Persistence of decidual NK cells and KIR genotypes in healthy pregnant and preeclamptic women: a case-control study in the third trimester of gestation

Background  

Natural Killer (NK) cells are the most abundant lymphocytes in the decidua during early gestation. The interactions of NK cells with the extravillous cytotrophoblast have been associated with a normal spiral artery remodeling process, an essential event for a successful pregnancy. Recent data indicate that alterations in the amount of decidual NK (dNK) cells contribute to the development of preeclampsia (PE). Moreover, genetic studies suggest that Killer Immunoglobulin-like Receptors (KIR) expressed in dNK cells influence the susceptibility to PE. Although dNK cells have been well characterized during early pregnancy, they have been scarcely studied in the third trimester of gestation. The aim of this work was to characterize dNK cells at the last trimester of gestation and to analyze the KIR genotype of healthy and PE women.     

Hypoxia-Induced Changes in the Bioactivity of Cytotrophoblast-Derived Exosomes

Migration of extravillous trophoblasts (EVT) into decidua and myometrium is a critical process in the conversion of maternal spiral arterioles and establishing placenta perfusion. EVT migration is affected by cell-to-cell communication and oxygen tension. While the release of exosomes from placental cells has been identified as a significant pathway in materno-fetal communication, the role of placental-derived exosomes in placentation has yet to be established. The aim of this study was to establish the effect of oxygen tension on the release and bioactivity of cytotrophoblast (CT)-derived exosomes on EVT invasion and proliferation. CT were isolated from first trimester fetal tissue (n = 12) using a trypsin-deoxyribonuclease-dispase/Percoll method. CT were cultured under 8%, 3% or 1% O2 for 48 h. Exosomes from CT-conditioned media were isolated by differential and buoyant density centrifugation. The effect of oxygen tension on exosome release (µg exosomal protein/10⁶cells/48 h) and bioactivity were established. HTR-8/SVneo (EVT) were used as target cells to establish the effect (bioactivity) of exosomes on invasion and proliferation as assessed by real-time, live-cell imaging (Incucyte™). The release and bioactivity of CT-derived exosomes were inversely correlated with oxygen tension (p<0.001). Under low oxygen tensions (i.e. 1% O2), CT-derived exosomes promoted EVT invasion and proliferation. Proteomic analysis of exosomes identified oxygen-dependent changes in protein content. We propose that in response to changes in oxygen tension, CTs modify the bioactivity of exosomes, thereby, regulating EVT phenotype. Exosomal induction of EVT migration may represent a normal process of placentation and/or an adaptive response to placental hypoxia.     

Human Cytomegalovirus Infection Elicits New Decidual Natural Killer Cell Effector Functions

During the first trimester of pregnancy the uterus is massively infiltrated by decidual natural killer cells (dNK). These cells are not killers, but they rather provide a microenvironment that is propitious to healthy placentation. Human cytomegalovirus (HCMV) is the most common cause of intrauterine viral infections and a known cause of severe birth defects or fetal death. The rate of HCMV congenital infection is often low in the first trimester of pregnancy. The mechanisms controlling HCMV spreading during pregnancy are not yet fully revealed, but evidence indicating that the innate immune system plays a role in controlling HCMV infection in healthy adults exists. In this study, we investigated whether dNK cells could be involved in controlling viral spreading and in protecting the fetus against congenital HCMV infection. We found that freshly isolated dNK cells acquire major functional and phenotypic changes when they are exposed to HCMV-infected decidual autologous fibroblasts. Functional studies revealed that dNK cells, which are mainly cytokines and chemokines producers during normal pregnancy, become cytotoxic effectors upon their exposure to HCMV-infected autologous decidual fibroblasts. Both the NKG2D and the CD94/NKG2C or 2E activating receptors are involved in the acquired cytotoxic function. Moreover, we demonstrate that CD56^pos dNK cells are able to infiltrate HCMV-infected trophoblast organ culture ex-vivo and to co-localize with infected cells in situ in HCMV-infected placenta. Taken together, our results present the first evidence suggesting the involvement of dNK cells in controlling HCMV intrauterine infection and provide insights into the mechanisms through which these cells may operate to limit the spreading of viral infection to fetal tissues.     

