NOTCH3 signalling controls human trophoblast stem cell expansion and differentiation.

Failures in growth and differentiation of the early human placenta are associated with severe pregnancy disorders such as pre-eclampsia and fetal growth restriction. However, regulatory mechanisms controlling development of placental epithelial cells, the trophoblasts, remain poorly elucidated. Using trophoblast stem cells (TSCs), trophoblast organoids (TB-ORGs) and primary cytotrophoblasts (CTBs) of early pregnancy, we herein show that autocrine NOTCH3 signalling controls human placental expansion and differentiation. The NOTCH3 receptor was specifically expressed in proliferative CTB progenitors and its active form, the nuclear NOTCH3 intracellular domain (NOTCH3-ICD), interacted with the transcriptional co-activator mastermind-like 1 (MAML1). Doxycycline-inducible expression of dominant-negative MAML1 in TSC lines provoked cell fusion and upregulation of genes specific for multinucleated syncytiotrophoblasts, which are the differentiated hormone-producing cells of the placenta. However, progenitor expansion and markers of trophoblast stemness and proliferation were suppressed. Accordingly, inhibition of NOTCH3 signalling diminished growth of TB-ORGs, whereas overexpression of NOTCH3-ICD in primary CTBs and TSCs showed opposite effects. In conclusion, the data suggest that canonical NOTCH3 signalling plays a key role in human placental development by promoting self-renewal of CTB progenitors.

Transforming growth factor-ß signaling governs the differentiation program of extravillous trophoblasts in the developing human placenta.

Abnormal placentation has been noticed in a variety of pregnancy complications such as miscarriage, early-onset preeclampsia, and fetal growth restriction. Defects in the developmental program of extravillous trophoblasts (EVTs), migrating from placental anchoring villi into the maternal decidua and its vessels, is thought to be an underlying cause. Yet, key regulatory mechanisms controlling commitment and differentiation of the invasive trophoblast lineage remain largely elusive. Herein, comparative gene expression analyses of HLA-G-purified EVTs, isolated from donor-matched placenta, decidua, and trophoblast organoids (TB-ORGs), revealed biological processes and signaling pathways governing EVT development. In particular, bioinformatics analyses and manipulations in different versatile trophoblast cell models unraveled transforming growth factor-ß (TGF-ß) signaling as a crucial pathway driving differentiation of placental EVTs into decidual EVTs, the latter showing enrichment of a secretory gene signature. Removal of Wingless signaling and subsequent activation of the TGF-ß pathway were required for the formation of human leukocyte antigen-G+ (HLA-G+) EVTs in TB-ORGs that resemble in situ EVTs at the level of global gene expression. Accordingly, TGF-ß-treated EVTs secreted enzymes, such as DAO and PAPPA2, which were predominantly expressed by decidual EVTs. Their genes were controlled by EVT-specific induction and genomic binding of the TGF-ß downstream effector SMAD3. In summary, TGF-ß signaling plays a key role in human placental development governing the differentiation program of EVTs.

WNT and NOTCH signaling in human trophoblast development and differentiation.

Correct development of the human placenta and its differentiated epithelial cells, syncytial trophoblasts (STBs) and extravillous trophoblasts (EVTs), is crucial for a successful pregnancy outcome. STBs develop by cell fusion of mononuclear cytotrophoblasts (CTBs) in placental floating villi, whereas migratory EVTs originate from specialized villi anchoring to the maternal decidua. Defects in trophoblast differentiation have been associated with severe pregnancy disorders such as early-onset preeclampsia and fetal growth restriction. However, the evolutionary pathways underlying normal and adverse placentation are poorly understood. Herein, we discuss Wingless (WNT) and NOTCH signaling, two pathways that play pivotal roles in human placenta and trophoblast development. Whereas WNT is necessary for expansion of trophoblast progenitors and stem cells, NOTCH1 is required for proliferation and survival of EVT precursors. Differentiation of the latter is orchestrated by a switch in NOTCH receptor expression as well as by changes in WNT ligands and their downstream effectors.

Pivotal role of the transcriptional co-activator YAP in trophoblast stemness of the developing human placenta.

