Interstitial Extravillous Trophoblast Research Articles

Immune-regulatory properties of endovascular extravillous trophoblast cells in human placenta

IntroductionUterine spiral artery remodeling is the prerequisite for ensuring adequate blood supply to the maternal-fetal interface during human pregnancy. One crucial cellular event in this process involves the extensive replacement of the spiral artery endothelial cells by endovascular extravillous trophoblasts (enEVTs), a subtype of extravillous trophoblasts (EVTs). However, our understanding of the properties of enEVTs remains limited. MethodsHuman enEVTs in decidual tissues during early pregnancy was purified using flow sorting by specific makers, NCAM1 and HLA-G. The high-throughput RNA sequencing analysis as well as the cytokine antibody array experiments were carried out to analyze for cell properties. Gene ontology (GO) enrichment, kyoto encyclopedia of genes and genomes (KEGG) enrichment, and gene set enrichment analysis (GSEA) were performed on differentially expressed genes of enEVTs. Immunofluorescent assays were used to verify the analysis results. ResultsBoth enEVTs and interstitial EVTs (iEVTs) exhibited gene expression patterns typifying EVT characteristics. Intriguingly, enEVTs displayed gene expression associated with immune responses, particularly reminiscent of M2 macrophage characteristics. The active secretion of multiple cytokines and chemokines by enEVTs provided partial validation for their expression pattern of immune-regulatory genes. DiscussionOur study reveals the immune-regulatory properties of human enEVTs and provides new insights into their functions and mechanisms involved in spiral artery remodeling.

Similarities and differences in placental development between humans and cynomolgus monkeys.

The placenta is an extraembryonic organ, which is essential to maintain a normal pregnancy. However, placental development in humans is poorly understood because of technical and ethical reasons. We analyzed the anatomical localization of each trophoblastic subtype in the cynomolgus monkey placenta by immunohistochemistry in the early second trimester. Histological differences among the mouse, cynomolgus monkey, and human placenta were compared. The PubMed database was used to search for studies on placentation in rodents and primates. The anatomical structures and subtypes of the placenta in cynomolgus monkeys are highly similar to those in humans, with the exception of fewer interstitial extravillous trophoblasts in cynomolgus monkeys. The cynomolgus monkey appears to be a good animal model to investigate human placentation.

Dominance of Pro-Inflammatory Cytokines Over Anti-Inflammatory Ones in Placental Bed of Creta Cases.

The objective of the study is to investigate changes occurring in key inflammatory cytokines at molecular level (including genetic and protein) in placental bed of placenta creta compared to that of normal placenta and their correlation to interstitial extravillous trophoblasts (EVT) number. Case-control study including placentas of patients with invasive placentation (creta placentas, n = 19) compared with those of normal placentation (n = 19). Besides routine histology and immunocytochemistry detection (cytokeratin-7 [CK-7]), addition to biochemical evaluation of expression of various cytokines including tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), IL6, IL-1RA, IL-8, IL-10, and IL-13 was carried out. Routine histological examination of placentas of creta cases revealed CK-7+ extravillous trophoblasts (EVT) penetrating deeply the myometrium with various histopathological arrangements and trophoblastic vascular invasion of the deep myometrial blood vessels. A significant increase (P < 0.05) in the mRNA expression of TNF-α, IL-1 β, and IL6 with an insignificant decrease in placental bed IL-1RA, IL-8, IL-10, and IL-13 was observed in creta cases compared to the control ones. A corresponding significant increase was detected in the protein levels of TNF-α, IL-1 β, and IL-6 as well as an insignificant decrease in placental bed IL-1RA, IL-8, IL-10, and IL-13 in creta cases compared to the normal ones. Moreover, we displayed a significant positive correlation (P < 0.05) between interstitial EVT number and mRNA expression of almost all pro-inflammatory cytokines with negative but insignificant correlation with anti-inflammatory cytokines in creta cases. The upregulated pro-inflammatory cytokines and the correlation of their expression with the increased interstitial EVT provide a supporting evidence of their potentially more relevant role in the development of placenta creta than the anti-inflammatory ones.