Three-dimensional spheroidal culture of cytotrophoblast cells mimics the phenotype and differentiation of cytotrophoblasts from normal and preeclamptic pregnancies

Normal placental development is dependent on the orchestrated differentiation of cytotrophoblast (CTB) cells. This study was aimed at studying cytotrophoblast cells from normal and preeclamptic pregnancies in a three-dimensional spheroid-based cell culture model. First trimester cytotrophoblast cells cultured as spheroids maintain their high proliferative and invasive phenotype and respond to different cytokines upon stimulation in a three-dimensional invasion assay. In contrast, third trimester cytotrophoblast spheroids maintain their quiescent nonproliferating phenotype and invasion can only be induced by EGF. Contrasting the regular spheroidal arrangement of cytotrophoblast cells from normal third trimester pregnancies, spheroidal organization of preeclamptic cytotrophoblast cells is disturbed and the cells downregulate CD105 in vivo and in vitro. Furthermore, the invasion of both normal and preeclamptic third trimester, but not first trimester cytotrophoblast cells, is inhibited by pro-inflammatory cytokines. Plasma samples from pregnant women with preeclampsia significantly stimulate the invasion of first trimester cytotrophoblast cells and the sprouting of human umbilical vein endothelial cells (HUVECs) compared to plasma samples from healthy pregnant women. Taken together, the data establish the spheroidal cytotrophoblast model as a powerful system to mimic the in vivo phenotype of first and third trimester and preeclamptic cytotrophoblast cells and demonstrate that plasma-derived factors modulate the differentiation of cytotrophoblast cells.     

Serine peptidase HTRA3 is closely associated with human placental development and is elevated in pregnancy serum

HTRA3 is a newly identified serine peptidase of the mammalian HTRA (high-temperature requirement factor A) family, that is upregulated dramatically during mouse placental development. The current study determined whether HTRA3 was involved in human placentation. During the menstrual cycle, HTRA3 was expressed primarily in the endometrial glands, being significantly upregulated toward the mid- to late secretory phases; prominent expression in the stroma detected only in the decidual cells in the late secretory phase. Thus, overall endometrial HTRA3 expression was highest in the late secretory phase, when the endometrium is prepared for maternal-trophoblast interaction. During the first trimester of pregnancy, both glandular and decidual HTRA3 expression increased further with the decidual upregulation being highly significant. The strong link between HTRA3 expression and endometrial stromal cell decidualization was further established in an in vitro model using primary endometrial stromal cells. HTRA3 was also expressed by certain trophoblast subtypes in the first-trimester placenta: strongly in the villous syncytiotrophoblast, trophoblast shell, and endovascular trophoblast and weakly in the distal portion of the trophoblast cell columns but not in villous cytotrophoblast, the proximal region of the cell columns, or interstitial trophoblast. Upregulation of HTRA3 expression in association with placental development was revealed by a significant elevation of this protein in the maternal serum during the first trimester. We thus propose that HTRA3 is a previously unrecognized factor closely associated with and potentially important for human placentation. This study established crucial groundwork for future investigations toward establishing the physiological roles of HTRA3 in human placentation.     

Decidual-secreted factors alter invasive trophoblast membrane and secreted proteins implying a role for decidual cell regulation of placentation

Inadequate or inappropriate implantation and placentation during the establishment of human pregnancy is thought to lead to first trimester miscarriage, placental insufficiency and other obstetric complications. To create the placental blood supply, specialized cells, the ‘extravillous trophoblast’ (EVT) invade through the differentiated uterine endometrium (the decidua) to engraft and remodel uterine spiral arteries. We hypothesized that decidual factors would regulate EVT function by altering the production of EVT membrane and secreted factors. We used a proteomics approach to identify EVT membrane and secreted proteins regulated by decidual cell factors. Human endometrial stromal cells were decidualized in vitro by treatment with estradiol (10⁻⁸ M), medroxyprogesterone acetate (10⁻⁷ M) and cAMP (0.5 mM) for 14 days. Conditioned media (CM) was collected on day 2 (non-decidualized CM) and 14 (decidualized CM) of treatment. Isolated primary EVT cultured on Matrigel™ were treated with media control, non-decidualized or decidualized CM for 16 h. EVT CM was fractionated for proteins <30 kDa using size-exclusion affinity nanoparticles (SEAN) before trypsin digestion and HPLC-MS/MS. 43 proteins produced by EVT were identified; 14 not previously known to be expressed in the placenta and 12 which had previously been associated with diseases of pregnancy including preeclampsia. Profilin 1, lysosome associated membrane glycoprotein 1 (LAMP1), dipeptidyl peptidase 1 (DPP1/cathepsin C) and annexin A2 expression by interstitial EVT in vivo was validated by immunhistochemistry. Decidual CM regulation in vitro was validated by western blotting: decidualized CM upregulated profilin 1 in EVT CM and non-decidualized CM upregulated annexin A2 in EVT CM and pro-DPP1 in EVT cell lysate. Here, non-decidualized factors induced protease expression by EVT suggesting that non-decidualized factors may induce a pro-inflammatory cascade. Preeclampsia is a pro-inflammatory condition. Overall, we have demonstrated the potential of a proteomics approach to identify novel proteins expressed by EVT and to uncover the mechanisms leading to disease states.     

