Trimester Decidua Research Articles

Decidual Cell Regulation of Natural Killer Cell–Recruiting Chemokines: Implications for the Pathogenesis and Prediction of Preeclampsia

First trimester human decidua is composed of decidual cells, CD56(bright)CD16(-) decidual natural killer (dNK) cells, and macrophages. Decidual cells incubated with NK cell-derived IFN-γ and either macrophage-derived TNF-α or IL-1β synergistically enhanced mRNA and protein expression of IP-10 and I-TAC. Both chemokines recruit CXCR3-expressing NK cells. This synergy required IFN-γ receptor 1 and 2 mediation via JAK/STAT and NFκB signaling pathways. However, synergy was not observed on neutrophil, monocyte, and NK cell-recruiting chemokines. Immunostaining of first trimester decidua localized IP-10, I-TAC, IFN-γR1, and -R2 to vimentin-positive decidual cells versus cytokeratin-positive interstitial trophoblasts. Flow cytometry identified high CXCR3 levels on dNK cells and minority peripheral CD56(bright)CD16(-) pNK cells and intermediate CXCR3 levels on the majority of CD56(dim)CD16(+) pNK cells. Incubation of pNK cells with either IP-10 or I-TAC elicited concentration-dependent enhanced CXCR3 levels and migration of both pNK cell subsets that peaked at 10 ng/mL, whereas each chemokine at a concentration of 50 ng/mL inhibited CXCR3 expression and pNK cell migration. Deciduae from women with preeclampsia, a leading cause of maternal and fetal morbidity and mortality, displayed significantly lower dNK cell numbers and higher IP-10 and I-TAC levels versus gestational age-matched controls. Significantly elevated IP-10 levels in first trimester sera from women eventually developing preeclampsia compared with controls, identifying IP-10 as a novel, robust early predictor of preeclampsia.

Chorioallantoic Placentation in Viperid Snakes

Chorioallantoic Placentation in Viperid Snakes

Do decidual natural killer cells have a role in vascular smooth muscle cell dedifferentiation during spiral artery remodelling?

Adequate extravillous trophoblast (EVT) invasion is essential for successful placentation. Although miR-520c-3p plays an important role in CD44-mediated invasion in cancer cells, there is little information on whether miR-520c-3p is involved in the regulatory mechanisms of CD44-mediated EVT invasion.We screened first trimester trophoblast cells and trophoblast cell lines for expression of miR-520c-3p using real-time polymerase chain reaction. The cell invasion assay was performed using EVT cell lines, HTR8/SVneo and HChEpC1b, to investigate the capability of suppressing EVT invasion by miR-520c-3p. Laser microdissection analysis was then used to determine whether miR-520c-3p was present in the first trimester decidua. Finally, the possibility of chorionic villous trophoblast (CVT)-EVT communication via exosomal miR-520c-3p was determined using an in vitro model based on BeWo exosomes and the EVT cell lines as recipient cells.The miR-520c-3p level was significantly downregulated in EVT cell lines and EVTs. Cell invasion was significantly inhibited in miR-520c-3p-overexpressing cell lines, involving a significant reduction of CD44. Laser microdissection analysis showed that miR-520c-3p in the periarterial area of the decidua was significantly higher than that in the non-periarterial area. Using an in vitro model system, BeWo exosomal miR-520c-3p was internalized into the EVT cells with subsequently reduced cell invasion via CD44 repression.EVT invasion is synergistically enhanced by the reciprocal expression of endogenous miR-520c-3p and CD44. The present study supports a novel model involving a placenta-associated miRNA function in cell-cell communication in which CVT exosomal miR-520c-3p regulates cell invasion by targeting CD44 in EVTs.

Corin, an enzyme with a putative role in spiral artery remodeling, is up-regulated in late secretory endometrium and first trimester decidua