LMO2 plays differential roles in trophoblast subtypes and is associated with preeclampsia

Preeclampsia (PE) is a common obstetric disease caused by placenta development abnormality, typically characterized as inadequate trophoblast invasion and spiral artery remodeling. In this study, we found that LMO2 level was decreased in both cytotrophoblast (CTB) and interstitial extravillous trophoblast (iEVT) in human PE placentas, and LMO2 selectively promoted cell migration in iEVT derived HTR-8/SVneo cells whereas increased proliferation in CTB derived JEG-3 cells. In mechanism, LMO2 interacted with NCKAP1, leading to destruction of WAVE regulatory complex and increased lamellipodia formation in HTR-8/SVneo cells, whereas interacted with β-catenin and up-regulated a number of core Wnt/Hippo pathway target genes in JEG-3 cells. This study revealed the differentially functional patterns of LMO2 in different trophoblast subtypes, and suggested LMO2 as a novel target for PE prediction, prevention and treatment in clinical.

Human embryonic development: from peri-implantation to gastrulation.

The basic body plan of the mammalian embryo is established through gastrulation, a pivotal early postimplantation event during which the three major germ layers (endoderm, ectoderm, and mesoderm) are specified with cellular and spatial diversity. Despite its basic and clinical importance, human embryo development from peri-implantation to gastrulation remains shrouded in mystery. Recent advances in the elongated in vitro culture of rodent and non-primate embryos and the construction of embryo-like structures have helped to improve understanding of the mechanisms of human early embryonic development. Here, we review the recent advances and possible future directions in the development of in vitro models to better understand human embryogenesis from peri-implantation to gastrulation.

Abnormal expression of the costimulatory molecule B7-H4 in placental chorionic villous and decidual basalis tissues of patients with preeclampsia and HELLP syndrome.

B7-H4, a checkpoint molecule of the B7 family, regulates a broad spectrum such as T-cell activation, cytokine secretion, tumour progression, and invasion capacities. Our previous data revealed that soluble B7-H4 (sB7-H4) blood serum levels are elevated in women at high risk for the hypertensive pregnancy disorder preeclampsia (PE) in the first trimester, as well as in patients with confirmed early/late-onset PE. We here aim to investigate the expression pattern of B7-H4 in placental tissues of PE and HELLP Syndrome versus control group. B7-H4 protein expression and localization were investigated by immunoblotting and co-immunohistochemistry in placental chorionic villous and decidual basalis tissues. B7-H4 protein was prominently expressed at the cell membrane, in the cytoplasm of the syncytiotrophoblast (STB) and interstitial extravillous trophoblast (EVT). B7-H4 protein levels in placental chorionic villous tissue were significantly higher in women with early-onset/late-onset PE and HELLP, while it was decreased in decidual basalis tissues of early-onset PE and HELLP compared with controls. B7-H4 was inversely expressed in placental chorionic villous and decidual basalis tissues of PE and HELLP patients. The increase in B7-H4 in the STB in PE and HELLP may lead to excessive apical expression and release of soluble B7-H4 in the maternal circulation. In contrast, the decrease in B7-H4 in decidual basalis tissues could be related to the decrease in invasion ability of the EVT in PE. Thus, the current results strongly suggest that B7-H4 is involved in the pathogenesis of PE and HELLP.

Alpha\u2010actinin\u20104 is essential for maintaining normal trophoblast proliferation and differentiation during early pregnancy