Immunophenotype and cytokine profiles of rhesus monkey CD56bright and CD56dim decidual natural killer cells

The primate endometrium is characterized in pregnancy by a tissue-specific population of CD56(bright) natural killer (NK) cells. These cells are observed in human, rhesus, and other nonhuman primate decidua. However, other subsets of NK cells are present in the decidua and may play distinct roles in pregnancy. The purpose of this study was to define the surface marker phenotype of rhesus monkey decidual NK (dNK) cell subsets, and to address functional differences by profiling cytokine and chemokine secretion in contrast with decidual T cells and macrophages. Rhesus monkey decidual leukocytes were obtained from early pregnancy tissues, and were characterized by flow cytometry and multiplex assay of secreted factors. We concluded that the major NK cell population in rhesus early pregnancy decidua are CD56(bright) CD16(+)NKp30(-) decidual NK cells, with minor CD56(dim) and CD56(neg) dNK cells. Intracellular cytokine staining demonstrated that CD56(dim) and not CD56(bright) dNK cells are the primary interferon-gamma (IFNG) producers. In addition, the profile of other cytokines, chemokines, and growth factors secreted by these two dNK cell populations was generally similar, but distinct from that of peripheral blood NK cells. Finally, analysis of multiple pregnancies from eight dams revealed that the decidual immune cell profile is characteristic of an individual animal and is consistently maintained across successive pregnancies, suggesting that the uterine immune environment in pregnancy is carefully regulated in the rhesus monkey decidua.     

Toxoplasma gondii Infection Regulates the Balance of Activating and Inhibitory Receptors on Decidual Natural Killer Cells

Inhibitory receptors and activating receptor expressed on decidual natural killer (dNK) cells are generally believed to be important in abnormal pregnancy outcomes and induced adverse pregnancy. However, if Toxoplasma gondii (T. gondii) infection induced abnormal pregnancy was related to dNK cells changes is not clear. In this study, we used human dNK cells co-cultured with human extravillous cytotrophoblast (EVT) cells following YFP-Toxoplasma gondii (YFP-T. gondii) infection in vitro and established animal pregnant infection model. Levels of inhibitory receptors KIR2DL4 and ILT-2, their ligand HLA-G, and activating receptor NKG2D in human decidua, and NKG2A and its ligand Qa-1 and NKG2D in mice uterine were analyzed by real-time PCR and flow cytometry with levels of NKG2D significantly higher than those of KIR2DL4 and ILT-2 in vitro and in invo. The level of NKG2D was positively correlated with cytotoxic activity of dNK cells in vitro. Numbers of abnormal pregnancies were significantly greater in the infected group than in the control group. This result demonstrated that the increased NKG2D expression and imbalance between inhibitory receptors of dNK cells and HLA-G may contribute to abnormal pregnancy outcomes observed upon maternal infection with T. gondii.     