What is the nature of cellular Corin expression in human gestational tissues? CORIN is expressed in non-pregnant late secretory phase endometrium, first trimester human implantation sites and is up-regulated with decidualization ex vivo. Adequate trophoblast invasion and spiral artery remodeling/transformation is critical for successful implantation. CORIN, best known for its role in activating atrial natruietic peptide (ANP) to regulate blood pressure, has recently been proposed to be centrally involved in trophoblast invasion and spiral artery remodeling. It is postulated that ANP, activated by CORIN, promotes trophoblast invasion and that a deficiency causes pre-eclampsia. Mice deficient in either Corin or ANP displayed poor trophoblast invasion, impaired spiral artery remodeling and phenocopied human pre-eclampsia. However, the precise cellular localization of CORIN within human gestational tissues has not been well characterized. We measured CORIN protein localization in a number of human gestational tissues relevant to early embryo/placental implantation: non-pregnant (NP) endometrial biopsies (n = 5 per phase of the menstrual cycle), first trimester placental bed biopsies (n = 12) and pre-term control (n = 10) and severe early onset preeclamptic placentas (n = 15). Endometrial stromal cells were isolated from human endometrial biopsies (n = 5) and induced to decidualize ex vivo. Finally, CORIN concentrations were measured in serum obtained from pregnant women during the first trimester of whom, 56 subsequently ended up with a healthy term delivery (controls), 18 developed fetal growth restriction (FGR) and 21 had a miscarriage. We performed immunohistochemistry to assess CORIN localization. Changes in Corin mRNA expression in human endometrial stromal cells decidualized ex vivo were measured by quantitative RT-PCR, and levels of CORIN within human sera were measured by ELISA. CORIN was expressed in both NP late secretory phase endometrium and first trimester decidua within placental bed biopsies. Importantly, decidualization of primary human endometrial cells ex vivo significantly increased Corin expression (P < 0.05). CORIN was also detected within the villous cytotrophoblast, but there was no change in mRNA levels in placentas complicated by severe preterm pre-eclampsia when compared with pre-term controls. Although CORIN was detected in first trimester serum, levels did not change across gestation, nor could they predict miscarriage or FGR (other disorders of impaired placental invasion). Owing to the fact that we utilized early pregnancy human specimens, this is mainly a descriptive study with a limited amount of functional experiments. This is the first study to thoroughly characterize Corin mRNA and protein expression in human gestational tissue. Our findings support recent data from murine studies collectively suggesting that CORIN plays a critical role in trophoblast migration and spiral artery remodeling during early pregnancy in humans. Therefore, further studies of CORIN biology in early pregnancy may identify new therapeutic targets to improve implantation quality in early pregnancy and potentially reduce the rates of pregnancy complications caused by inadequate implantation (pre-eclampsia, FGR and miscarriage). This study was supported by The National Health and Medical Research Council of Australia (Salary support #490970, #490995). The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript. The authors declare that no competing interests exist.

Trophoblast-Induced Changes in C-X-C Motif Chemokine 10 Expression Contribute to Vascular Smooth Muscle Cell Dedifferentiation During Spiral Artery Remodeling

During pregnancy, fetal trophoblast disrupt endothelial cell and vascular smooth muscle cell (VSMC) interactions in spiral arteries of the maternal decidua to enable increased nutritional and oxygen delivery to the fetus. Little is known regarding this transformation because of difficulties of studying human pregnancy in vivo. This study investigated how trophoblast-secreted factors affect the interactions of vascular cells and the differentiation status of VSMC during spiral arteries remodeling using 3-dimensional vascular spheroid coculture. Endothelial cell and VSMC were cocultured in hanging droplets to form spheroids representing an inverted vessel lumen. Control or conditioned media from an extravillous trophoblast (EVT) cell line was incubated with vascular spheroids for 24 hours. Spheroid RNA was then analyzed by Illumina Sentrix BeadChip array. Spheroids incubated with EVT conditioned medium showed significant up/downregulation of 101 genes (>1.5-fold; P<0.05), including an upregulation of C-X-C motif chemokine 10 (IP-10). C-X-C motif chemokine 10 expression was confirmed by qualitative real-time PCR and Western blot analysis of spheroids, and immunohistochemistry of first trimester decidua and ex vivo dissected nonplacental bed spiral arteries. EVT conditioned medium reduced VSMC expression of differentiation markers, and both EVT conditioned medium and C-X-C motif chemokine 10 increased motility of VSMC indicating dedifferentiation of VSMC. EVT-induced C-X-C motif chemokine 10 expression may contribute to spiral arteries remodeling during pregnancy by altering the motility and differentiation status of the VSMC in the vessel.