BackgroundProper differentiation of trophoblasts in the human placenta is essential for a successful pregnancy, whereas abnormal regulation of this process may lead to adverse pregnancy outcomes, especially preeclampsia (PE). However, the underlying mechanism of trophoblast differentiation remains unclear. Previous studies have reported the involvement of alpha-actinin-4 (ACTN4) in the actin cytoskeleton dynamics and motility. Hence, we hypothesized that ACTN4 may act as an important regulator in the normal proliferation and differentiation of trophoblasts during early pregnancy.Method:To test this hypothesis, we collected villous tissues from women undergoing a legal pregnancy termination during 6–10 weeks of gestation and explanted them for cell culture and siRNA transfection. We also obtained placental tissues from PE patients and healthy pregnant women and isolated the primary cytotrophoblast (CTB) cells. The expression of ACTN4 in the CTBs of placental villi and during the differentiation of CTBs into STBs was detected by immunofluorescence, immunohistochemistry (IHC), and EdU proliferation assays. Besides, villous explant, Matrigel invasion, transwell migration assay, and Wound-healing assay were performed to identify the possible role of ACTN4 in the outgrowth of explants and the invasion, migration, and proliferation of cell column trophoblasts (CCTs). Western blot analysis was carried out to compare the protein expression level of AKT, Snail activities, and epithelial-to-mesenchymal transition (EMT) in the villi or HTR8/SVneo cells with ACTN4 knockdown.ResultsACTN4 was highly expressed in CTB cells and interstitial extravillous trophoblast (iEVT) cells but not found in the syncytiotrophoblast (STB) cells in the first trimester villi. Downregulation of ACTN4 led to reduced trophoblast proliferation and explant outgrowth ex vivo, as well as iEVT invasion and migration in vitro due to disrupt of actin filaments organization. Such ACTN4 inhibition also decreased AKT and Snail activities and further impeded the EMT process. In addition, ACTN4 expression was found to be downregulated in the iEVTs from preeclamptic placentas.ConclusionsOur findings suggest that ACTN4 may act as an important regulator of trophoblast proliferation and differentiation during early pregnancy, and dysregulation of this protein may contribute to preeclampsia pathogenesis.

Placental endovascular extravillous trophoblasts (enEVTs) educate maternal T-cell differentiation along the maternal-placental circulation.

ObjectivesDuring human pregnancy, the endothelial cells of the uterine spiral arteries (SPA) are extensively replaced by a subtype of placental trophoblasts, endovascular extravillous trophoblasts (enEVTs), thus establishing a placental‐maternal circulation. On this pathway, foetus‐derived placental villi and enEVTs bath into the maternal blood that perfuses along SPA being not attacked by the maternal lymphocytes. We aimed to reveal the underlying mechanism of such immune tolerance.MethodsIn situ hybridization, immunofluorescence, ELISA and FCM assay were performed to examine TGF‐β1 expression and distribution of regulatory T cells (Tregs) along the placental‐maternal circulation route. The primary enEVTs, interstitial extravillous trophoblasts (iEVTs) and decidual endothelial cells (dECs) were purified by FACS, and their conditioned media were collected to treat naïve CD4+ T cells. Treg differentiation was measured by FLOW and CFSE assays.ResultsWe found that enEVTs but not iEVTs or dECs actively produced TGF‐β1. The primary enEVTs significantly promoted naïve CD4+ T‐cell differentiation into immunosuppressive FOXP3+ Tregs, and this effect was dependent on TGF‐β1. In recurrent spontaneous abortion (RSA) patients, an evidently reduced proportion of TGF‐β1–producing enEVTs and their ability to educate Tregs differentiation were observed.ConclusionsOur findings demonstrate a unique immune‐regulatory characteristic of placental enEVTs to develop immune tolerance along the placental‐maternal circulation. New insights into the pathogenesis of RSA are also suggested.

PLAC8, a new marker for human interstitial extravillous trophoblast cells, promotes their invasion and migration.

ABSTRACTProper differentiation of trophoblast cells in the human placenta is a prerequisite for a successful pregnancy, and dysregulation of this process may lead to malignant pregnancy outcomes, such as preeclampsia. Finding specific markers for different types of trophoblast cells is essential for understanding trophoblast differentiation. Here, we report that placenta-specific protein 8 (PLAC8) is specifically expressed in the interstitial extravillous trophoblast cells (iEVTs) on the fetomaternal interface. Using model systems, including placental villi-decidua co-culture, iEVTs induction by using primary trophoblast cells or explants, etc., we found that PLAC8 promotes invasion and migration of iEVTs. Mechanistically, time-lapse imaging, GTPase activity assay, co-immunoprecipitation and RNA-seq studies show that PLAC8 increases the Cdc42 and Rac1 activities, and further induces the formation of filopodia at the leading edge of the migratory trophoblast cells. More interestingly, PLAC8 is significantly upregulated under hypoxia and expression of PLAC8 is higher in iEVTs from preeclamptic placentas when compared with those from the normal control placentas. Together, PLAC8 is a new marker for iEVTs and plays an important role in promoting trophoblast invasion and migration.