Various pregnancy complications, such as severe forms of preeclampsia or intrauterine growth restriction, are thought to arise from failures in the differentiation of human placental trophoblasts. Progenitors of the latter either develop into invasive extravillous trophoblasts, remodeling the uterine vasculature, or fuse into multinuclear syncytiotrophoblasts transporting oxygen and nutrients to the growing fetus. However, key regulatory factors controlling trophoblast self-renewal and differentiation have been poorly elucidated. Using primary cells, three-dimensional organoids, and CRISPR-Cas9 genome-edited JEG-3 clones, we herein show that YAP, the transcriptional coactivator of the Hippo signaling pathway, promotes maintenance of cytotrophoblast progenitors by different genomic mechanisms. Genetic or chemical manipulation of YAP in these cellular models revealed that it stimulates proliferation and expression of cell cycle regulators and stemness-associated genes, but inhibits cell fusion and production of syncytiotrophoblast (STB)-specific proteins, such as hCG and GDF15. Genome-wide comparisons of primary villous cytotrophoblasts overexpressing constitutively active YAP-5SA with YAP KO cells and syncytializing trophoblasts revealed common target genes involved in trophoblast stemness and differentiation. ChIP-qPCR unraveled that YAP-5SA overexpression increased binding of YAP-TEAD4 complexes to promoters of proliferation-associated genes such as CCNA and CDK6 Moreover, repressive YAP-TEAD4 complexes containing the histone methyltransferase EZH2 were detected in the genomic regions of the STB-specific CGB5 and CGB7 genes. In summary, YAP plays a pivotal role in the maintenance of the human placental trophoblast epithelium. Besides activating stemness factors, it also directly represses genes promoting trophoblast cell fusion.

Human diamine oxidase cellular binding and internalization in vitro and rapid clearance in vivo are not mediated by N-glycans but by heparan sulfate proteoglycan interactions.

Human diamine oxidase (hDAO) rapidly inactivates histamine by deamination. No pharmacokinetic data are available to better understand its potential as a new therapeutic modality for diseases with excess local and systemic histamine, like anaphylaxis, urticaria or mastocytosis. After intravenous administration of recombinant hDAO to rats and mice, more than 90% of the dose disappeared from the plasma pool within 10 min. Human DAO did not only bind to various endothelial and epithelial cell lines in vitro, but was also unexpectedly internalized and visible in granule-like structures. The uptake of rhDAO into cells was dependent on neither the asialoglycoprotein-receptor (ASGP-R) nor the mannose receptor (MR) recognizing terminal galactose or mannose residues, respectively. Competition experiments with ASGP-R and MR ligands did not block internalization in vitro or rapid clearance in vivo. The lack of involvement of N-glycans was confirmed by testing various glycosylation mutants. High but not low molecular weight heparin strongly reduced the internalization of rhDAO in HepG2 cells and HUVECs. Human DAO was readily internalized by CHO-K1 cells, but not by the glycosaminoglycan- and heparan sulfate-deficient CHO cell lines pgsA-745 and pgsD-677, respectively. A docked heparin hexasaccharide interacted well with the predicted heparin binding site 568RFKRKLPK575. These results strongly imply that rhDAO clearance in vivo and cellular uptake in vitro is independent of N-glycan interactions with the classical clearance receptors ASGP-R and MR, but is mediated by binding to heparan sulfate proteoglycans followed by internalization via an unknown receptor.

PRG2 and AQPEP are misexpressed in fetal membranes in placenta previa and percreta†.

The obstetrical conditions placenta accreta spectrum (PAS) and placenta previa are a significant source of pregnancy-associated morbidity and mortality, yet the specific molecular and cellular underpinnings of these conditions are not known. In this study, we identified misregulated gene expression patterns in tissues from placenta previa and percreta (the most extreme form of PAS) compared with control cases. By comparing this gene set with existing placental single-cell and bulk RNA-Seq datasets, we show that the upregulated genes predominantly mark extravillous trophoblasts. We performed immunofluorescence on several candidate molecules and found that PRG2 and AQPEP protein levels are upregulated in both the fetal membranes and the placental disk in both conditions. While this increased AQPEP expression remains restricted to trophoblasts, PRG2 is mislocalized and is found throughout the fetal membranes. Using a larger patient cohort with a diverse set of gestationally aged-matched controls, we validated PRG2 as a marker for both previa and PAS and AQPEP as a marker for only previa in the fetal membranes. Our findings suggest that the extraembryonic tissues surrounding the conceptus, including both the fetal membranes and the placental disk, harbor a signature of previa and PAS that is characteristic of EVTs and that may reflect increased trophoblast invasiveness.

STAT3-dependent analysis reveals PDK4 as independent predictor of recurrence in prostate cancer.