Maternal Nodal inversely affects NODAL and STOX1 expression in the fetal placenta

Nodal, a secreted signaling protein from the transforming growth factor beta (TGF-β)-super family plays a vital role during early embryonic development. Recently, it was found that maternal decidua-specific Nodal knockout mice show intrauterine growth restriction (IUGR) and preterm birth. The chromosomal location of NODAL is in the same linkage area as the placental (fetal) pre-eclampsia (PE) susceptibility gene STOX1, which is associated with the familial form of early-onset, IUGR-complicated PE. As the STOX1 linkage was originally identified in women being born from a pre-eclamptic pregnancy as well as suffering from PE themselves, the linkage could in part be caused by NODAL, which is why the potential maternal–fetal interaction between STOX1 and NODAL was investigated. In the PE families with the STOX1 susceptibility allele carried by the children born from pre-eclamptic pregnancies, it was found that the pre-eclamptic mothers themselves all carried the NODAL H165R SNP, which causes a 50% reduced activity. Surprisingly, in decidua-specific Nodal knockout mice the fetal placenta showed up-regulation of STOX1 and NODAL expression. Conditioned media of human first trimester decidua and a human endometrial stromal cell line (T-HESC) treated with siRNAs against NODAL or carrying the H165R SNP were also able to induce NODAL and STOX1 expression when added to SGHPL-5 first trimester extravillous trophoblast cells. Finally, a human TGF-β/BMP signaling pathway PCR-array on decidua and the T-HESC cell line with Nodal knockdown revealed upregulation of Activin-A, which was confirmed in conditioned media by ELISA. We show that maternal decidua Nodal knockdown gives upregulation of NODAL and STOX1 mRNA expression in fetal extravillous trophoblast cells, potentially via upregulation of Activin-A in the maternal decidua. As both Activin-A and Nodal have been implicated in PE, being increased in serum of pre-eclamptic women and upregulated in pre-eclamptic placentas respectively, this interaction at the maternal–fetal interface might play a substantial role in the development of PE.

FUNCTIONAL PLASTICITY OF DECIDUAL NATURAL KILLER CELLS DURING MATERNAL HUMAIN CYTOMEGALOVIRUS INFECTION

Event Abstract Back to Event FUNCTIONAL PLASTICITY OF DECIDUAL NATURAL KILLER CELLS DURING MATERNAL HUMAIN CYTOMEGALOVIRUS INFECTION Nabila Jabrane-Ferrat1* and Johan Siewiera1 1 Inserm UMR1043 - CNRS UMR5282 - Université Toulouse III, Immunology, France Circulating Natural Killer cells constitute the first line of defense against invading pathogens. These effectors of the innate immune system recognize and kill tumor or viral-infected cells. In addition to peripheral blood and lymphoid organs, NK cells (dNK) are found in the decidua basalis of the human pregnant uterus. Decidual NK cells secrete a large variety of cytokines, chemokines and growth factors but are poorly cytotoxic. We previously showed that dNK cells freshly isolated from the 1st trimester decidua basalis are able to kill target cells upon ligation of NKp46 activating receptor. This cytotoxic capacity is under the control of CD94/NKG2A inhibiting receptor. We challenged the hypothesis that upon viral infection, dNK cells receive activating signals that overcome the inhibitory signal and trigger their cytotoxic effector function. Using an ex vivo autologous system, we describe a mechanism by which dNK cells kill HCMV infected targets. We show that dNK cells engage activating immune synapse with HCMV-infected autologous targets. dNK cells stimulated with HCMV-infected targets are able to reorganize rapidly both their microtubule organizing center and their Golgi apparatus. These changes in cell morphology result in focused delivery of lytic granules and subsequent killing of target cells. Furthermore, dNK cells stimulated with infected autologous fibroblasts acquired a cytotoxic phenotype. Taken together our data provide the first evidence that dNK cells may be involved in the protection of the fetus against maternal viral infections. Immune synapse between dNK cell (perforin in red) and uterin fibroblast that was infected with human cytomegalovirus. Actin in green, alpha tubulin in blue. Images were taken on a Zeiss confocal microscope (x63) at the U1043 imaging core facility. Figure 1 References Siewiera J, El Costa H, Tabiasco J, Berrebi A, Cartron G, Le Bouteiller P, Jabrane-Ferrat N. Human cytomegalovirus infection elicits new decidual natural killer cell effector functions.PLoS Pathog. 2013 Apr;9(4):e1003257. Keywords: Natural Killer cells, Pregnancy, congenital infection, Cytomegalovirus, Cytotoxicity Conference: 15th International Congress of Immunology (ICI), Milan, Italy, 22 Aug - 27 Aug, 2013. Presentation Type: Abstract Topic: Innate immunity Citation: Jabrane-Ferrat N and Siewiera J (2013). FUNCTIONAL PLASTICITY OF DECIDUAL NATURAL KILLER CELLS DURING MATERNAL HUMAIN CYTOMEGALOVIRUS INFECTION. Front. Immunol. Conference Abstract: 15th International Congress of Immunology (ICI). doi: 10.3389/conf.fimmu.2013.02.01137 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 17 Jul 2013; Published Online: 22 Aug 2013. * Correspondence: Dr. Nabila Jabrane-Ferrat, Inserm UMR1043 - CNRS UMR5282 - Université Toulouse III, Immunology, Toulouse, Midi-Pyrenees, 31000, France, nabila.jabrane-ferrat@inserm.fr Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Nabila Jabrane-Ferrat Johan Siewiera Google Nabila Jabrane-Ferrat Johan Siewiera Google Scholar Nabila Jabrane-Ferrat Johan Siewiera PubMed Nabila Jabrane-Ferrat Johan Siewiera Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