Failure of physiologic transformation of spiral arteries, endothelial and trophoblast cell activation, and acute atherosis in the basal plate of the placenta

Failure of physiologic transformation of spiral arteries has been reported in preeclampsia, fetal growth restriction, fetal death, and spontaneous preterm labor with intact or ruptured membranes. Spiral arteries with failure of physiologic transformation are prone to develop atherosclerotic-like lesions of atherosis. There are striking parallels between preeclampsia and atherosclerotic disease, and between lesions of atherosis and atherosclerosis. Endothelial activation, identified by intercellular adhesion molecule-1 expression, is present in atherosclerotic-like lesions of heart transplantation, and is considered a manifestation of rejection. Similarly, endothelial activation/dysfunction has been implicated in the pathophysiology of atherosclerosis and preeclampsia. Intercellular adhesion molecule-1-overexpressing-activated endothelial cells are more resistant to trophoblast displacement than nonactivated endothelium, and may contribute to shallow spiral artery trophoblastic invasion in obstetrical syndromes having failure of physiologic transformation. We sought to determine whether failure of spiral artery physiologic transformation was associated with activation of interstitial extravillous trophoblasts and/or spiral artery endothelium and presence of acute atherosis in the placental basal plate. A cross-sectional study of 123 placentas (19-42 weeks' gestation) obtained from normal pregnancies (n= 22), preterm prelabor rupture of membranes (n= 26), preterm labor (n= 23), preeclampsia (n= 27), intrauterine fetal death (n= 15), and small for gestational age (n= 10) was performed. Failure of spiral artery physiologic transformation and presence of cell activation was determined using immunohistochemistry of placental basal plates containing a median of 4 (minimum: 1; maximum: 9) vessels per placenta. Endothelial/trophoblast cell activation was defined by the expression of intercellular adhesion molecule-1. Investigators examining microscopic sections were blinded to clinical diagnosis. Pairwise comparisons among placenta groups were performed with Fisher exact test and Wilcoxon rank sum test using a Bonferroni-adjusted level of significance (.025). We found that 87% (94/108) of placentas having spiral arteries with failure of physiologic transformation (actin-positive and cytokeratin-negative) in the basal plate, and 0% (0/15) of placentas having only spiral arteries with complete physiologic transformation (cytokeratin-positive and actin-negative), had arterial endothelial and/or interstitial extravillous trophoblasts reactive with the intercellular adhesion molecule-1 activation marker (P < .001). A significant correlation (R2= 0.84) was found between expression of spiral artery endothelial and interstitial extravillous trophoblast intercellular adhesion molecule-1 (P < .001) in activated placentas. Lesions of atherosis were found in 31.9% (30/94) of placentas with complete and/or partial failure of physiologic transformation of spiral arteries that were intercellular adhesion molecule-1-positive, in none of the 14 placentas with failure of physiologic transformation that were intercellular adhesion molecule-1-negative, and in none of the 15 placentas with complete spiral artery physiologic transformation without failure (P= .001). All placentas (30/30, 100%) with atherosis were identified in placentas having concomitant spiral artery endothelial and interstitial extravillous trophoblast activation. Failure of spiral artery physiologic transformation in the placental basal plate is associated with interstitial extravillous trophoblast and arterial endothelial activation along with increased frequency of spiral artery atherosis. These findings may be used to improve the characterization of different disorders of the placental bed such as in refining the existing tools for the early prediction of risk for preterm, preeclamptic, and other abnormal pregnancies.

Properties of Uterine Natural Killer Cells in Human Pregnancy, Major Receptors Involved, and Routes of Trophoblast Invasion

Suppression of the maternal immune system to tolerate the fetal alloantigens during implantation translates into an immunological paradox. The initial process of implantation is marked by the migration of interstitial extravillous trophoblast (EVT) into the endometrial wall and is assisted by maternal uterine natural killer (uNK) cells. Any impairment in uNK assistance can ultimately lead to loss of pregnancy. In this review, we discuss current information regarding the role of uNK cells in healthy human pregnancy and their importance in sustaining a controlled invasion of the endometrium by EVT.

