Cytotrophoblast Research Articles

Transcriptional Dynamics of Cultured Human Villous Cytotrophoblasts.

During human pregnancy, cytotrophoblasts (CTBs) play key roles in uterine invasion, vascular remodeling, and anchoring of the feto-placental unit. Due to the challenges associated with studying human placentation in utero, cultured primary villous CTBs are used as a model of the differentiation pathway that leads to invasion of the uterine wall. In vitro, CTBs emulate in vivo cell behaviors, such as migration, aggregation, and substrate penetration. Although some of the molecular features related to these cell behaviors have been described, the underlying mechanisms, at a global level, remain undefined at midgestation. Thus, in this study, we characterized second-trimester CTB differentiation/invasion in vitro, correlating the major morphological transitions with the transcriptional changes that occurred at these steps. After plating on Matrigel as individual cells, CTBs migrated toward each other and formed multicellular aggregates. In parallel, using a microarray approach, we observed differentially expressed (DE) genes across time, which were enriched for numerous functions, including cell migration, vascular remodeling, morphogenesis, cell communication, and inflammatory signaling. DE genes encoded several molecules that we and others previously linked to critical CTB function in vivo, suggesting that the novel DE molecules we discovered played important roles. Immunolocalization confirmed that CTBs in situ gave a signal for two of the most highly expressed genes in vitro. In summary, we characterized, at a global level, the temporal dynamics of primary human CTB gene expression in culture. These data will enable future analyses of various types of in vitro perturbations-for example, modeling disease processes and environmental exposures.

VariableDAXXgene methylation is a common feature of placental trophoblast differentiation, preeclampsia, and response to hypoxia

Placental functioning relies on the appropriate differentiation of progenitor villous cytotrophoblasts (CTBs) into extravillous cytotrophoblasts (EVCTs), including invasive EVCTs, and the multinucleated syncytiotrophoblast (ST) layer. This is accompanied by a general move away from a proliferative, immature phenotype. Genome-scale expression studies have provided valuable insight into genes that are associated with the shift to both an invasive EVCT and ST phenotype, whereas genome-scale DNA methylation analysis has shown that differentiation to ST involves widespread methylation shifts, which are counteracted by low oxygen. In the current study, we sought to identify DNA methylation variation that is associated with transition from CTB to ST in vitro and from a noninvasive to invasive EVCT phenotype after culture on Matrigel. Of the several hundred differentially methylated regions that were identified in each comparison, the majority showed a loss of methylation with differentiation. This included a large differentially methylated region (DMR) in the gene body of death domain-associated protein 6 (DAXX ), which lost methylation during both CTB syncytialization to ST and EVCT differentiation to invasive EVCT. Comparison to publicly available methylation array data identified the same DMR as among the most consistently differentially methylated genes in placental samples from preeclampsia pregnancies. Of interest, in vitro culture of CTB or ST in low oxygen increases methylation in the same region, which correlates with delayed differentiation. Analysis of combined epigenomics signatures confirmed DAXX DMR as a likely regulatory element, and direct gene expression analysis identified a positive association between methylation at this site and DAXX expression levels. The widespread dynamic nature of DAXX methylation in association with trophoblast differentiation and placenta-associated pathologies is consistent with an important role for this gene in proper placental development and function.-Novakovic, B., Evain-Brion, D., Murthi, P., Fournier, T., Saffery, R. Variable DAXX gene methylation is a common feature of placental trophoblast differentiation, preeclampsia, and response to hypoxia.

Up-regulation of CD81 inhibits cytotrophoblast invasion and mediates maternal endothelial cell dysfunction in preeclampsia