PLASTICITY OF DECIDUAL NATURAL KILLER CELLS DURING MATERNAL HCMV INFECTION

Event Abstract Back to Event PLASTICITY OF DECIDUAL NATURAL KILLER CELLS DURING MATERNAL HCMV INFECTION Nabila Jabrane-Ferrat1* and Johan Siewiera1 1 INSERM 1043 / CNRS 5282, Centre de Physiopathologie de Toulouse-Purpan, France Circulating Natural Killer cells constitute the first line of defense against invading pathogens. These effectors of the innate immune system recognize and kill tumor or viral-infected cells. In addition to peripheral blood and lymphoid organs, NK cells are found in the decidua basalis of the human pregnant uterus. Decidual NK cells secrete a large variety of cytokines, chemokines and growth factors but are poorly cytotoxic. We previously showed that dNK cells freshly isolated from the 1st trimester decidua basalis are able to kill target cells upon ligation of NKp46 activating receptor. This cytotoxic capacity is under the control of CD94/NKG2A inhibiting receptor. We challenged the hypothesis that upon viral infection, dNK cells receive activating signals that overcome the inhibitory signals and trigger their cytotoxic effector function. Using an ex vivo autologous system, we describe a mechanism by which dNK cells kill HCMV infected targets. We show that dNK cells engage activating immune synapse with HCMV-infected autologous targets. dNK cells stimulated with HCMV-infected targets are able to reorganize rapidly both their microtubule organizing center and their Golgi apparatus. These changes in cell morphology result in focused delivery of lytic granules and subsequent killing of target cells. Furthermore, dNK cells stimulated with infected autologous fibroblasts acquired a cytotoxic phenotype. Taken together our data provide the first evidence that dNK cells may be involved in the protection of the fetus against maternal viral infections. Figure 1 References Siewiera J, El Costa H, Tabiasco J, Berrebi A, Cartron G, Bouteiller P, Jabrane-Ferrat N. Human cytomegalovirus infection elicits new decidual natural killer cell effector functions. PLoS Pathog. 2013 Apr;9(4):e1003257. doi: 10.1371/journal.ppat.1003257. Epub 2013 Apr 4. Keywords: NK cells, Cytomegalovirus, preganancy, Nk receptors, decidua basalis Conference: 15th International Congress of Immunology (ICI), Milan, Italy, 22 Aug - 27 Aug, 2013. Presentation Type: Abstract Topic: Innate immunity Citation: Jabrane-Ferrat N and Siewiera J (2013). PLASTICITY OF DECIDUAL NATURAL KILLER CELLS DURING MATERNAL HCMV INFECTION. Front. Immunol. Conference Abstract: 15th International Congress of Immunology (ICI). doi: 10.3389/conf.fimmu.2013.02.00542 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 04 Jun 2013; Published Online: 22 Aug 2013. * Correspondence: Dr. Nabila Jabrane-Ferrat, INSERM 1043 / CNRS 5282, Centre de Physiopathologie de Toulouse-Purpan, Toulouse, Midi-Pyrenees, 31000, France, nabila.jabrane-ferrat@inserm.fr Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Nabila Jabrane-Ferrat Johan Siewiera Google Nabila Jabrane-Ferrat Johan Siewiera Google Scholar Nabila Jabrane-Ferrat Johan Siewiera PubMed Nabila Jabrane-Ferrat Johan Siewiera Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Toll‐Like Receptor 4 Expression in Decidual Cells and Interstitial Trophoblasts Across Human Pregnancy