Prostate cancer (PCa) has a broad spectrum of clinical behavior; hence, biomarkers are urgently needed for risk stratification. Here, we aim to find potential biomarkers for risk stratification, by utilizing a gene co-expression network of transcriptomics data in addition to laser-microdissected proteomics from human and murine prostate FFPE samples. We show up-regulation of oxidative phosphorylation (OXPHOS) in PCa on the transcriptomic level and up-regulation of the TCA cycle/OXPHOS on the proteomic level, which is inversely correlated to STAT3 expression. We hereby identify gene expression of pyruvate dehydrogenase kinase 4 (PDK4), a key regulator of the TCA cycle, as a promising independent prognostic marker in PCa. PDK4 predicts disease recurrence independent of diagnostic risk factors such as grading, staging, and PSA level. Therefore, low PDK4 is a promising marker for PCa with dismal prognosis.

Matrix metalloproteinase 15 plays a pivotal role in human first trimester cytotrophoblast invasion and is not altered by maternal obesity.

Adequate anchoring of the placenta in the uterus through invasion of first trimester cytotrophoblasts (CTB) is required for a successful pregnancy. This process is mediated by matrix metalloproteinases (MMPs) and regulated by the maternal environment. Obesity is known to alter the intrauterine milieu and has been related to impaired invasion. We hypothesized that placental MMP15, a novel membrane-type MMP, is involved in CTB invasion and regulated by maternal obesity in early pregnancy. Thus, in this study MMP15 was immunolocalized to invasive extravillous and interstitial CTB. MMP15 silencing in chorionic villous explants using two different siRNAs reduced trophoblast outgrowth length (-35%, P ≤ .001 and -26%, P < .05) and area (-43%, P ≤ .001 and -36%, P ≤ .01) without altering trophoblast proliferation or apoptosis. Short-term treatment of primary first trimester trophoblasts with IL-6 (10 ng/mL), interleukin 10 (IL-10) (50 ng/mL), and tumor necrosis factor α (TNF-α) (10 ng/mL) did not affect MMP15 protein levels. Likewise, MMP15 mRNA and protein levels were unaltered between human first trimester placentas from control pregnancies vs those complicated with maternal obesity. Overall, our results suggest that the role of MMP15 in placental development and function in early pregnancy is limited to CTB invasion without being affected by short- and long-term inflammation.

Genome amplification and cellular senescence are hallmarks of human placenta development.

Genome amplification and cellular senescence are commonly associated with pathological processes. While physiological roles for polyploidization and senescence have been described in mouse development, controversy exists over their significance in humans. Here, we describe tetraploidization and senescence as phenomena of normal human placenta development. During pregnancy, placental extravillous trophoblasts (EVTs) invade the pregnant endometrium, termed decidua, to establish an adapted microenvironment required for the developing embryo. This process is critically dependent on continuous cell proliferation and differentiation, which is thought to follow the classical model of cell cycle arrest prior to terminal differentiation. Strikingly, flow cytometry and DNAseq revealed that EVT formation is accompanied with a genome-wide polyploidization, independent of mitotic cycles. DNA replication in these cells was analysed by a fluorescent cell-cycle indicator reporter system, cell cycle marker expression and EdU incorporation. Upon invasion into the decidua, EVTs widely lose their replicative potential and enter a senescent state characterized by high senescence-associated (SA) ß-galactosidase activity, induction of a SA secretory phenotype as well as typical metabolic alterations. Furthermore, we show that the shift from endocycle-dependent genome amplification to growth arrest is disturbed in androgenic complete hydatidiform moles (CHM), a hyperplastic pregnancy disorder associated with increased risk of developing choriocarinoma. Senescence is decreased in CHM-EVTs, accompanied by exacerbated endoreduplication and hyperploidy. We propose induction of cellular senescence as a ploidy-limiting mechanism during normal human placentation and unravel a link between excessive polyploidization and reduced senescence in CHM.

Densities of decidual high endothelial venules correlate with T-cell influx in healthy pregnancies and idiopathic recurrent pregnancy losses.