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Objectives Autophagy is an intracellular degradation system for energy production under stress. We have studied the role of autophagy in extravillous trophoblast (EVT) invasion and vascular remodeling under hypoxia. Methods EVT cell lines, HTR8/ SVneo and HchEpC1b, were used. We have established autophagy deficient EVT cell lines by infection of ATG4BC74A mutant expression vector. Autophagy was evaluated by the detection of LC3 dot formation by immunohistochemistry and L3C-IIby western blotting. Vascular remodeling was evaluated by tube formation assay using EVT cells and endothelial cells co-culture system. This study was approved by ethics committee of University of Toyama. Results Autophagy was observed in EVT cells under hypoxia or CoCl2 treatment. In wild type EVT cells, the invasions of EVT were enhanced under hypoxic condition. On the other hand, the invasion and vascular remodeling were significantly reduced in autophagy deficient EVT cells compared with wild type EVT cells under hypoxia. Importantly, soluble endoglin (sEng) suppressed EVT invasion and vascular remodeling under hypoxia by inhabitation of autophagy formation. Soluble Flt-1 and TNFα did not affect the autophagy formation under hypoxia. CoCl2, which induces hypoxia inducible factor 1α (HIF-1α) over-expression, activated autophagy in EVT cells even in normoxic condition. Cell invasion in autophagy deficient EVT cells were reduced by CoCl2 treatment through the suppression of MMP-9 level and cellular ATP levels. Decreased invasiveness was neutralized by ATP supplementation. P62 which is selectively degraded by autophagy accumulated in interstitial EVT and vascular EVT in placental biopsy samples of preeclampsia (n = 10), but not in FGR cases, suggesting that autophagy is impaired in EVT of preeclampsia, but not in FGR. Conclusions Impaired autophagy by sEng, through decreased EVT invasion and vascular remodeling, is one of the etiologies of poor placentation in preeclampsia. Disclosures S. Saito: None. A. Nakashima: None.

Interferon-γ Protects First-Trimester Decidual Cells against Aberrant Matrix Metalloproteinases 1, 3, and 9 Expression in Preeclampsia

Human extravillous trophoblast (EVT) invades the decidua via integrin receptors and subsequently degrades extracellular matrix proteins. In preeclampsia (PE), shallow EVT invasion elicits incomplete spiral artery remodeling, causing reduced uteroplacental blood flow. Previous studies show that preeclamptic decidual cells, but not interstitial EVTs, display higher levels of extracellular matrix-degrading matrix metalloproteinase (MMP)-9, but not MMP-2. Herein, we extend our previous PE-related assessment of MMP-2 and MMP-9 to include MMP-1, which preferentially degrades fibrillar collagens, and MMP-3, which can initiate a local proteolytic cascade. In human first-trimester decidual cells incubated with estradiol, tumor necrosis factor-α (TNF-α) significantly enhanced MMP-1, MMP-3, and MMP-9 mRNA and protein levels and activity measured by real-time quantitative RT-PCR, ELISA, immunoblotting, and zymography, respectively. In contrast, interferon γ (IFN-γ) reversed these effects and medroxyprogesterone acetate elicited further reversal. Immunoblotting revealed that p38 mitogen-activated protein kinase signaling mediated TNF-α enhancement of MMP-1, MMP-3, and MMP-9, whereas IFN-γ inhibited p38 mitogen-activated protein kinase phosphorylation. Unlike highly regulated MMP-1, MMP-3, and MMP-9, MMP-2 mRNA and protein expression was constitutive in decidual cells. Because inflammation underlies PE-associated shallow EVT invasion, these results suggest that excess macrophage-derived TNF-α augments expression of MMP-1, MMP-3, and MMP-9 in decidual cells tointerfere with normal stepwise EVT invasion of the decidua. In contrast, decidual natural killer cell-derived IFN-γ reverses such TNF-α-induced MMPs to protect against PE.

