends on their location: high in number of cells in the perivillous region and low within the villi. The strongest expression of ICAM-1 on immune cells attached to areas of trophoblastic rupture suggests that the loss of trophoblast can lead to an amplification of the inflammatory response.     

Oxygen regulates human cytotrophoblast migration by controlling chemokine and receptor expression

IntroductionPlacental development involves the variation of oxygen supply due to vascular changes and cytotrophoblast invasion. Chemokines and their receptors play an important role during placental formation. Herein, the analysis of the chemokine/receptor pair CXCL12/CXCR4 and further chemokine receptors, such as CCR1, CCR7 and CXCR6 expression in human cytotrophoblasts was conducted.MethodsHuman cytotrophoblasts were examined directly after isolation or after incubation with different oxygen tensions and a chemical HIF-stimulator for 12 h with realtime PCR, immunoblot, immunohistochemistry. Conditioned media of placental villi, decidua, and endothelial cells was used for ELISA analysis of CXL12. Cytotrophoblast migration assays were conducted applying conditioned media of endothelial cells, a CXCL12 gradient, and different oxygen level. Endometrial and decidual tissue was stained for CXCL12 expression.ResultsAn upregulation of CXCL12, CXCR4, CCR1, CCR7 and CXCR6 was observed after cytotrophoblast differentiation. Low oxygen supply upregulated CXCR4, CCR7 and CXCR6, but downregulated CXCL12 and CCR1. In contrast to the HIF associated upregulation of the aforementioned proteins, downregulation of CXCL12 and CCR1 seemed to be HIF independent. Cytotrophoblast migration was stimulated by low oxygen, the application of a CXCL12 gradient and endothelial cell conditioned media. CXCL12 was detected in endometrial vessels, glands and conditioned media of placental and decidual tissue, but not decidual vessels.Discussion/conclusionTaken together, oxygen supply and cytotrophoblast differentiation seem to be regulators of chemokine and receptor expression and function in human cytotrophoblasts. Therefore, this system seems to be involved in placental development, directed cytotrophoblast migration in the decidual compartment and a subsequent sufficient supply of the growing fetus.     

Effect of oxygen on the expression of renin–angiotensin system components in a human trophoblast cell line

During the first trimester, normal placental development occurs in a low oxygen environment that is known to stimulate angiogenesis via upregulation of vascular endothelial growth factor (VEGF). Expression of the placental renin–angiotensin system (RAS) is highest in early pregnancy. While the RAS and oxygen both stimulate angiogenesis, how they interact within the placenta is unknown. We postulated that low oxygen increases expression of the proangiogenic RAS pathway and that this is associated with increased VEGF in a first trimester human trophoblast cell line (HTR-8/SVneo). HTR-8/SVneo cells were cultured in one of three oxygen tensions (1%, 5% and 20%). RAS and VEGF mRNA expression were determined by qPCR. Prorenin, angiotensin converting enzyme (ACE) and VEGF protein levels in the supernatant, as well as prorenin and ACE in cell lysates, were measured using ELISAs. Low oxygen significantly increased the expression of both angiotensin II type 1 receptor (AGTR1) and VEGF (both P < 0.05). There was a positive correlation between AGTR1 and VEGF expression at low oxygen (r = 0.64, P < 0.005). Corresponding increases in VEGF protein were observed with low oxygen (P < 0.05). Despite no change in ACE1 mRNA expression, ACE levels in the supernatant increased with low oxygen (1% and 5%, P < 0.05). Expression of other RAS components did not change. Low oxygen increased AGTR1 and VEGF expression, as well as ACE and VEGF protein levels, suggesting that the proangiogenic RAS pathway is activated. This highlights a potential role for the placental RAS in mediating the proangiogenic effects of low oxygen in placental development.     

Placental concentrations of bisphenol A and birth weight from births in the Southeastern U.S.

IntroductionBisphenol A (BPA) is a weakly estrogenic compound that has been detected in a wide variety of food products and biological matrices (saliva, blood, urine, etc). Despite the potential risk of human exposure to BPA, little information exists concerning maternal and fetal exposure to BPA during pregnancy. The aim of this study is to evaluate the correlation between placental BPA concentration, infant birth weight and calculated birth weight centile, and several other maternal and infant parameters.MethodsPlacental sample were collected from 200 subjects. BPA levels were measured by isotope dilution GC–MS. Additional maternal and infant data were gathered from medical charts and were potential correlates with placental BPA levels.ResultsPlacental BPA concentrations ranged from 4.4 ng/g to 273.9 ng/g in oven-dried tissue (average 103.4 ± 61.8 ng/g). There was a significant negative correlation between calculated birth weight centile and levels of placental BPA (p < 0.05). Low birth weight and small for gestational age infants also had significantly greater placental BPA concentrations as compared to normal weight infants and average/large for gestational age infants. Infants born to African American mothers also had greater placental BPA concentrations as compared to infants born to Hispanic mothers.DiscussionPlacental BPA concentrations are correlated with the growth potential of the fetus and may play a role in reduced fetal growth.     