STUDY QUESTION: Do high endothelial venules (HEVs) appear in the uterus of healthy and pathological pregnancies? SUMMARY ANSWER: Our study reveals that HEVs are present in the non-pregnant endometrium and decidua parietalis (decP) but decline upon placentation in decidua basalis (decB) and are less abundant in decidual tissues from idiopathic, recurrent pregnancy losses (RPLs). WHAT IS KNOWN ALREADY: RPL is associated with a compromised decidual vascular phenotype. STUDY DESIGN, SIZE, DURATION: Endometrial (n = 29) and first trimester decidual (n = 86, 6-12th week of gestation) tissue samples obtained from endometrial biopsies or elective pregnancy terminations were used to determine the number of HEVs and T cells. In addition, quantification of HEVs and immune cells was performed in a cohort of decidual tissues from RPL (n = 25). PARTICIPANTS/MATERIALS, SETTING, METHODS: Position and frequency of HEVs were determined in non-pregnant endometrial as well as decidual tissue sections using immunofluorescence (IF) staining with antibodies against E-selectin, intercellular adhesion molecule, von Willebrand factor, ephrin receptor B4, CD34 and a carbohydrate epitope specific to HEVs (MECA-79). Immune cell distribution and characterization was determined by antibodies recognizing CD45 and CD3 by IF staining- and flow cytometry-based analyses. Antibodies against c-c motif chemokine ligand 21 (CCL21) and lymphotoxin-beta were used in IF staining and Western blot analyses of decidual tissues. MAIN RESULTS AND THE ROLE OF CHANCE: Functional HEVs are found in high numbers in the secretory endometrium and decP but decline in numbers upon placentation in decB (P ≤ 0.001). Decidua parietalis tissues contain higher levels of the HEV-maintaining factor lymphotoxin beta and decP-associated HEVs also express CCL21 (P ≤ 0.05), a potent T-cell chemoattractant. Moreover, there is a positive correlation between the numbers of decidual HEVs and the abundance of CD3+ cells in decidual tissue sections (P ≤ 0.001). In-depth analysis of a RPL tissue collection revealed a decreased decB (P ≤ 0.01) and decP (P ≤ 0.01) HEV density as well as reduced numbers of T cells in decB (P ≤ 0.05) and decP (P ≤ .001) sections when compared with age-matched healthy control samples. Using receiver-operating characteristics analyses, we found significant predictive values for the ratios of CD3/CD45 (P < 0.001) and HEVs/total vessels (P < 0.001) for the occurrence of RPL. LIMITATIONS, REASONS FOR CAUTION: Analyses were performed in first trimester decidual tissues from elective terminations of pregnancy or non-pregnant endometrium samples from patients diagnosed with non-endometrial pathologies including cervical polyps, ovarian cysts and myomas. First trimester decidual tissues may include pregnancies which potentially would have developed placental disorders later in gestation. In addition, our cohort of non-pregnant endometrium may not reflect the endometrial vascular phenotype of healthy women. Finally, determination of immune cell distributions in the patient cohorts studied may be influenced by the different modes of tissue derivation. Pregnancy terminations were performed by surgical aspiration, endometrial tissues were obtained by biopsies and RPL tissues were collected after spontaneous loss of pregnancy. WIDER IMPLICATIONS OF THE FINDINGS: In this study, we propose an inherent mechanism by which the endometrium and in particular the decidua control T-cell recruitment. By demonstrating reduced HEV densities and numbers of T cells in decB and decP tissues of RPL samples we further support previous findings reporting an altered vascular phenotype in early pregnancy loss. Altogether, the findings provide important information to further decipher the etiologies of unexplained RPL. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by the Austrian Science Fund (P31470 B30 to M.K.) and by the Austrian National Bank (17613ONB to J.P.). There are no competing interests to declare. TRIAL REGISTRATION NUMBER: N/A.

Human placenta and trophoblast development: key molecular mechanisms and model systems.

Abnormal placentation is considered as an underlying cause of various pregnancy complications such as miscarriage, preeclampsia and intrauterine growth restriction, the latter increasing the risk for the development of severe disorders in later life such as cardiovascular disease and type 2 diabetes. Despite their importance, the molecular mechanisms governing human placental formation and trophoblast cell lineage specification and differentiation have been poorly unravelled, mostly due to the lack of appropriate cellular model systems. However, over the past few years major progress has been made by establishing self-renewing human trophoblast stem cells and 3-dimensional organoids from human blastocysts and early placental tissues opening the path for detailed molecular investigations. Herein, we summarize the present knowledge about human placental development, its stem cells, progenitors and differentiated cell types in the trophoblast epithelium and the villous core. Anatomy of the early placenta, current model systems, and critical key regulatory factors and signalling cascades governing placentation will be elucidated. In this context, we will discuss the role of the developmental pathways Wingless and Notch, controlling trophoblast stemness/differentiation and formation of invasive trophoblast progenitors, respectively.

Metabolism of cholesterol and progesterone is differentially regulated in primary trophoblastic subtypes and might be disturbed in recurrent miscarriages.