Preeclampsia (PE) is initiated by abnormal placentation in the early stages of pregnancy, followed by systemic activation of endothelial cells of the maternal small arterioles in the late second or third trimester (TM) of pregnancy. During normal pregnancy, placental cytotrophoblasts (CTBs) invade the maternal uterine wall and spiral arteries, whereas this process is interrupted in PE. However, it is not known how the malformed placenta triggers maternal endothelial crisis and the associated manifestations. Here, we have focused on the association of CD81 with PE. CD81, a member of the tetraspanin superfamily, plays significant roles in cell growth, adhesion, and motility. The function of CD81 in human placentation and its association with pregnancy complications are currently unknown. In the present study, we have demonstrated that CD81 was preferentially expressed in normal first TM placentas and progressively down-regulated with gestation advance. In patients with early-onset severe PE (sPE), CD81 expression was significantly up-regulated in syncytiotrophoblasts (STBs), CTBs and the cells in the villous core. In addition, high levels of CD81 were observed in the maternal sera of patients with sPE. Overexpressing CD81 in CTBs significantly decreased CTB invasion, and culturing primary human umbilical vein endothelial cells (HUVECs) in the presence of a high dose of exogenous CD81 resulted in interrupted angiogenesis and endothelial cell activation in vitro. Importantly, the phenotype of human PE was mimicked in the CD81-induced rat model.

Severe pre-eclampsia is associated with alterations in cytotrophoblasts of the smooth chorion.

Pre-eclampsia (PE), which affects ∼8% of first pregnancies, is associated with faulty placentation. Extravillous cytotrophoblasts (CTBs) fail to differentiate properly, contributing to shallow uterine invasion and deficient spiral artery remodeling. We studied the effects of severe PE (sPE) on the smooth chorion portion of the fetal membranes. The results showed a significant expansion of the CTB layer. The cells displayed enhanced expression of stage-specific antigens that extravillous CTBs normally upregulate as they exit the placenta. Transcriptomics revealed the dysregulated expression of many genes (e.g. placental proteins, markers of oxidative stress). We confirmed an sPE-related increase in production of PAPPA1, which releases IGF1 from its binding protein. IGF1 enhanced proliferation of smooth chorion CTBs, a possible explanation for expansion of this layer, which may partially compensate for the placental deficits.

Cinobufotalin impedes Sw.71 cytotrophoblast cell line function via cell cycle arrest and apoptotic signaling.

Preeclampsia (preE) is a hypertensive disorder of pregnancy. Cardiotonic steroids (CTS) are endogenous inhibitors of Na+/K+ ATPase, and at least one CTS, marinobufagenin (MBG), is elevated in a rat model of preE prior to the development of the syndrome. MBG and ouabain impair cytotrophoblast (CTB) cell function, which is critical for placental development. We evaluated the effect of a CTS, cinobufotalin (CINO), on CTB cell function in vitro. CINO at ≥1nM inhibited CTB cell proliferation, migration, and invasion (p<0.05), but had no effect on cell viability. There was a higher (p<0.05) percentage of G0/G1 phase cells in groups treated with CINO at ≥1nM. CINO caused an increase in stress signaling p38 MAPK and a positive annexin-V staining in CTB cells, indicating the activation of apoptotic signaling. However, the CINO-induced apoptotic signaling was prevented by p38 inhibition. These data demonstrate that CINO impairs CTB cell function via cell cycle arrest and apoptotic signaling.

DNA methylation-associated repression of MEST/PEG1 expression contributes to the invasion of extravillous trophoblast cells

IntroductionThe invasion of extravillous cytotrophoblasts (EVTs) into the maternal uterine decidua and vasculature is critical for human placenta development and pregnancy maintenance. The imprinted gene MEST/PEG1 has been implicated in trophoblast development; however, the role of MEST in EVT invasion and the accompanying early pregnancy complications are not fully understood. MethodsWestern blot, immunofluorescence and immunohistochemistry were used to detect MEST protein expression and localization by using antibodies recognize 2 reported isoforms. Specific small interference RNA (siRNA) targeting both of the MEST isoforms was applied to silence MEST expression in extravillous explants and HTR8/SVneo cells. Cell invasion and migration were assessed using the Matrigel invasion, Transwell migration assay and the xCELLigence system. Promoter DNA methylation was examined using bisulfite-sequencing polymerase chain reaction (BSP). ResultsMEST protein was highly expressed in EVTs in the first trimester placenta and in the invasive EVT cell lines HTR-8/Svneo and HPT-8. Weak MEST expression was found in cytotrophoblasts (CTBs) and the choriocarcinoma-derived CTB cell line JEG-3. The specific siRNA knockdown of MEST expression significantly reduced HTR-8/Svneo cell invasion and migration as well as extravillous explant outgrowth, which were associated with the downregulation of Twist, N-cadherin and Vimentin. Decreased MEST protein expression with isoform 2 promoter hypermethylation was observed in the placentas of missed abortions, suggesting a possible pathological mechanism of missed abortion. ConclusionsSuppressed expression of MEST was associated with its isoform 2 promoter hypermethylation ex vivo placenta tissues and in vitro cultured EVT cell lines. The present results provide a possible pathological mechanism of missed abortion.