Toll-like receptor-4 (TLR-4) protects against Gram-negative bacteria expressed lipopolysaccharide and 'danger signals' from injured or dying cells. Although decidual cells (DCs) and interstitial trophoblasts (ITs) are in close contact, TLR-4 has been studied extensively only in ITs. Formalin-fixed, paraffin-embedded serial sections of endometrium in follicular and luteal phases and deciduas from first and second trimester elective terminations and third trimester normal deliveries were immunostained for TLR-4, trophoblast-specific cytokeratin, and DC-specific vimentin. HSCORE assessed TLR-4 immunostaining in DCs versus ITs. TLR-4 HSCORES were significantly higher in: (i) first trimester DCs than luteal phase pre-decidual stromal cells; (ii) first and third versus second trimester DCs, but similar between third trimester deciduas parietalis and basalis; (iii) first versus second trimester ITs; (iv) DCs versus ITs across gestation. Higher TLR-4 in DCs than ITs suggests DCs as primary targets for Gram-negative bacteria and/or inflammation-related danger signals.

Sexual intercourse with pyospermia decreases uterine CD16-/CD56 bright natural killer cells in women with infertility

PROBLEM: In the uterine endometrium and the first trimester decidua, there are a number of immune cells derived from the bonemarrow that are capable of responding to alloimmune stimulations from the embryo. Themain population of endometrial NK cell is cytokine producing CD16-/CD56bright NK cells, whereas the most abundant population in peripheral blood NK cells is cytotoxic CD16+/CD56dim NK cells. We previously reported that the increase in cytotoxic NK cells in peripheral blood and the endometrium have a beneficial effect on the therapeutic results of in vitro fertilization andembryo transfer (IVF-ET). Wehave also reported that seminal plasmaplays an important role in the recruitment of CD56bright NK cells into the endometrium. The present study was aimed to investigate the influence of sexual intercourse onNK cell subsets in the mid-secretory endometrium using flow cytometry. METHODS: This study was designed as a prospective study. All study subjects had signed informed consents prior to entry to this study, in accordance with local IRB protocol. All subjects who were planning to have invitro fertilization and embryo transfer (IVF-ET) had sexual intercourse before the endometrial sampling without any contraceptive devices. The mid-secretary endometrium was obtained from 234 women, and then submitted for flow cytometric analysis. Endomerial NK cell subpopulation was analyzed for the expression of CD16 and CD56 using 3-color flow cytometry. Semen analysis was performed at the time of oocytes pick up and was classified according to the World Health Organization (WHO) 2010 guideline. Of 234 semen samples, 10 were diagnosed as pyospermia (pyospermia group), and 224 as not-pyospermia (non-pyospermia group). RESULTS: There were no differences between 2 groups in the total sperm concentration and the motile sperm concentration, or in the percentage of CD56+ NK cells. The percentage of CD16-/CD56+ cells, however, was lower in pyospermia group (25.9±21.7%) compared with that in non-pyospermia group (35.4±18.2%). The percentage of CD16-/CD56bright cells was significantly lower in pyospermia group (28.0±27.8%) compared with that in not-pyospermia group (42.9±22.1%, p< 0.01). CONCLUSIONS: Our study firstly demonstrated that pyospermia has a harmful influence on endometrial NK cell subsets. The immunologically advantageous condition for successful pregnancies after sexual intercourse may give support to the hypothesis that seminal fluid may act as a regulator of endometrial immune cells and an inducer of feto-maternal immune tolerance. Further research should be undertaken to reveal the biological actions of seminal munology 94 (2012) 5–130

Vascular Endothelial Expression of Indoleamine 2,3-Dioxygenase 1 Forms a Positive Gradient towards the Feto-Maternal Interface

We describe the distribution of indoleamine 2,3-dioxygenase 1 (IDO1) in vascular endothelium of human first-trimester and term placenta. Expression of IDO1 protein on the fetal side of the interface extended from almost exclusively sub-trophoblastic capillaries in first-trimester placenta to a nearly general presence on villous vascular endothelia at term, including also most bigger vessels such as villous arteries and veins of stem villi and vessels of the chorionic plate. Umbilical cord vessels were generally negative for IDO1 protein. In the fetal part of the placenta positivity for IDO1 was restricted to vascular endothelium, which did not co-express HLA-DR. This finding paralleled detectability of IDO1 mRNA in first trimester and term tissue and a high increase in the kynurenine to tryptophan ratio in chorionic villous tissue from first trimester to term placenta. Endothelial cells isolated from the chorionic plate of term placenta expressed IDO1 mRNA in contrast to endothelial cells originating from human umbilical vein, iliac vein or aorta. In first trimester decidua we found endothelium of arteries rather than veins expressing IDO1, which was complementory to expression of HLA-DR. An estimation of IDO activity on the basis of the ratio of kynurenine and tryptophan in blood taken from vessels of the chorionic plate of term placenta indicated far higher values than those found in the peripheral blood of adults. Thus, a gradient of vascular endothelial IDO1 expression is present at both sides of the feto-maternal interface.