During pregnancy, extravillous trophoblasts (EVTs) invade the maternal decidua and remodel the local vasculature to establish blood supply for the growing fetus. Compromised EVT function has been linked to aberrant pregnancy associated with maternal and fetal morbidity and mortality. However, metabolic features of this invasive trophoblast subtype are largely unknown. Using primary human trophoblasts isolated from first trimester placental tissues, we show that cellular cholesterol homeostasis is differentially regulated in EVTs compared with villous cytotrophoblasts. Utilizing RNA-sequencing, gene set-enrichment analysis, and functional validation, we provide evidence that EVTs display increased levels of free and esterified cholesterol. Accordingly, EVTs are characterized by increased expression of the HDL-receptor, scavenger receptor class B type I, and reduced expression of the LXR and its target genes. We further reveal that EVTs express elevated levels of hydroxy-delta-5-steroid dehydrogenase 3 beta- and steroid delta-isomerase 1 (HSD3B1) (a rate-limiting enzyme in progesterone synthesis) and are capable of secreting progesterone. Increasing cholesterol export by LXR activation reduced progesterone secretion in an ABCA1-dependent manner. Importantly, HSD3B1 expression was decreased in EVTs of idiopathic recurrent spontaneous abortions, pointing toward compromised progesterone metabolism in EVTs of early miscarriages. Here, we provide insights into the regulation of cholesterol and progesterone metabolism in trophoblastic subtypes and its putative relevance in human miscarriage.

Notch1 controls development of the extravillous trophoblast lineage in the human placenta.

Development of the human placenta and its different epithelial trophoblasts is crucial for a successful pregnancy. Besides fusing into a multinuclear syncytium, the exchange surface between mother and fetus, progenitors develop into extravillous trophoblasts invading the maternal uterus and its spiral arteries. Migration into these vessels promotes remodelling and, as a consequence, adaption of blood flow to the fetal-placental unit. Defects in remodelling and trophoblast differentiation are associated with severe gestational diseases, such as preeclampsia. However, mechanisms controlling human trophoblast development are largely unknown. Herein, we show that Notch1 is one such critical regulator, programming primary trophoblasts into progenitors of the invasive differentiation pathway. At the 12th wk of gestation, Notch1 is exclusively detected in precursors of the extravillous trophoblast lineage, forming cell columns anchored to the uterine stroma. At the 6th wk, Notch1 is additionally expressed in clusters of villous trophoblasts underlying the syncytium, suggesting that the receptor initiates the invasive differentiation program in distal regions of the developing placental epithelium. Manipulation of Notch1 in primary trophoblast models demonstrated that the receptor promotes proliferation and survival of extravillous trophoblast progenitors. Notch1 intracellular domain induced genes associated with stemness of cell columns, myc and VE-cadherin, in Notch1- fusogenic precursors, and bound to the myc promoter and enhancer region at RBPJκ cognate sequences. In contrast, Notch1 repressed syncytialization and expression of TEAD4 and p63, two regulators controlling self-renewal of villous cytotrophoblasts. Our results revealed Notch1 as a key factor promoting development of progenitors of the extravillous trophoblast lineage in the human placenta.

Human trophoblast invasion: new and unexpected routes and functions.

Until recently, trophoblast invasion during human placentation was characterized by and restricted to invasion into uterine connective tissues and the uterine spiral arteries. The latter was explained to connect the arteries to the intervillous space of the placenta and to guarantee the blood supply of the mother to the placenta. Today, this picture has dramatically changed. Invasion of endoglandular trophoblast into uterine glands, already starting at the time of implantation, enables histiotrophic nutrition of the embryo prior to perfusion of the placenta with maternal blood. This is followed by invasion of endovenous trophoblasts into uterine veins to guarantee the drainage of fluids from the placenta back into the maternal circulation throughout pregnancy. In addition, invasion of endolymphatic trophoblasts into the lymph vessels of the uterus has been described. Only then, invasion of endoarterial trophoblasts into spiral arteries takes place, enabling hemotrophic nutrition of the fetus starting with the second trimester of pregnancy. This new knowledge paves the way to identify changes that may occur in pathological pregnancies, from tubal pregnancies to recurrent spontaneous abortions.

Granulocytic Myeloid-Derived Suppressor Cells Accumulate in Human Placenta and Polarize toward a Th2 Phenotype.

Tolerance induction toward the semiallogeneic fetus is crucial to enable a successful pregnancy; its failure is associated with abortion or preterm delivery. Skewing T cell differentiation toward a Th2-dominated phenotype seems to be pivotal in maternal immune adaption, yet underlying mechanisms are incompletely understood. Myeloid-derived suppressor cells (MDSCs) are innate immune cells that mediate T cell suppression and are increased in cord blood of healthy newborns and in peripheral blood of pregnant women. In this study, we demonstrate that granulocytic MDSCs (GR-MDSCs) accumulate in human placenta of healthy pregnancies but are diminished in patients with spontaneous abortions. Placental GR-MDSCs effectively suppressed T cell responses by expression of arginase I and production of reactive oxygen species and were activated at the maternal-fetal interface through interaction with trophoblast cells. Furthermore, GR-MDSCs isolated from placenta polarized CD4(+) T cells toward a Th2 cytokine response. These results highlight a potential role of GR-MDSCs in inducing and maintaining maternal-fetal tolerance and suggest them as a promising target for therapeutic manipulation of pregnancy complications.