First trimester infection with Listeria monocytogenes is characterized by decidual and placental inflammation in pregnant cynomolgus macaques

First trimester infection with Listeria monocytogenes is characterized by decidual and placental inflammation in pregnant cynomolgus macaques

Human pluripotent stem cells as a model of trophoblast differentiation in both normal development and disease

Trophoblast is the primary epithelial cell type in the placenta, a transient organ required for proper fetal growth and development. Different trophoblast subtypes are responsible for gas/nutrient exchange (syncytiotrophoblasts, STBs) and invasion and maternal vascular remodeling (extravillous trophoblasts, EVTs). Studies of early human placental development are severely hampered by the lack of a representative trophoblast stem cell (TSC) model with the capacity for self-renewal and the ability to differentiate into both STBs and EVTs. Primary cytotrophoblasts (CTBs) isolated from early-gestation (6-8 wk) human placentas are bipotential, a phenotype that is lost with increasing gestational age. We have identified a CDX2(+)/p63(+) CTB subpopulation in the early postimplantation human placenta that is significantly reduced later in gestation. We describe a reproducible protocol, using defined medium containing bone morphogenetic protein 4 by which human pluripotent stem cells (hPSCs) can be differentiated into CDX2(+)/p63(+) CTB stem-like cells. These cells can be replated and further differentiated into STB- and EVT-like cells, based on marker expression, hormone secretion, and invasive ability. As in primary CTBs, differentiation of hPSC-derived CTBs in low oxygen leads to reduced human chorionic gonadotropin secretion and STB-associated gene expression, instead promoting differentiation into HLA-G(+) EVTs in an hypoxia-inducible, factor-dependent manner. To validate further the utility of hPSC-derived CTBs, we demonstrated that differentiation of trisomy 21 (T21) hPSCs recapitulates the delayed CTB maturation and blunted STB differentiation seen in T21 placentae. Collectively, our data suggest that hPSCs are a valuable model of human placental development, enabling us to recapitulate processes that result in both normal and diseased pregnancies.

Evidence for Differential Glycosylation of Trophoblast Cell Types

Human placental villi are surfaced by the syncytiotrophoblast (STB), with a layer of cytotrophoblasts (CTB) positioned just beneath the STB. STB in normal term pregnancies is exposed to maternal immune cells in the placental intervillous space. Extravillous cytotrophoblasts (EVT) invade the decidua and spiral arteries, where they act in conjunction with natural killer (NK) cells to convert the spiral arteries into flaccid conduits for maternal blood that support a 3-4 fold increase in the rate of maternal blood flow into the placental intervillous space. The functional roles of these distinct trophoblast subtypes during pregnancy suggested that they could be differentially glycosylated. Glycomic analysis of these trophoblasts has revealed the expression of elevated levels of biantennary N-glycans in STB and CTB, with the majority of them bearing a bisecting GlcNAc. N-glycans terminated with polylactosamine extensions were also detected at low levels. A subset of the N-glycans linked to these trophoblasts were sialylated, primarily with terminal NeuAcα2-3Gal sequences. EVT were decorated with the same N-glycans as STB and CTB, except in different proportions. The level of bisecting type N-glycans was reduced, but the level of N-glycans decorated with polylactosamine sequences were substantially elevated compared with the other types of trophoblasts. The level of triantennary and tetraantennary N-glycans was also elevated in EVT. The sialylated N-glycans derived from EVT were completely susceptible to an α2-3 specific neuraminidase (sialidase S). The possibility exists that the N-glycans associated with these different trophoblast subpopulations could act as functional groups. These potential relationships will be considered.

Integrin α4-positive human trophoblast progenitors: functional characterization and transcriptional regulation.