Location of cell cycle regulators cyclin B1, cyclin A, PCNA, Ki67 and cell cycle inhibitors p21, p27 and p57 in human first trimester placenta and deciduas

Although placental development and implantation depend on the coordination of trophoblast proliferation, differentiation and invasion, little is known about the cell cycle regulators that govern the control of these events. The hypothesis that the coordinated expression of cell cycle progression and inhibition factors will determine whether cytotrophoblasts proliferate or undergo cell cycle arrest or cell cycle exit allowing subsequent differentiation was tested. The cell cycle promotors cyclin A, cyclin B1, PCNA, Ki67 and the cell cycle inhibitors p21, p27 and p57 were immunolocalized in tissue sections of first trimester pregnancies (weeks 6 and 9–12). Double staining with cytokeratin 7 allowed unambiguous identification of extravillous cytotrophoblast (EVT) in the decidua. Villous cytotrophoblasts were immunolabelled for Ki67 and cyclin A but only few were stained with anti-cyclin B1. The syncytiotrophoblast was devoid of immunoreactivity for any of the cell cycle progression factors. It expressed especially p21, whereas p27 and p57 were predominantly found in villous cytotrophoblasts. PCNA, Ki67, cyclin A and cyclin B1 were immunolocalized in proximal and distal EVTs of anchoring villi and in EVT which had invaded the upper decidual segments. All EVTs strongly expressed p27 and p57, but not p21. These data clearly suggest different functions for p21, p27 and p57 in placental development with distinct roles for p21 and p57 in syncytiotrophoblast and EVT differentiation, respectively. p27 appears to be involved in both the processes. The results may also challenge the concept of differential mitotic activity in the proximal and distal parts of the first trimester cytotrophoblast cell column, but more functional studies are clearly needed. The presence of p27 and p57 in EVT cells, which invade the deciduas deeply, may account for the loss of mitogenic potential of these cells.     

Tim-3 Is Upregulated in NK Cells during Early Pregnancy and Inhibits NK Cytotoxicity toward Trophoblast in Galectin-9 Dependent Pathway

NK cells accumulate at the maternal-fetal interface (MFI) and play essential roles in maintaining immune tolerance during pregnancy. The mechanisms that facilitate NK cells tolerance to fetal tissue are largely unknown. T cell Ig and mucin domain-containing protein 3 (Tim-3) is a newly defined molecule with essential immunological function in many physiological and pathological processes. Recent study showed that Tim-3 was involved in the regulation of immune tolerance at MFI. However, whether Tim-3 regulates NK cells cytotoxicity toward trophoblasts is unclear. Here, we showed Tim-3 was mainly expressed by decidual NK cells (dNK) and Tim-3 level in dNK was higher than peripheral NK cells (pNK). Tim-3⁺ dNK expressed more levels of mature markers CD94 and CD69 than Tim-3^- dNK cells and blocking Tim-3 significantly inhibited dNK IFN-γ and TNF-α secretion. Furthermore, we found TGF-β1 may contribute to such up-regulation of Tim-3 in NK cells. Interestingly, blocking Tim-3 enhanced NK cytotoxicity toward trophoblast cell line HTR-8 but not K562. We found HTR-8 expressed Tim-3 ligand Galectin-9, in contrast K562 did not. Small interfering RNA-mediated silencing of Galectin-9 expression enhanced NK cytotoxicity toward HTR-8. We further showed Tim-3/Galecin-9 inhibited NK cytotoxicity toward trophoblast partially via impairing the degranulation process. In addition, clinical data showed that abnormal Tim-3 level on pNK might be associated with recurrent spontaneous abortion (RSA). Thus, our data demonstrate Tim-3/Galectin-9 pathway maintains local tolerance by suppressing NK cytotoxicity toward trophoblasts which may represent a new immunologic tolerance mechanism at MFI.     

Immunohistochemical identification of the extravillous trophoblast during the placentation of the degu (Octodon degus)

Recent data indicate that placentation in Octodon degus is similar to that in humans, making it a potential animal model for studies in human placental pathologies related to alterations in the migration of the extravillous trophoblast (EVT). Our objective was to immunohistochemically identify degu EVT during placentation by using cytoskeletal protein markers to establish the normal migratory pattern of the EVT. Fifteen O.degus were divided into three equal groups: day 27, 60, and 84 of gestation. The placentas were immunostained for cytokeratin (CK) and alpha smooth muscle actin (SMA). At day 27, the migrating EVT immunostained for SMA but not for CK. Once the EVT was incorporated in the maternal vessels (day 60) it was positive for CK but negative for SMA. The smooth muscle cells of the mesometrial arteries that remained after EVT invasion were positive for SMA. At day 84, the media muscular layer had partially regenerated but some EVT was still present. Furthermore, at day 27 cyclooxygenase-1 (COX-1) was detected in the endothelium of the maternal decidual vessels. Our results suggest that during the early stages of placentation, the cytoskeletal organization of the actin network of the migrating EVT corresponds to that of a cell with motile behavior. Once the EVT invaded the spiral arteries, the cytoskeleton reorganized, adopting the structure of an epithelial-like cell, expressing CK intermediate filaments. The media muscle layer regenerated near the end of gestation but some EVT remained. During EVT formation the endothelium of the maternal decidual vessels immunostained for COX-1.     