Neuregulin-1-mediated ErbB2-ErbB3 signalling protects human trophoblasts against apoptosis to preserve differentiation.

During placentation, foetal trophoblasts invade deeply into maternal tissue to establish a foeto-maternal circulation. We have previously shown that extravillous trophoblast (EVT) lineage cells express ErbB2 and ErbB3, of which the potential as an oncogenic unit is well established. However, a physiological function of this receptor combination in humans remains a puzzling question. Here, we demonstrate neuregulin 1 (NRG1) expression and secretion by human decidual stromal cells. Stimulation of human primary trophoblasts with exogenous NRG1 induced phosphorylation of ErbB2, ErbB3 and related downstream effectors. Co-immunoprecipitation experiments confirmed the formation of ErbB2-ErbB3 dimers upon ligand engagement. Along this line, receptor knockdown and ErbB3 neutralization strongly diminished NRG1-dependent activation of the signalling complex. Functional studies revealed that NRG1 promotes EVT formation in placental explant cultures. Although, in the presence of NRG1, basal and camptothecin-induced trophoblast apoptosis was significantly repressed, this effect was abolished upon ErbB3 inhibition. Notably, camptothecin provoked a strong reduction of trophoblast cell column size, whereas NRG1-treated explants were refractory to the compound. Taken together, our findings newly identify a physiological function of the NRG1-ErbB2-ErbB3 axis in trophoblast survival during human placental development.

Extravillous trophoblast invasion of venous as well as lymphatic vessels is altered in idiopathic, recurrent, spontaneous abortions.

STUDY QUESTION: Do extravillous trophoblasts (EVTs) invade non-arterial decidual vessels in healthy and pathological pregnancies? SUMMARY ANSWER: Our results reveal that trophoblast invasion of venous and lymphatic vessels is a frequent event during the first trimester of pregnancy and is compromised in  recurrent spontaneous abortion (RSA). In addition, the present data suggest that EVTs populate regional lymph nodes during pregnancy. WHAT IS ALREADY KNOWN: Human trophoblasts remodel and invade decidual spiral arteries. In addition, a recent report demonstrates that trophoblasts contact and invade decidual veins. STUDY DESIGN, SIZE, DURATION: Tissue samples of human first trimester deciduae basalis (n = 54, 6th-13th weeks of gestation) obtained from elective pregnancy terminations were used to study trophoblast invasion into veins and lymphatics, in comparison to arteries. Age-matched cases of idiopathic, recurrent spontaneous abortions tissue samples (n = 23) were assessed for cell numbers of EVTs in these decidual vessels. In addition, lymph nodes of four pregnant women were analysed for the presence of EVTs. PARTICIPANTS/MATERIALS, SETTING, METHODS: Localization, frequency and EVT-mediated targeting and invasion of arterial, venous as well as lymphatic vessels were determined in first trimester decidua basalis tissue sections using immunofluorescence staining with antibodies against CD31, CD34, ephrin B2 (EFNB2), ephrin receptor B4 (EPHB4), HLA-G, podoplanin, prospero-related homeobox 1 (Prox-1), alpha-smooth muscle actin 2 (ATCTA2), von willebrand factor (vWF) and proteoglycan 2 (PRG2). Arterial, venous and lymphatic-associated EVTs were further characterized according to their position in the vascular structure and classified as intramural (im) or intraluminal (il). MAIN RESULTS AND THE ROLE OF CHANCE: EVTs, specifically expressing PRG2, target and invade veins and lymphatics in first trimester decidua basalis since HLA-G+ trophoblast were detected in the vascular wall (intramural EVT, imEVTs) and in the lumen of these vessels (intraluminal EVT, ilEVTs). In total, 276 arteries, 793 veins and 113 lymphatics were analysed. While EVTs contact and invade arteries and veins to a similar extent we found that lymphatics are significantly less affected by EVTs (P = 0.001). Moreover, ilEVTs were detected in the lumen of venous and lymphatic vessels, whereas ilEVTs were only found occasionally in the lumen of arteries. Interestingly, RSA tissue sections contained significantly more arterial (P = 0.037), venous (P = 0.002) and lymphatic vessels (P < 0.001), compared to healthy controls. However, while RSA-associated arterial remodeling was unchanged (P = 0.39) the ratios of EVT-affected versus total number of veins (P = 0.039) and lymphatics (P < 0.001) were significantly lower in RSA compared to age-matched healthy decidual sections. Finally, HLA-G+/PRG2+/CD45-EVTs can be detected in regional lymph nodes of pregnant women diagnosed with cervical cancer. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: In this study, first trimester decidual tissues from elective terminations of pregnancies have been examined and used as a reference for healthy pregnancy. However, this collective may also include pregnancies which would have developed placental disorders later in gestation. Due to limitations in tissue availability our staining results for EVT-specific marker expression in regional lymph nodes of pregnant women are based on four cases only. WIDER IMPLICATIONS OF THE FINDINGS: In this study, we propose migration of HLA-G+ cells into regional lymph nodes during pregnancy suggesting that the human EVT is capable of infiltrating maternal tissues via the blood stream. Moreover, the description of compromised EVT invasion into the venous and lymphatic vasculature in RSA may help to better understand the pathological characteristics of idiopathic recurrent pregnancy loss. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by the Austrian Science Fund (grant P-25187-B13 to J.P. and grant P-28417-B30 to M.K.). There are no competing interests to declare.