The chrondroitin sulfate proteoglycan (CSPG4) regulates human trophoblast function

IntroductionTrophoblast growth and invasion of the uterine endometrium are critical events during placentation and are tightly regulated by locally produced factors. Abnormal placentation can result in early miscarriage or preeclampsia and intrauterine growth restriction, leading to impaired fetal and/or maternal health. Chondroitin sulfate proteoglycan 4 (CSPG4) is involved in cancer cell migration and invasion, processes which are critical during placentation but unlike in cancer, trophoblast invasion is highly regulated. CSPG4 expression and function in trophoblast is unknown. We determined CSPG4 expression in human first trimester placenta and implantation sites, and investigated whether CSPG4 influenced proliferation, migration and invasion of a human extravillous trophoblast (EVT) cell line (HTR8/SVneo cells) as a model for extravillous trophoblast (EVT). Methods and resultsImmunoreactive CSPG4 localized to EVT cells in the trophoblast shell, subpopulations of interstitial EVT cells within the decidua and cytotrophoblast cells in placental villi. In HTR8/SVneo cells, siRNA knockdown of CSPG4 stimulated proliferation and decreased migration/invasion. In primary first trimester placental villi explants two cytokines, interleukin 11 (IL11) and leukemia inhibitory factor (LIF) with known roles in trophoblast function, stimulated CSPG4 mRNA expression and immunoreactive protein in the cyotrophoblast. Discussion and conclusionThis is the first demonstration of the production and function of CSPG4 in human placentation. These data suggest that locally produced CSPG4 stimulates human EVT migration and invasion and suggests that IL11 and LIF regulate villous cytotrophoblast differentiation towards the invasive phenotype at least in part via CSPG4.

Altered expression of interleukin-6, interleukin-8 and their receptors in decidua of women with sporadic miscarriage

Are alterations in decidual expression of interleukin (IL)-6 and IL-8 associated with sporadic miscarriage? IL-6 and IL-8 secretion from decidual uterine natural killer (uNK) cells and macrophages isolated from women with spontaneous miscarriage was reduced compared with normal controls. Miscarriage is a common gynaecological problem with huge financial and personal implications. Eleven to twenty per cent of all clinically recognized pregnancies are lost before the 20th week of gestation, with miscarriages often being divided into early (≤ 12 completed weeks from last menstrual period) and late (≥ 13 weeks). Spiral artery remodelling is a key feature of early pregnancy; failure of this process has been implicated in sporadic miscarriage. The molecular triggers that initiate spiral artery remodelling are not clear, although cytokines such as IL-6 and IL-8 may play a role. This was a laboratory-based study using decidual and placental bed biopsy samples from women with sporadic miscarriage (n = 30) and termination of pregnancy controls (n = 30). Total adherent decidual cells, CD10(+) stromal cells, CD14(+) macrophages and CD56(+) uNK cells were isolated from decidua from apparently normal pregnancies that were terminated at either 8-10 or 12-14 weeks' gestation. In addition, CD14(+) macrophages and CD56(+) uNK cells were isolated from decidua from sporadic miscarriage at 8-10 weeks' gestation. Secreted IL-8 was measured in all isolated cell populations, while IL-6 was measured in CD14(+) macrophages and CD56(+) uNK cells from both sporadic miscarriage and normal controls. Placental bed biopsies were taken from women after sporadic miscarriage or termination of pregnancy at ≤ 12 completed weeks' or >13 weeks' gestational age, formalin-fixed, paraffin-embedded and immunostained for IL-6, IL-6Rα, GP130, IL-8, CXCR1, CXCR2 and CD13 (aminopeptidase N). Staining intensity for each factor was assessed in extravillous trophoblast cell populations, myometrial and decidual stroma, myometrial and decidual spiral arteries and decidual glandular epithelium. A CPA model was used to assess the potential role of IL-6 and IL-8 in spiral artery remodelling. IL-8 was secreted by total adherent decidual cells, CD10(+) stromal cells and CD14(+) macrophages at both 8-10 and 12-14 weeks' gestation, with CD14(+) cells secreting the highest levels. Both CD14(+) and CD56(+) cells isolated from decidua of early sporadic miscarriage produced lower IL-6 (P = 0.04, P = 0.01, respectively) and IL-8 levels (P = 0.0007, P = 0.002, respectively) compared with normal cases. In addition, altered expression of IL-6, IL-8 and their receptors was observed in various cell types in placental bed (myometrial stroma, glandular epithelium, interstitial extravillous trophoblast cells, vascular smooth muscle cells and endothelial cells) in sporadic miscarriage, particularly from later gestational ages. IL-6 and IL-8 disrupted vascular smooth muscle morphology and organization in an in vitro model of spiral artery remodelling. By the nature of sampling at the time of miscarriage, it was not possible to ascertain the cause or effect in the observed alterations of levels of IL-6 and IL-8 in sporadic miscarriage. Alterations in the expression of IL-6, IL-8 and their receptors may be associated with the aetiology of sporadic miscarriage, especially given the potential role of these cytokines in the regulation of trophoblast invasion and spiral artery remodelling. This project was supported by funding from Wellbeing of Women (RG1000). The authors have no competing interests to declare. Not applicable.