What are the functional characteristics and transcriptional regulators of human trophoblast progenitor cells (TBPCs)? TBPC lines established from the human smooth chorion by cell sorting for integrin α4 expressed markers of stemness and trophoblast (TB) stage-specific antigens, invaded Matrigel substrates and contributed to the cytotrophoblasts (CTBs) layer of smooth chorion explants with high-mobility group protein HMGI-C (HMGA2) and transcription factor GATA-4 (GATA4) controlling their progenitor state and TB identity. Previously, we reported the derivation of TBPC lines by trypsinization of colonies that formed in cultures of chorionic mesenchyme cells that were treated with an activin nodal inhibitor. Microarray analyses showed that, among integrins, α4 was most highly expressed, and identified HMGA2 and GATA4 as potential transcriptional regulators. The aim of this study was to streamline TBPC derivation across gestation. High-cell surface expression of integrin α4 enabled the use of a fluorescence-activated cell sorter (FACS) approach for TBPC isolation from the human smooth chorion (n = 6 lines). To confirm their TBPC identity, we profiled their expression of stemness and TB markers, and growth factor receptors. At a functional level, we assayed their invasive capacity (n = 3) and tropism for the CTB layer of the smooth chorion (n = 3). At a molecular level, we studied the roles of HMGA2 and GATA4. Cells were enzymatically disassociated from the human smooth chorion across gestation. FACS was used to isolate the integrin α4-positive population. In total, we established six TBPC lines, two per trimester. Their identity was determined by immunolocalization of a suite of antigens. Function was assessed via Matrigel invasion and co-culture with explants of the human smooth chorion. An siRNA approach was used to down-regulate HMGA2 and GATA4 expression and the results were confirmed by immunoblotting and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analyses. The endpoints analyzed included proliferation, as determined by 5-bromo-2'-deoxyuridine (BrDU) incorporation, and the expression of stage-specific antigens and hormones, as determined by qRT-PCR and immunostaining approaches. As with the original cell lines, the progenitors expressed a combination of human embryonic stem cell and TB markers. Upon differentiation, they primarily formed CTBs, which were capable of Matrigel invasion. Co-culture of the cells with smooth chorion explants enabled their migration through the mesenchyme after which they intercalated within the chorionic CTB layer. Down-regulation of HMGA2 showed that this DNA-binding protein governed their self-renewal. Both HMGA2 and GATA4 had pleitropic effects on the cells' progenitor state and TB identity. This study supported our hypothesis that TBPCs from the chorionic mesenchyme can contribute to the subpopulation of CTBs that reside in the smooth chorion. In the absence of in vivo data, which is difficult to obtain in humans, the results have the limitations common to all in vitro studies. The accepted view is that progenitors reside among the villous CTB subpopulation. Here, we show that TBPCs also reside in the mesenchymal layer of the smooth chorion throughout gestation. We theorize that they can contribute to the CTB layer in this region. This phenomenon may be particularly important in pathological situations when CTBs of the smooth chorion might provide a functional reserve for CTBs of the placenta proper. Research reported in this publication was supported by the Eunice Kennedy Shriver National Institute of Child Health and Human Development of the National Institutes of Health under award P50HD055764. O.G., N.L., K.O., A.P., T.G.-G., M.K., A.B., M.G. have nothing to disclose. S.J.F. received licensing fees and royalties from SeraCare Life Sciences for trisomic TBPC lines that were derived according to the methods described in this manuscript. N/A.

Expression and clinical significance of calcitonin receptor stimulating peptide-3 (CRSP-3) in placenta tissue of preeclampsia patients

Objective To study clinical significance and the difference of calcitonin receptor stimulating peptide-3 (CRSP-3) expression in placenta tissue between normal pregnancies and patients with preeclampsia. Methods CRSP-3 expression position in placenta tissues of 50 cases with preeclampsia (preeclampsia group: 25 cases of mild preeclampsia and 25 cases of severe preeclampsia) and 30 cases of normal late pregnancy (control group) was detected using immunohistochemical methods. CRSP-3 mRNA and protein expression in placenta tissues was detected using Real-time PCR and Western blotting method. Results Syncytiotrophoblast and cytotrophoblast cells expressed CRSP-3 in placenta tissue of preeclampsia and normal pregnancy patients. The expression levels of CRSP-3 mRNA and protein in placenta tissue of preeclampsia patients were higher than those in control group (P 0.05). Conclusion The up-regulation of CRSP-3 in placenta of preeclamptic patients may be associated with the etiology of preeclampsia, but may have no relation with progress and degree of preeclampsia.