Endothelin-1 down-regulates matrix metalloproteinase 14 and 15 expression in human first trimester trophoblasts via endothelin receptor type B.

STUDY QUESTION: Does endothelin-1 (ET-1) regulate matrix metalloproteinase (MMP) 14 and 15 production and invasion of human first trimester trophoblasts? SUMMARY ANSWER: ET-1 in pathophysiological concentrations down-regulates MMP14 and MMP15 expression via endothelin receptor (ETR) type B and decreases trophoblast migration and invasion. WHAT IS KNOWN ALREADY: MMP14 and MMP15 are involved in trophoblast invasion. Impairment of invasion has been linked to pregnancy complications such as pre-eclampsia (PE). ET-1 is up-regulated in PE. STUDY DESIGN, SIZE, DURATION: In vitro study using primary human trophoblasts from 50 first trimester placentas (gestational week 7-12). PARTICIPANTS/MATERIALS, SETTING, METHODS: Trophoblasts were cultured in the absence or presence of 10-100 nM ET-1. MMP14 and MMP15 mRNA and protein were quantified by RT-qPCR and Western blotting, respectively. Selective antagonists for ETRA (BQ-123) or ETRB (BQ-788) were used to identify ETR subtypes involved. Functional ET-1 effects were tested in first trimester chorionic villous explants and transwell invasion assays. The roles of tumor necrosis factor (TNF)-α (25 ng/ml) and oxygen (1%) in ET-1 regulation of MMP14 and 15 expression were assessed by Western blotting. MAIN RESULTS AND THE ROLE OF CHANCE: ET-1 down-regulated MMP14 and MMP15 mRNA (-21% and -26%, respectively, P < 0.05) and protein levels (-18% and -22%, respectively, P < 0.05). This effect was mediated via ETRB. ET-1 decreased trophoblast outgrowth in placental explants (-24%, P < 0.05) and trophoblast invasion (-26%, P ≤ 0.01). TNF-α enhanced ET-1 mediated MMP15 down-regulation (by 10%, P < 0.05), whereas hypoxia abolished the effect of ET-1 on both MMPs. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: Only primary trophoblasts were used in this study. Since trophoblast yield from first trimester placental material is limited, further aspects of MMP14 and 15 regulation could not be characterized. Other anti-invasive factors may be altered by ET-1 in trophoblasts and, thus, contribute to the reduced invasion, but have not been investigated. Oxygen levels similar to those found in the decidua (5-8% O2) were not analyzed in this study. WIDER IMPLICATIONS OF THE FINDINGS: ET-1 modifies placental function already during the first trimester of pregnancy, the time-window when the placental changes implicated in PE occur. Thus, our results improve the understanding of the placental mechanisms underlying trophoblast invasion and PE. STUDY FUNDING/COMPETING INTERESTS: The study was funded by the Oesterreichische Nationalbank (Anniversary Fund, project number: 14796) and the Herzfelder'sche Familienstiftung (to J.P.; number: 00685). AMM received funding from the Austrian Science Fund FWF (W1241) and the Medical University Graz through the PhD Program Molecular Fundamentals of Inflammation (DK-MOLIN). The authors have no conflict of interest.

Trophoblast subtype-specific EGFR/ERBB4 expression correlates with cell cycle progression and hyperplasia in complete hydatidiform moles.