Gestational regulation of granulocyte-colony stimulating factor receptor expression in the human placenta

A number of cytokines and their receptors are abundantly expressed at the materno-fetal interface and are thought to have a function in the regulation of placentation. Granulocyte-colony stimulating factor (G-CSF) is expressed by stromal cells in both placental tissue and maternal decidua throughout placentation. In this study, we examined the expression of placental G-CSF receptor (G-CSFR) mRNA and protein throughout gestation by ribonuclease protection assays, Western blotting, and immunohistochemistry. The major placental form of G-CSFR mRNA, corresponding to a membrane-bound form of the protein, was present in first-trimester placental tissues; levels decreased in second- and were highest in third-trimester placental tissues. Two placental G-CSFR molecules, 120 kDa and 150 kDa, were detected in first- and third-, but not second-, trimester tissues. The level of the 150-kDa G-CSFR was greater in the third- than in first-trimester samples. These differences were irrespective of whether or not the patients had received prostaglandin E1 analogues, prostaglandin E1 analogues and oxytocin, oxytocin alone, or mifepristone before labor. We demonstrated by immunohistochemistry that interstitial cytotrophoblast in first- and second-trimester decidual tissue and cytotrophoblast in term fetal membranes express G-CSFR. These data demonstrate that the expression of specific forms of placental G-CSFR is strictly cell type- and developmental stage-specific, and they suggest that G-CSFR may be important in decidual invasion of cytotrophoblast and in trophoblast function during placentation.     

Decorin is a novel VEGFR-2-binding antagonist for the human extravillous trophoblast

Extravillous trophoblasts (EVT) of the human placenta invade the uterine decidua and its arteries to ensure successful placentation. We previously identified two decidua-derived molecules, TGF-β and a TGF-β-binding proteoglycan decorin (DCN), as negative regulators of EVT proliferation, migration, and invasiveness and reported that DCN acts via multiple tyrosine kinase receptors [epidermal growth factor-receptor (EGF-R), IGF receptor-1 (IGFR1), and vascular endothelial growth factor 2 receptor (VEGFR-2)]. Because binding of DCN to VEGFR-2 has never been reported earlier, present study explored this binding, the approximate location of VEGFR-2-binding site in DCN, and its functional role in our human first trimester EVT cell line HTR-8/SVneo. Based on far-Western blotting and coimmunoprecipitation studies, we report that DCN binds both native (EVT expressed) and recombinant VEGFR-2 and that this binding is abrogated with a VEGFR-2 blocking antibody, indicating an overlap between the ligand-binding and the DCN-binding domains of VEGFR-2. We determined that (125)I-labeled VEGF-E (a VEGFR-2 specific ligand) binds EVT with a dissociation constant (K(d)) of 566 pM, and DCN displaced this binding with an inhibition constant (K(i)) of 3.93-5.78 nM, indicating a 7- to 10-fold lower affinity of DCN for VEGFR-2. DCN peptide fragments derived from the leucine rich repeat 5 domain that blocked DCN-VEGFR-2 interactions or VEGF-E binding in EVT cells also blocked VEGF-A- and VEGF-E-induced EVT cell proliferation and migration, indicative of functional VEGFR-2-binding sites of DCN. Finally, DCN inhibited VEGF-E-induced EVT migration by interfering with ERK1/2 activation. Our findings reveal a novel role of DCN as an antagonistic ligand for VEGFR-2, having implications for pathophysiology of preeclampsia, a trophoblast hypoinvasive disorder in pregnancy, and explain its antiangiogenic function.     