First Trimester Pregnancy Decidual Natural Killer Cells Contain and Spontaneously Release High Quantities of Granulysin

Granulysin (GNLY) is a novel cytolytic protein lytic against a variety of tumor cells and microbes. The role of GNLY during pregnancy has not been extensively explored. The aim of this study is to examine GNLY expression and distribution in the first trimester pregnancy peripheral blood (PB) and decidua, the ability of decidual and PB natural killer (NK) cells to secrete GNLY spontaneously, and the role of antigen-presenting cells (APC) in the regulation of GNLY expression in decidual NK cells. GNLY expression was analyzed using cell permeabilization method, flow cytometry, and immunohistochemistry. GNLY secretion by purified NK cells was detected by ELISA method. GNLY is abundantly expressed at the maternal-fetal interface in the first trimester pregnancy. Decidual T lymphocytes express significantly higher levels of GNLY (58%) then PB T lymphocytes (11%). Over 85% of decidual CD56(+) cells express GNLY and when cultured spontaneously release high quantities of GNLY. Decidual APC participate in the control of GNLY expression in CD56(+) cells. Abundant expression of GNLY in the decidual immunocompetent cells and the capacity of decidual CD56(+) cells to spontaneously secrete high quantities of GNLY point to important protective and immunomodulatory role that this molecule could play at the maternal-fetal interface.

Prokineticin 1 induces Dickkopf 1 expression and regulates cell proliferation and decidualization in the human endometrium

Prokineticin 1 (PROK1) signalling via prokineticin receptor 1 (PROKR1) regulates the expression of several genes with important roles in endometrial receptivity and implantation. This study investigated PROK1 regulation of Dickkopf 1 (DKK1) expression, a negative regulator of canonical Wnt signalling, and its function in the non-pregnant endometrium and first trimester decidua. DKK1 mRNA expression is elevated during the mid-secretory phase of the menstrual cycle and expression increases further in first trimester decidua. DKK1 protein expression is localized to glandular epithelial and stromal cells during the proliferative, early- and mid-secretory phases, whereas expression is confined to the stroma in the late-secretory phase and first trimester decidua. PROK1 induces the expression of DKK1 in endometrial epithelial cells stably expressing PROKR1 and in first trimester decidua explants, via a Gq-calcium-calcineurin-nuclear factor of activated T-cells-mediated pathway. Endometrial epithelial cell proliferation is negatively regulated by PROK1-PROKR1 signalling. We demonstrate that this effect on cell proliferation occurs via DKK1 expression, as siRNA targeted against DKK1 reduces the PROK1-induced decrease in proliferation. Furthermore, decidualization of primary human endometrial stromal cells with progesterone and cyclic adenosine monophosphate is inhibited by miRNA knock down of PROK1 or DKK1. These data demonstrate important roles for PROK1 and DKK1 during endometrial receptivity and early pregnancy, which include regulation of endometrial cell proliferation and decidualization.

P95 Do we deliver too soon for preterm pre-eclampsia?

Interactions between the highly invasive trophoblasts and the maternal uterine decidual extracellular matrix (ECM) are crucial in the determination of a successful pregnancy. Fibulin-5 (FBLN5) is a member of the fibulin family that alters cell adhesive and invasive properties and is expressed in human villous cytotrophoblasts.We aimed to determine the expression and immunolocalization of FBLN5 in human first trimester decidua and examine the effect of FBLN5 in trophoblast invasion in vitro using a first trimester placental villous outgrowth assay. We demonstrated that FBLN5 mRNA expression is upregulated in response to cAMP-mediated decidualization of primary human endometrial stromal cells, although FBLN5 itself does not enhance decidualization. We reported for the first time, FBLN5 protein production in first trimester decidual cells and also co-localization to HLAG-positive EVTs in first trimester decidua. Consequently, we investigated the effects of exogenous FBLN5 on placental villous outgrowth in vitro and demonstrated that FBLN5 promotes EVT migration/invasion. This is the first study to identify FBLN5 in decidualized human endometrial stromal cells, first trimester decidua and EVT and determine a functional role for FBLN5 in human EVTs, suggesting that decidual and or EVT-derived FBLN5 regulates EVT invasion and placentation in women.