Comparison of syncytiotrophoblast generated from human embryonic stem cells and from term placentas

Human embryonic stem cells (ESCs) readily commit to the trophoblast lineage after exposure to bone morphogenetic protein-4 (BMP-4) and two small compounds, an activin A signaling inhibitor and a FGF2 signaling inhibitor (BMP4/A83-01/PD173074; BAP treatment). During differentiation, areas emerge within the colonies with the biochemical and morphological features of syncytiotrophoblast (STB). Relatively pure fractions of mononucleated cytotrophoblast (CTB) and larger syncytial sheets displaying the expected markers of STB can be obtained by differential filtration of dispersed colonies through nylon strainers. RNA-seq analysis of these fractions has allowed them to be compared with cytotrophoblasts isolated from term placentas before and after such cells had formed syncytia. Although it is clear from extensive gene marker analysis that both ESC- and placenta-derived syncytial cells are trophoblast, each with the potential to transport a wide range of solutes and synthesize placental hormones, their transcriptome profiles are sufficiently dissimilar to suggest that the two cell types have distinct pedigrees and represent functionally different kinds of STB. We propose that the STB generated from human ESCs represents the primitive syncytium encountered in early pregnancy soon after the human trophoblast invades into the uterine wall.

Anti‐MBG antibodies attenuate MBG‐induced anti‐proliferative and anti‐angiogenic milieu in cytotrophoblast cell model

Preeclampsia (PreE) is a hypertensive pregnancy disorder, which occurs in approximately 10% of all gestations. Recently, a digitalis‐like factor, marinobufagenin (MBG) has been implicated as a causative factor in preE. We demonstrated that MBG inhibits the proliferation, migration, and invasion of cytotrophoblast (CTB) cells. We have identified a novel human monoclonal antibody with higher specificity than Digibind for MBG. We assessed the attenuation of MBG‐induced CTBs dysfunction by three anti‐MBG antibodies: 206–208, H1L2, and 3e9.MethodsA panel of anti‐MBG antibodies with potential as human therapeutic agents was developed by Panorama Research, Inc.; Sunnyvale, CA (206–208, H1L2). H1L2 was a humanized version of previously described anti‐MBG murine antibody 3e9. 206–208 was identified in a human phage antibody library. Human CTBs were treated with DMSO (vehicle) or 0.1, 1, 10 or 100 nM of MBG for 48 h. Some cells were pretreated with 206–208, H1L2, or 3e9 for 2h, while others were co‐treated with these antibodies prior to MBG treatment. Culture media were collected for analysis of pro‐angiogenic and anti‐angiogenic factors. Cell viability was measured using a CellTiter Assay kit. Cell lysates were utilized to evaluate the expression of proliferating cell nuclear antigen (PCNA) and p38 MAPK phosphorylation by western blot. Levels of vascular endothelial growth factor (VEGF), placental growth factor (PlGF), soluble fms‐like tyrosine kinase‐1 (sFlt‐1), and soluble endoglin (sEng) were measured in culture media using ELISA kits. Statistical comparisons were performed using analysis of variance with Duncan's post hoc test.ResultsMBG down‐regulated PCNA and up‐regulated p38 phosphorylation in CTBs treated with ≥1 nM MBG compared to basal (DMSO treatment) (*p &lt; 0.05 for each). Secretion of sFlt‐1 and sEng were increased, while VEGF and PIGF were decreased in CTBs treated with ≥1.0 nM MBG (*p &lt; 0.05 for each). Both anti‐MBG antibodies pretreatment and co‐treatment significantly up‐regulated PCNA and down‐regulated p38 phosphorylation, and corrected the angiogenic profile of CTBs (p &lt; 0.05 for each). The anti‐proliferative and anti‐angiogenic effects of MBG were not due to a cytotoxic effect, as evaluated by a cell viability assay. MBG at 0.1 nM had no effect.ConclusionsWe found that all 3 anti‐MBG antibodies attenuate MBG‐induced anti‐proliferative and anti‐angiogenic milieu in cultured CTBs. Here we describe for the first time two fully human anti‐MBG antibodies, which can be targeted as therapeutic agents for the development of innovative treatment strategies for preE and potentially other disorders involving aberrant MBG signaling.Support or Funding InformationNIH Small Business Innovation Research (SBIR) : 1R43HD079238‐01