Dynamic maternal and fetal Notch activity and expression in placentation

IntroductionMurine placentation requires trophoblast Notch2, while the Notch ligand, JAGGED1, is reduced in invasive trophoblasts from women with preeclampsia. However, the placental cells with active Notch signaling and expression of other Notch proteins and ligands in placentation have yet to be defined. We sought to identify endothelial cell and trophoblast subtypes with canonical Notch signaling in the decidua and placenta and correlate this to expression of Notch proteins and ligands.MethodsNotch reporter transgenic mice were used to define canonical Notch activity and immunofluorescence staining performed to characterize expression of Notch1, 2, 3, 4 and ligands, Delta-like 4 (Dll4) and Jagged1 (Jag1) during early placentation and in the mature placenta.ResultsNotch signaling is active in maternal and fetal endothelial cells and trophoblasts during early placentation and in the mature placenta. Dll4, Jag1, Notch1, and Notch4 are expressed in maternal vasculature in the decidua. Dll4, Jag1 and Notch1 are expressed in fetal vasculature in the labyrinth. Dll4, Notch2 and Notch4 are co-expressed in the ectoplacental cone. Notch2 and Notch4 are expressed in parietal-trophoblast giant cells and junctional zone trophoblasts with active canonical Notch signaling and in labyrinthine syncytiotrophoblasts and sinusoidal-trophoblast giant cells.DiscussionCanonical Notch activity and distinct expression patterns for Notch proteins and ligands was evident in endothelium and trophoblasts, suggesting Notch1, Notch2, Notch4, Dll4, and Jag1 have distinct and overlapping functions in placentation. Characterization of Notch signaling defects in existing mouse models of preeclampsia may shed light on the role of Notch in developing the preeclampsia phenotype.     

Measurement of serum estradiol/progesterone ratio on the day of embryo transfer to predict clinical pregnancies in intracytoplasmic sperm injection (ICSI) cycles. Is this of real clinical value?

Study objectiveTo assess the value of measuring serum estradiol/progesterone (E₂/P) ratio on the day of embryo transfer (ET) in intracytoplasmic sperm injection (ICSI) cycles to predict clinical pregnancies.DesignA prospective study.SettingThe assisted reproduction unit of a large University Maternity Hospital.Materials and methodsFifty seven women consecutively treated by ICSI for male factor infertility were included in the study. Early luteal serum E₂ and P were measured on the day of ET and serum E₂/P ratios were calculated for clinical pregnancies and non-clinical pregnancy cycles.Main outcome measureClinical pregnancy rate.ResultsThirty four women failed to have clinical pregnancy (Non-clinical pregnancy group) while 23 women had clinical pregnancies (Clinical pregnancy group). There were no statistically significant differences between the two outcome groups regarding the median values for E₂ levels, P levels and E₂/P ratios (655 pmol/l, 172.5 nmol/l and 2.8 for non-clinical pregnancy cycles versus 814 pmol/l, 180 nmol/l and 2.9 for clinical pregnancy cycles, respectively). Receiver-operating characteristic (ROC) curve for E₂/P ratio was constructed to predict clinical pregnancies, the area under the curve (AUC) was 0.513 (95% confidence interval, 0.377–0.648; P, 0.865) and the best cut-off value was an E₂/P ratio of 2.5 (sensitivity of 69.57%, specificity of 44.12%, positive predictive value of 45.7% and negative predictive value of 68.2%).Major conclusionsMeasurement of E₂/P ratio on the day of embryo transfer in ICSI cycles is not of clinical value to predict clinical pregnancies.     

Porphyromonas gingivalis induces IL-8 and IFN-gamma secretion and apoptosis in human extravillous trophoblast derived HTR8/SVneo cells via activation of ERK1/2 and p38 signaling pathways

IntroductionPreterm birth is a major cause for infant mortality and morbidity. A large number of studies have suggested a link between periodontal disease and preterm birth. The purpose of this study was to investigate the interaction between a periodontopathic bacterium Porphyromonas gingivalis and human extravillous trophoblast derived HTR8/SVneo cells.MethodsProduction of cytokines in HTR8 cells was measured via ELISA. Annexin V/PI flow cytometry was performed to assess apoptosis. Protein expression was measured by western blot. Specific pharmacological inhibitors were used to inactivate relevant signaling pathways (p38 MAPK, SB203580; ERK1/2, U0126; JNK, SP600125; NF-κB, JSH-23) to determine their roles in inflammation and apoptosis.ResultsHTR8 cells released significant amounts of IL-8 and IFN-γ during exposure to P. gingivalis. Meanwhile, the percentages of both early and late apoptotic cells increased significantly in response to P. gingivalis. The most significant effect on inflammation was found using SB203580 and U0126, followed by SP600125 and JSH-23. Moreover, U0126 and SB203580 both partially but significantly suppressed P. gingivalis-induced apoptosis, with a large effect by U0126. Additionally, both heat-killed P. gingivalis and P. gingivalis lipopolysaccharide significantly induced IL-8 production.ConclusionP. gingivalis induces inflammation and apoptosis in HTR8 cells, and we demonstrated for the first time that activation of ERK1/2 and p38 MAPK pathways participates in P. gingivalis-induced inflammation and apoptosis. The abnormal regulation of inflammation and apoptosis in human trophoblasts by P. gingivalis infection may give new insights into how maternal periodontal disease and periodontal pathogens might be linked to preterm birth.