Killer Ig-like receptor expression in uterine NK cells is biased toward recognition of HLA-C and alters with gestational age

Immunogenetic studies suggest that interactions between maternal killer Ig-like receptor (KIR) expressed by uterine NK (uNK) cells, and fetal HLA-C molecules on trophoblast, influence the success of human placentation. However, the exact functional response of fresh uNK cells to trophoblast HLA-C molecules is unknown. In this study, we show by quantitative RT-PCR and FACS that both activating and inhibitory KIR specific for HLA-C are expressed at higher levels and on an increased proportion of NK cells in the human decidua compared with blood. In contrast, expression of KIR3DL1/S1, which is specific for HLA-B, is similar in both NK cell populations. Remarkably, there is also a temporal change in the expression pattern of HLA-C-specific KIR, with a decline in both intensity of expression and frequency on uNK cells throughout the first trimester of pregnancy. This selective up-regulation of KIR has functional consequences because uNK cells show increased binding of HLA-C tetramers compared with blood NK cells. Ab cross-linking shows that these KIR are functional and results in increased cytokine secretion. uNK cells, therefore, exhibit a unique KIR profile that enhances their ability to recognize trophoblast cells expressing HLA-C at the materno-fetal interface. This is the first report to demonstrate selective regulation of KIR expression over time in vivo in a normal physiological situation and suggests that KIR expression by uNK cells is regulated by the tissue microenvironment in the decidua.     

Intact caveolae are required for proper extravillous trophoblast migration and differentiation

Caveolae constitute membrane domains critical for the organization and synchronization of different signaling molecules related to numerous cell processes such as cell migration, invasion, and differentiation. Caveolin-1 (Cav-1) is the main integral membrane protein of these domains. Recently, it was found that a normal expression of aquaporin-3 (AQP3) is required for extravillous trophoblast (EVT) cell migration. Our aim was to investigate the role of caveolae in the migration, invasion, and endovascular differentiation of human EVT cells during placentation and its interaction with AQP3. EVT cells (Swan 71 cell line) were cultured in complete Dulbecco's modified Eagle's medium–nutrient mixture F12 and treated with 5 mM methyl-β-cyclodextrin (MβCD) to disrupt caveolae. We found that after MβCD treatment, Cav-1 protein was undetectable. In this condition, the ability of the cells to migrate was significantly decreased compared with the control cells, while no differences were observed in the number of invading cells and the metalloproteinases activity between control and MβCD-treated cells. Surprisingly, the disruption of caveolae significantly enhanced EVT endovascular differentiation. On the contrary, the silencing of AQP3, negatively affected tube-like formation. The theoretical analysis of the primary sequence of AQP3 protein revealed a putative Cav-1-binding site. In addition, immunoprecipitation and double immunofluorescence assays showed that AQP3 colocalized with Cav-1. These results showed that during placentation an intact caveola in EVT cells may be necessary for AQP3 and Cav-1 interaction and any perturbations might result in serious pregnancy disorders.     

Differential expression of the receptors for epidermal growth factor and insulin in the developing human placenta

The detailed cellular distribution of epidermal growth factor (EGF) receptors and insulin receptors during the development of the human placenta was examined. We show that EGF receptors are expressed by villous cytotrophoblast cells in first trimester human placentae. However, where these cells proliferate to form extravillous cytotrophoblast cell columns, there is a dramatic decrease in EGF receptor expression. There is no such differential expression of insulin receptors on this cell population. In contrast, both EGF-and insulin-receptors are present throughout gestation on the microvillous membrane of the terminally differentiated and non-proliferative syncytiotrophoblast although, at term, EGF-but not insulin-receptors are also found on the basolateral membrane of this epithelium. We further show that EGF receptors isolated from first trimester and term human placentae have functional tyrosine kinase activities but differ in their extent of glycosylation. These results suggest that EGF receptors probably play several distinct functional roles in these epithelial cells depending on their proliferative capacity and differentiation status.     