Expression of the ciliary neurotrophic factor and its receptor α in human placenta of first and third trimester of gestation

The ciliary neurotrophic factor (CNTF) is a member of the IL-6 family of cytokines along with cardiotrophin-1, IL-11, leukemia inhibitory factor, oncostatin-M and IL-6 itself. These cytokines play an important role in the regulation of cellular processes such as gene activation and cell proliferation and differentiation. CNTF is a pleiotropic cytokine which effects are mediated via CNTF receptor α (CNTFRα). CNTF increases differentiation and/or survival in neuronal cells but it also has different effects on other cell types such as muscle cells, bone cells, adipocytes, retinal cells and pancreatic β-cells (1, 2). In addition, recent studies demonstrate that CNTF plays an important role in weight control since exogenously administration of CNTF has an anorectic effect in mice (3,4). Although many studies proved that CNTF plays different roles in many cell types, its role in the development of human placenta has never been investigated. In this study we investigated the expression of CNTF and CNTFRα in human trophoblast by, immunohistochemistry, immunocytochemistry and Western Blot analysis using normal first and third trimester human placentas and HTR-8/SVneo cell lines. Interestingly, using immunohistochemistry CNTF and CNTFRα were expressed in the cytotrophoblast and syncytiotrophoblast in the first and third trimester of gestation respectively. Moreover, the immunofluorescence analyses by confocal microscopy showed that CNTF is expressed in the cytoplasm and nuclei whereas CNTFRα is mainly expressed in the cell membrane and cytoplasm of HTR-8/SVneo cell line. In this study we demonstrated that CNTF and CNTFRα are normally expressed in human placenta and they may play an important role during placental development.

Expression of PAPPA2 in human fetomaternal interface and involvement in trophoblast invasion and migration.

Pregnancy-associated plasma protein-A 2 (PAPPA2) is a placental-enriched gene that is important for normal human placentation and defects in the gene can cause complications in pregnancy. Yet the exact expression pattern and role of PAPPA2 in the human fetomaternal interface are not clear. In this study, in situ hybridization (ISH) and immunohistochemistry (IHC) were employed to examine the spatial and temporal expression of PAPPA2 in the human fetomaternal interface. IHC results exhibited wide expression of PAPPA2 in the fetomaternal interface, with placental syncytiatrophoblast (STB) and extravillous trophoblast (EVT) showing strong expression and the cytotrophoblast (CTB) showing weak expression of PAPPA2. These results were confirmed by ISH. Quantitative reverse transcription-polymerase chain reaction and western blot showed the elevation of PAPPA2 in first trimester EVT differentiation and term CTB spontaneous syncytialization. PAPPA2-siRNA transfection significantly depressed the invasion and migration ability of a trophoblast cell line (HTR8/SVneo) in a transwell migration and Matrigel invasion model compared to a negative control siRNA (P < 0.05), also revealing that matrix metalloproteinase 9 (MMP9) secretion is downregulated. This was confirmed using a human first trimester placental villi explant culture model. Our results reveal the spatial and temporal expression of PAPPA2 in the human fetomaternal interface and show the positive regulatory role of PAPPA2 in human trophoblast invasion and migration through the secretion of MMP9.

Differential Spatiotemporal Patterns of Galectin Expression are a Hallmark of Endotheliochorial Placentation.

Galectins influence the progress of pregnancy by regulating key processes associated with embryo-maternal cross talk, including angiogenesis and placentation. Galectin family members exert multiple roles in the context of hemochorial and epitheliochorial placentation; however, the galectin prolife in endotheliochorial placenta remains to be investigated. Here, we used immunohistochemistry to analyze galectin (gal)-1, gal-3 and gal-9 expression during early and late endotheliochorial placentation in two different species (dogs and cats). We found that during early feline gestation, all three galectin members were more strongly expressed on trophoblast and maternal vessels compared to the decidua. This was accompanied by an overall decrease of gal-1, gal-3 and gal-9 expressions in late feline gestation. In canine early pregnancy, we observed that gal-1 and gal-9 were expressed strongly in cytotrophoblast (CTB) cells compared to gal-3, and no galectin expression was observed in syncytiotrophoblast (STB) cells. Progression of canine gestation was accompanied by increased gal-1 and gal-3 expressions on STB cells, whereas gal-9 expression remained similar in CTB and STB. These data suggest that both the maternal and fetal compartments are characterized by a spatiotemporal regulation of galectin expression during endotheliochorial placentation. This strongly suggests the involvement of the galectin family in important developmental processes during gestation including immunemodulation, trophoblast invasion and angiogenesis. A conserved functional role for galectins during mammalian placental development emerges from these studies.