Differential expression of angiotensin II type 1 and type 2 receptors at the maternal–fetal interface: potential roles in early placental development

Angiotensin II (Ang II) is locally generated in the placenta and regulates syncytial transport, vascular contractility and trophoblast invasion. It acts through two receptor subtypes, AGTR1 and AGTR2 (AT1 and AT2), which typically mediate antagonising actions. The objectives of this study are to characterise the cellular distribution of AGTR1 and AGTR2 at the maternal-fetal interface and explore the effects on cytotrophoblast turnover. Low levels of AGTR2 mRNA were detected in first trimester placental homogenates using real-time PCR. Immunohistochemistry using polyclonal antibodies against AGTR1 and AGTR2 detected the receptors in first trimester placenta, decidua basalis and villous tip outgrowths in culture. Serial staining with cytokeratin-7 was used to identify extravillous trophoblasts (EVTs). AGTR1 was found in the syncytiotrophoblast microvillous membrane, in a subpopulation of villous cytotrophoblasts, and in Hofbauer cells. AGTR1 was strongly upregulated in cytotrophoblasts in cell columns and villous tip outgrowths, but was absent in interstitial and endovascular EVTs within the decidua. AGTR2 immunostaining was present in Hofbauer cells and villous cytotrophoblasts, but was absent from syncytiotrophoblast. Faint staining was detected in cell column cytotrophoblasts and villous outgrowths, but not in EVTs within the decidua. Both receptors were detected in placental homogenates by western blotting. Ang II significantly increased proliferation of cytotrophoblasts in both villous explants and villous tip outgrowths, but did not affect apoptosis. Blockade of AGTR1 and AGTR2 together abrogated this effect. This study shows specific expression patterns for AGTR1 and AGTR2 in distinct trophoblast populations at the maternal-fetal interface and suggests that Ang II plays a role in placental development and generation of EVTs.

Chorionic gonadotrophin regulates CXCR4 expression in human endometrium via E-series prostanoid receptor 2 signalling to PI3K-ERK1/2: implications for fetal-maternal crosstalk for embryo implantation

Murine knock-out models and blastocyst co-culture studies have identified prostaglandin-endoperoxide synthase (PTGS) 2, prostaglandin (PG) E receptor 2 (PTGER2) and the chemokine receptor CXCR4 as important regulators of early pregnancy events. In vitro studies and studies in non-human primates have shown that these proteins are regulated in the endometrium by the early embryonic signal, chorionic gonadotrophin (CG). Here we show that expressions of PTGER2 and CXCR4 are elevated during the mid-secretory phase of the menstrual cycle and decidua of early pregnancy in humans. Using first trimester decidua explants, we show that CG induces expression of PTGS2 and biosynthesis of PGE2, and expression of PTGER2. Subsequently, PGE2via PTGER2 induces expression of CXCR4. Using an in vitro model system of Ishikawa endometrial epithelial cells stably expressing PTGER2 and human first trimester decidua explants, we demonstrate that CXCR4 expression is regulated by PTGER2 via the epidermal growth factor receptor (EGFR)-phosphatidylinositol-3-kinase (PI3K)-extracellular signal-regulated kinase (ERK1/2) pathway.Taken together, our data suggest that early embryonic signals may regulate fetal–maternal crosstalk in the human endometrium by inducing CXCR4 expression via the PGE2–PTGER2-mediated induction of the EGFR, PI3K and ERK1/2 pathways.

First Trimester Histiotrophe Shows Altered Sialylation Compared with Secretory Phase Glycoconjugates in Human Endometrium

Objectives Histiotrophe is now recognized as being an important feature of early human pregnancy, providing nutrients and growth factors to the developing embryo. Our aim was to examine the glycan composition of histiotrophe from first trimester decidua and to compare it with secretions present in endometrial glands from the late secretory phase of the menstrual cycle. Study design Twenty samples of decidua from pregnancies between 8 and 11 weeks were processed into epoxy resin and sections stained with a panel of 22 lectins, together with six late secretory phase endometrial biopsies. Main outcome measures Specimens were analysed using a semi-quantitative ranking system and the density of lectin binding to the glandular secretions and the epithelium assessed. Results With the onset of pregnancy, β-galactose, α-N-acetyl galactosamine and N-Acetyl lactosamine bound by Arachis hypogaea, Glycine max, Helix pomatia and Erythrina crystagalli agglutinins appeared in terminal positions on oligosaccharide chains, suggesting loss of the capping sialic acid residues present in the non-pregnant state. Conclusions Suppression of terminal sialylation is evident during early pregnancy, suggesting that modifications to endometrial glandular activity occur in response to placental signals. The changes may facilitate absorption of histiotrophe by the trophoblast and enhance availability of substrates for degradation.