OP0011. Impaired autophagy in extravillous trophoblast may induce poor placentation in preeclampsia

Shallow invasion of extravillous trophoblast (EVT) and inadequate vascular remodeling by EVT are the major cause of poor placentation in preeclampsia. In early pregnancy period, trophoblast experience hypoxic and low-nutrient condition. Autophagy is a cellular bulk degradation system to maintain cellular homeostasis under stress. We examined whether autophagy sustained the EVT functions under the harsh condition. An enhancement of autophagy was observed in primary cultured EVT and EVT cell line under 2% oxygen condition or cobalt treatment which induce HIF1α. The invasion and vascular remodeling under hypoxic condition were significantly reduced in autophagy-deficient EVT cells compared with wild-type EVT cells. Treatment of ATP partially rescued the poor invation under hypoxic condition suggesting that getting energy using autophagy play important role for cell invasion under hypoxia. Soluble endoglin (sENG), that is increased in sera in preeclamptic cases, suppressed EVT invasion and vascular remodeling by EVT by inhibiting autophagy. Next we have examined the impaired autophagy by immunohistochemical staining for p62. p62 is selectively degradated in autophagosome, so if impaired autophagy is present, p62 is accumulated in the cytoplasma. Enhanced imunostainings for p62 in interstitial EVT and endovascular EVT in preeclamptic cases were observed showing impaired autophagy in EVT of preeclamptic cases. sENG is known to inhibit TGFβ that is a essential factor for differentiation of regulatory T cells. Impaired autophagy and decreased regulatory T cells by increased sENG may play important roles in pathophysiology of preeclampsia.

Effects of local decidua on trophoblast invasion and spiral artery remodeling in focal placenta creta – An immunohistochemical study

ObjectivesPlacenta creta is an increasingly prevalent cause of maternal morbidity/mortality. Decidua is at least focally defective and extravillous trophoblast (EVT) may be abnormal. The study aims to compare differences in migratory trophoblast and spiral artery remodeling between areas with and without decidua at the placental implantation site. Study designSixteen (12 creta, 4 non-creta) caesarean hysterectomy specimens were studied immunohistochemically. Invasive EVT and multinucleate trophoblast giant cells (MTGC) were quantified; confluent EVT (>5 opposed EVTs) and spiral artery remodeling were assessed semi-quantitatively. ResultsIn 6 cases, placenta creta was focal. Compared to placenta creta with local decidua, cases without local decidua had increased interstitial EVT cells (×200 field) (SEM 45.6 [4.9] vs. 80.5 [3.9], p < 0.0001), fewer multinucleate trophoblast giant cells (expressed as a percentage of total EVT) (0.8 [0.3] vs. 31.5 [2.2]% p < 0.0001) and EVT was more confluent (p < 0.0001). In contrast, placenta creta cases with local decidua had a greater degree of spiral artery remodeling (mean remodeling score 1.65 [0.07] vs. 1.13 [0.05], p < 0.0001) associated with increased intramural trophoblast (p = 0.0008). The only difference between placenta creta with local decidua and normal placentation cases was an increased number of interstitial EVT cells in creta cases (45.6 [4.9] vs. 24.8 [3.2], p = 0.04). ConclusionsNumbers of interstitial EVT are increased in placenta creta, more so in cases without local decidua. Despite this spiral artery modeling is reduced in placenta creta cases with no decidua. The results emphasize the crucial role of decidua in control of trophoblast invasion and spiral artery remodeling.