Differentiation of first trimester cytotrophoblast to extravillous trophoblast involves an epithelial–mesenchymal transition

The transformation of cytotrophoblast (CTB) to extravillous trophoblast (EVT) is an essential process for placental implantation. EVT generated at the tips of the anchoring villi migrate away from the placenta and invade the endometrium and maternal spiral arteries, where they modulate maternal immune responses and remodel the arteries into high-volume conduits to facilitate uteroplacental blood flow. The process of EVT differentiation has several factors in common with the epithelial-to-mesenchymal transition (EMT) observed in embryonic development, wound healing and cancer metastasis. We hypothesized that the generation of invasive EVT from CTB was a form of EMT. We isolated paired CTB and EVT from first trimester placentae, and compared their gene expression using a PCR array comprising probes for genes involved in EMT. Out of 84 genes, 24 were down-regulated in EVT compared to CTB, including epithelial markers such as E-cadherin (−11-fold) and occludin (−75-fold). Another 30 genes were up-regulated in EVT compared to CTB including mesenchymal markers such as vimentin (235-fold) and fibronectin (107-fold) as well as the matrix metalloproteinases, MMP2 and MMP9 (357-fold, 129-fold). These alterations also included major increases in the ZEB2 (zinc finger E-box binding homeobox 2, 198-fold) and TCF4 (transcription factor 4, 18-fold) transcription factors, suggesting possible stimulatory mechanisms. There was substantial up-regulation of the genes encoding TGFβ1 and TGFβ2 (48-fold, 115-fold), which may contribute to the maintenance of the mesenchymal-like phenotype. We conclude that transformation of CTB to EVT is consistent with an EMT, although the differences with other types of EMT suggest this may be a unique form.

Abstract P088: Pravastatin Protects Against Glucose-induced Anti-proliferative, Anti-invasive and Anti-angiogenic Milieu in Cytotrophoblasts

Objective: An increasing level of evidence supports the utility of pravastatin in prevention against preeclampsia (preE). We previously demonstrated that hyperglycemia induces cytotrophoblasts (CTBs) dysfunction characteristic of a preE-like phenotype. We sought to demonstrate the utility of pravastatin in rescuing CTBs from hyperglycemia induced dysfunction. Methods: Human CTBs were treated with 100, 150, 200, 300, or 400 mg/dL glucose for 48h. Some cells were pretreated with pravastatin (1ug/mL) for 2h, while others were co-treated with pravastatin (1ug/mL) prior to glucose treatment. Some cells were treated with D-Mannitol. Cell migration was performed by Matrigel migration assay kit according to manufacturer protocol. Cell lysates were utilized to evaluate the expression of urokinase plasminogen activator (uPA), plasminogen activator inhibitor 1 (PAI-1), proliferating cell nuclear antigen (PCNA) and p38 MAPK phosphorylation by western blot. Levels of vascular endothelial growth factor (VEGF), placental growth factor (PlGF), soluble fms-like tyrosine kinase-1 (sFlt-1), soluble endoglin (sEng) and interleukin 6 (IL-6) were measured in culture media using ELISA kits. Statistical comparisons were performed using analysis of variance with Duncan’s post hoc test. Results: Hyperglycemia inhibited CTBs migration, down-regulated uPA, PAI-1, PCNA and up-regulated p38 phosphorylation in CTBs treated with ≥150 mg/dL glucose compared to basal (100 mg/dL) (*p &lt; 0.05 for each). Secretion of sFlt-1, sEng and IL-6 were increased while VEGF and PIGF were decreased in CTBs treated ≥150 mg/dl of glucose (*p &lt; 0.05 for each). Both pravastatin pretreatment and co-treatment significantly rescued CTBs migration, up-regulated uPA, PAI-1, PCNA, down-regulated p38 phosphorylation, and corrected the angiogenic profile of CTBs (p &lt; 0.05 for each). D-Mannitol had no effect on CTBs. Conclusions: Pravastatin mitigates the hyperglycemia-induced dysfunction of CTBs by attenuating the glucose-induced anti-proliferative, anti-invasive and anti-angiogenic phenotype. These data should alleviate critical concerns regarding pravastatin use on CTBs development early in pregnancy and support the current research to use of pravastatin in preE prevention.