Endothelin Type A Receptor (ETA) Expression Is Regulated by HOXA10 in Human Endometrial Stromal Cells

Endothelin type A receptor (ET(A)) is a member of the superfamily of G protein-coupled receptors. Our laboratory conducted a microarray screen that identified ET(A) as target of HOXA10 transcriptional control in endometrium. Here, we confirm HOXA10-regulated ET(A) expression in endometrium. Endometrial biopsies were obtained from fertile reproductive-age individuals, and first trimester decidual samples were obtained at the time of elective termination. Immunohistochemistry (IHC) was used to identify ET(A) protein in endometrium as well as first trimester decidua. ET(A) was expressed in endometrial stromal cells throughout the menstrual cycle. ET(A) was also highly expressed in first trimester decidual cells. The regulatory relationship between HOXA10 and ET(A) was established by transient transfection analysis. The human endometrial stromal cell line (HESC) and the human endometrial epithelial cell line (Ishikawa) were transfected with pcDNA/HOXA10, HOXA10 small interfering RNA (siRNA), or respective controls. Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) was performed to determine expression levels of HOXA10 and ET(A) in each group. ET(A) gene expression increased 9-fold (P < .05) after pcDNA/HOXA10 transfection of HESC. ET(A) was not regulated by HOXA10 in Ishikawa cells. We conclude that ET(A) is expressed in normal endometrium and decidua. Expression of this receptor is regulated by an essential mediator of endometrial receptivity, HOXA10. ET(A) may enhance the proliferative potential of endometrial cells in a manner similar to that seen in vascular smooth muscle cells. ET( A) likely acts as a molecular mechanism by which HOXA10 promotes stromal cell growth and prostaglandin production in both the implantation window and decidua.

Endoglandular trophoblast, an alternative route of trophoblast invasion? Analysis with novel confrontation co-culture models

Routes of trophoblast invasion seem to be clear, whereas specific invasive pathways need further elucidation. Extravillous trophoblasts (EVTs) transform spiral arteries to guarantee appropriate blood flow to the placenta in the second trimester. Embryo nutrition during the first trimester is thought to be histiotrophic, whereas proof that EVTs also invade uterine glands is lacking. We developed novel three-dimensional confrontation co-culture models to elucidate invasion of EVTs into uterine glands. First trimester decidua parietalis and placental villous explants were directly confronted and co-cultured for 72 h, or confronted indirectly after 72 h pre-culture for re-epithelialization of decidua pieces. Cryosections were stained by immunohistochemistry or immunofluorescent/immunohistochemical double labelling and compared with first trimester placentation sites in situ. EVTs deeply invaded decidual tissues in direct confrontation assays and were found between the decidual epithelial cells and epithelial basement membrane. EVTs were also detected in the decidual stroma in direct proximity to glands, sometimes even replacing glandular epithelial cells. Similar observations were made in sections from the first trimester decidua/placental bed. In the invaded parts of sections of decidua basalis, 55% +/- 7% (mean +/- SEM; n = 10, range 6-11 weeks) of glandular cross sections were associated with or infiltrated by EVTs. Using novel confrontation co-culture assays, a potential new route of EVT invasion was detected. EVTs appear to break through the basement membrane of uterine glands to open their lumen towards the intervillous space. These data support the hypothesis of histiotrophic nutrition of the embryo prior to onset of maternal blood flow within the placenta.

Nuclear Translocation of Nuclear Factor Kappa B in First Trimester Deciduas and Chorionic Villi in Early Spontaneous Miscarriage Women

The nuclear factor kappa B is widely expressed in the distinct subpopulations of chorionic villi and deciduas of first-trimester pregnancies. We examined the cellular distribution and expression of nuclear factor kappa B in the human first-trimester chorionic villi and deciduas of women with early spontaneous miscarriage and viable pregnancy by confocal laser scanning microscope and immunohistochemistry. There is a greater nuclear translocation of nuclear factor kappa B is restricted to villous stromal cells, decidual stromal cells, glandular epithelial cells and vessel endothelial cells in early spontaneous miscarriage than in viable pregnancies. Collectively these observations suggest that over-activation of nuclear factor kappa B has a relationship with early spontaneous miscarriages.