STUDY QUESTION: Do trophoblast subtypes differ in their expression of erythroblastic leukaemia viral oncogene homologue (ERBB) receptor family members and responsiveness towards specific growth factor ligands? SUMMARY ANSWER: Our data reveal a reciprocal expression pattern of epidermal growth factor receptor (EGFR)/ERBB4 in proliferative and ERBB2/ERBB3 in invasive trophoblast subtypes, as well as a restricted responsiveness to epidermal growth factor (EGF) and heparin-binding (HB)-EGF. WHAT IS KNOWN ALREADY: EGFR is expressed by villous cytotrophoblasts (vCTBs), but absent from extravillous trophoblasts (EVTs), which specifically up-regulate ERBB2. STUDY DESIGN, SIZE, DURATION: Tissue samples of human first trimester placentae (n = 50) and deciduae (n = 5) obtained from elective pregnancy terminations were used to study trophoblast subtype-specific ERBB receptor expression and responsiveness to recombinant human EGF and HB-EGF. Age-matched complete hydatidiform mole (CHM) placentae (n = 12) were assessed for EGFR and ERBB4 expression in proliferation-competent regions. PARTICIPANTS/MATERIALS, SETTING, METHODS: ERBB receptor expression was analysed in primary trophoblast cell isolates by means of microarray, quantitative real-time PCR and western blotting, as well as immunofluorescence stainings of placental and decidual tissue sections. EGF and HB-EGF were tested for their potential to activate ERBB receptors in purified EGFR(+) and HLA-G(+) trophoblasts. 5-Ethynyl-2'-deoxyuridine incorporation assays were performed to study the effect of both ligands on the proliferative capacity of primary trophoblasts as well as of vCTBs and proximal cell column trophoblasts (pCCTs) in placental floating explants. Finally, the average number of EGFR(+) vCTB and pCCT layers was determined in CHM placentae and compared with healthy age-matched controls. MAIN RESULTS AND THE ROLE OF CHANCE: Proliferative vCTBs and pCCTs co-express EGFR and ERBB4, but are devoid of ERBB2 and ERBB3. In contrast, HLA-G(+) trophoblast subtypes exhibit an EGFR/ERBB4(-) and ERBB2/ERBB3(+) phenotype. EGF and HB-EGF activate EGFR, ERBB4, AKT and extracellular signal-regulated kinase 1/2 in EGFR(+) primary trophoblasts; however, they do not show an effect on HLA-G(+) EVTs. Both ligands strongly induce cell cycle progression in primary trophoblasts (P < 0.05) and placental explant-associated vCTBs (P < 0.05) and pCCTs (P < 0.05). Notably, EGFR(+) vCTB (P < 0.0001) and pCCT (P < 0.0001) layers are significantly expanded in CHM placentae when compared with healthy controls. LIMITATIONS, REASONS FOR CAUTION: Cells were removed from their physiological context and may therefore respond differently to various stimuli. WIDER IMPLICATIONS OF THE FINDINGS: In this study we define EGFR as a marker for proliferative trophoblast subtypes within the human placenta. Manipulation of EGFR signalling might thus offer a promising therapeutic avenue for the treatment of molar pregnancies associated with trophoblast hyperplasia. STUDY FUNDING/COMPETING INTERESTS: This study was supported by the Austrian Science Fund (grant P-25187-B13 to J.P.). There are no competing interests to declare.

Macrophage-derived IL-33 is a critical factor for placental growth.

IL-33, the most recently discovered member of the IL-1 superfamily and ligand for the transmembrane form of ST2 (ST2L), has been linked to several human pathologies including rheumatoid arthritis, asthma, and cardiovascular disease. Deregulated levels of soluble ST2, the natural IL-33 inhibitor, have been reported in sera of preeclamptic patients. However, the role of IL-33 during healthy pregnancy remains elusive. In the current study, IL-33 was detected in the culture supernatants of human placental and decidual macrophages, identifying them as a major source of secreted IL-33 in the uteroplacental unit. Because flow cytometry and immunofluorescence stainings revealed membranous ST2L expression on specific trophoblast populations, we hypothesized that IL-33 stimulates trophoblasts in a paracrine manner. Indeed, BrdU incorporation assays revealed that recombinant human IL-33 significantly increased proliferation of primary trophoblasts as well as of villous cytotrophoblasts and cell column trophoblasts in placental explant cultures. These effects were fully abolished upon addition of soluble ST2. Interestingly, Western blot and immunofluorescence analyses demonstrated that IL-33 activates AKT and ERK1/2 in primary trophoblasts and placental explants. Inhibitors against PI3K (LY294002) and MEK1/2 (UO126) efficiently blocked IL-33-induced proliferation in all model systems used. In summary, with IL-33, we define for the first time, to our knowledge, a macrophage-derived regulator of placental growth during early pregnancy.