The epithelial-mesenchymal transition in extravillous trophoblast differentiation: Role in abnormally invasive placenta

Objectives: One aspect of our studies of abnormally invasive placenta (AIP, placenta accreta) involves investigation of cytotrophoblast (CTB) differentiation into invasive extravillous trophoblast (EVT) in normal and AIP pregnancies. Differentiation involves a process with similarities to the epithelial-mesenchymal transition (EMT), observed in gastrulation, wound-healing and cancer metastasis. We asked whether the CTB to EVT change was an EMT process and whether it was further advanced in AIP. Methods: We compared CTB and EVT from normal, term pregnancies (n1⁄44,8) and EVT from placenta previa (EVT-PP; n1⁄44) and AIP pregnancies (EVT-AIP; n1⁄48) obtained in the mid-third trimester. All AIP pregnancies were placenta percreta as determined by histopathology. After EVT purification by Percoll gradient and immunomagnetic separation targeted to HLA-G, cellular RNA was extracted and analysed on targeted PCR arrays containing EMT-related genes (Qiagen). Results: The EVT/EVT-PP comparison showed no significant differences and EVT-PP results were included in the EVT group thereafter. CTB/EVT comparison showed alterations in 48 of 84 genes (p<0.05). Examples of the fold-change in specific genes illustrative of this process are shown in Table 1. By contrast the EVT/EVT-AIP comparison showed changes in only 14 genes, however this included up-regulation of several genes that have been shown to promote the EMT in other cell types (Table 2). Conclusions: The differentiation of CTB into EVT involves changes characteristic of EMT, including loss of cell-cell interaction proteins such as occludin, an integrin switch, up-regulation of proteases and inhibitors and alterations in other EMT-related cellular components. We conclude that CTB-EVT differentiation appears to be an EMT process. The EVT/EVT-AIP comparison shows clear signs of an enhanced EMT in the EVT-AIP, most notably in the up-regulation of the EMT master regulator, ZEB2 but also in expression changes observed for SNAI2, SPP1, MMP9, TIMP1 and TGFs. P1.29. VARIATIONS IN SUTURE TECHNIQUES AT CESAREAN SECTION AND ITS RELATION TO COMPLICATIONS IN SUBSEQUENT PREGNANCIES

The expressions of protein RhoA/RhoB ang Rock1/Rock2 in severe preeclampsia

Objective To investigate expressions of Ras homologue protein family A (RhoA)/RhoB and Rho-associated coiled coil Rho forming protein Kinase 1 (Rock1)/Rock2 in placenta tissues of severe preeclampsia (sPE) to clarify the molecular mechanisms of sPE. Methods The locations and expressions of RhoA/RhoB and Rock1/Rock2 between two groups were detected with immunohistochemistry. Results RhoA, RhoB, Rock1, and Rock2 were mainly distributed in cytoplasms of syncytiotrophoblasts, cytotrophoblast, endothelial cells, and endometrial stromal cells. Rock1 and Rock2 were less expressed in nucleus. The expression levels of RhoA, RhoB, Rock1, and Rock2 in sPE group were significantly higher than control group (P<0.05). Conclusions The signal pathway that consists of upstream RhoA /RhoB and downstream Rock1/Rock2 is up-regulated in sPE. It demonstrates that signal molecules including RhoA, RhoB, Rock1, and Rock2 may involve in the sPE pathogenesis through affecting shallow placenta implantation in preeclampsia, ischemia, hypoxia and apoptosis of trophoblasts, and injury of vascular endothelial cells and artery vasospasm. Key words: Pre-eclampsia/ME; Rho factor/ME; rho-associated kinases ME