Accreta complicating complete placenta previa is characterized by reduced systemic levels of vascular endothelial growth factor and by epithelial-to-mesenchymal transition of the invasive trophoblast

We sought to characterize serum angiogenic factor profile of women with complete placenta previa and determine if invasive trophoblast differentiation characteristic of accreta, increta, or percreta shares features of epithelial-to-mesenchymal transition. We analyzed gestational age-matched serum samples from 90 pregnant women with either complete placenta previa (n = 45) or uncomplicated pregnancies (n = 45). Vascular endothelial growth factor (VEGF), placental growth factor, and soluble form of fms-like-tyrosine-kinase-1 were immunoassayed. VEGF and phosphotyrosine immunoreactivity was surveyed in histological specimens relative to expression of vimentin and cytokeratin-7. Women with previa and invasive placentation (accreta, n = 5; increta, n = 6; percreta, n = 2) had lower systemic VEGF (invasive previa: median 0.8 [0.02-3.4] vs control 6.5 [2.7-10.5] pg/mL, P = .02). VEGF and phosphotyrosine immunostaining predominated in the invasive extravillous trophoblasts that coexpressed vimentin and cytokeratin-7, an epithelial-to-mesenchymal transition feature and tumorlike cell phenotype. Lower systemic free VEGF and a switch of the interstitial extravillous trophoblasts to a metastable cell phenotype characterize placenta previa with excessive myometrial invasion.

Distinct expression and localization of serine protease HtrA1 in human endometrium and first‐trimester placenta

Mammalian embryos cannot survive without the placenta. Development of the human placenta requires trophoblast proliferation, differentiation, and invasion as well as highly coordinated modulation of the maternal uterus. HtrA1 is a member of the recently identified mammalian HtrA (high temperature requirement factor A) serine protease family with a high level of expression in the placenta. In this study, we examined whether HtrA1 expression (mRNA and protein) is associated with placental development in the human. HtrA1 is up-regulated in both endometrial glands and decidual cells during endometrial preparation for embryo implantation and during first-trimester pregnancy at placentation. HtrA1 expression was also detected in certain trophoblast subtypes during early pregnancy. The villous syncytiotrophoblast and cytotrophoblast showed the strongest expression while the interstitial extravillous trophoblast showed the lowest or no expression of HtrA1. The distinct distribution of HtrA1 at the maternal-trophoblast interface suggests that HtrA1 may play a role in placental development.

Decreased expression of the angiogenic regulators CYR61 (CCN1) and NOV (CCN3) in human placenta is associatedwith pre-eclampsia

The pregnancy disorder pre-eclampsia (PE) is thought to be caused in part by shallow invasion of the extravillous trophoblast (EVT) leading to uteroplacental insufficiency and hypoxia. Here, we focused on the expressions of cysteine-rich 61 (CYR61, CCN1) and nephroblastoma overexpressed (NOV, CCN3), members of the CCN family of angiogenic regulators, in human placenta during normal pregnancy compared with pre-eclamptic and HELLP placentae using quantitative RT-PCR, western blotting and immunocytochemistry. During normal pregnancy, both proteins showed increasing expression levels and were strongly coexpressed in endothelial cells of vessels, stromal cells and interstitial EVT giant cells. However, NOV showed an earlier onset of expression in villous endothelial cells during gestation compared with CYR61, which may signify distinct roles of these proteins in placental angiogenesis. In early-onset pre-eclamptic placentae, both CYR61 and NOV were expressed at a significantly lower level compared with normal matched controls. This decrease of CYR61 and NOV in pre-eclamptic placentae is not associated with a decrease of the endothelial marker CD34 or vimentin. No obvious changes in the localization of CYR61 and NOV in pre-eclamptic placentae were detected but a change in the intracellular distribution in trophoblast giant cells. Our data point to a potential role of both molecules in the pathogenesis of early-onset PE.