Possible role of RKIP in cytotrophoblast migration: immunohistochemical and in vitro studies

Raf kinase inhibitor protein (RKIP) regulates growth and differentiation signaling of mitogen-activated protein kinases (MAPK), GRK2 and NF-kappaB pathways each of which regulates cytotrophoblast differentiation and normal placental development. We show here that RKIP is expressed in human normal and preeclampic placentas as detected by immunostaining. RKIP was detected in villous cytotrophoblast in normal placenta and switched to syncytiotrophoblast in pre-eclampsia (PE)-complicated pregnancies. RKIP was also localized in extravillous cytotrophoblast of cell islands and cell columns both in normal and in PE placentas, although staining was less uniform in the latter specimens. In order to test RKIP involvement in cytotrophoblast function, we performed in vitro studies on HTR-8/SVneo cells, a first trimester cytotrophoblast cell line. We show that the RKIP inhibitor locostatin reduces ERK phosphorylation and impairs HTR-8/SV neo cells motility in wound closure experiments. We also document the presence of GRK2 mRNA, the reduction of phosphorylated RKIP expression by locostatin and the induction of PAI mRNA expression in HTR-8/SV neo cells, suggesting the involvement of GRK2 and NF-kappaB pathways in these cells. In conclusion, our work provides evidence that RKIP is a novel factor expressed in cytotrophoblast cells where it likely regulates cell migration.     

CUL1 promotes trophoblast cell invasion at the maternal–fetal interface

Human trophoblast progenitor cells differentiate via two distinct pathways, to become the highly invasive extravillous cytotrophoblast (CTB) cells (EVT) or fuse to form syncytiotrophoblast. Inadequate trophoblast differentiation results in poor placenta perfusion, or even complications such as pre-eclampsia (PE). Cullin1 (CUL1), a scaffold protein in cullin-based ubiquitin ligases, plays an important role in early embryonic development. However, the role of CUL1 in trophoblast differentiation during placenta development has not been examined. Here we show that CUL1 was expressed in CTB cells and EVT in the first trimester human placentas by immunohistochemistry. CUL1 siRNA significantly inhibited outgrowth of extravillous explants in vitro, as well as invasion and migration of HTR8/SVneo cells of EVT origin. This inhibition was accompanied by decreased gelatinolytic activities of matrix metalloproteinase (MMP)-9 and increased expression of tissue inhibitors of MMPs (TIMP-1 and -2). Consistently, exogenous CUL1 promoted invasion and migration of HTR8/SVneo cells. Notably, CUL1 was gradually decreased during trophoblast syncytialization and CUL1 siRNA significantly enhanced forskolin-induced fusion of choriocarcinoma BeWo cells. CUL1 protein levels in human pre-eclamptic placental villi were significantly lower as compared to their matched control placentas. Taken together, our results suggest that CUL1 promotes human trophoblast cell invasion and dysregulation of CUL1 expression may be associated with PE.     

Promoter Hypomethylation of Maspin Inhibits Migration and Invasion of Extravillous Trophoblast Cells during Placentation

Objective

Extravillous trophoblast (EVT) cells invade the endometrium and the maternal spiral arterioles during the first trimester. Mammary Serine Protease Inhibitor (Maspin, SERPINB5) plays a putative role in regulating the invasive activity of cytotrophoblasts. The maspin gene is silenced in various cancers by an epigenetic mechanism that involves aberrant cytosine methylation. We investigated the effect of the methylation status of the maspin promoter on the maspin expression and the aggressiveness of EVT cells.

Methods

Western blotting was used to detect the maspin protein expression in EVT cells upon hypoxia. The proliferative ability, the apoptosis rate and the migration and invasiveness were measured with Cell Counting Kit-8 assay, Flow Cytometry technology and Transwell methods. Subsequently, we treated cells with recombinant maspin protein. The methylation degree of maspin promoter region upon hypoxia/ decitabine was detected by bisulfite sequencing PCR and methylation-specific PCR. Finally, we explored the effects of decitabine on maspin protein expression and the aggressiveness of EVT cells.

Results

Hypoxia effectively increased maspin protein expression in EVT cells and significantly inhibited their aggressiveness. The addition of recombinant maspin protein inhibited this aggressiveness. Decitabine reduced the methylation in the maspin promoter region and effectively increased the maspin protein expression, which significantly weakened the migration and invasiveness of EVT cells.

Discussion

The methylation status of the maspin promoter is an important factor that affects the migration and invasion of EVT cells during early pregnancy. A decrease in the methylation status can inhibit the migration and invasion of EVT cells to affect placentation and can result in the ischemia and hypoxia of placenta.     

Distribution of mosaicism in human placentae

Traditional first trimester chorionic villus sampling (CVS) for prenatal diagnosis can be performed by cytogenetic analysis of cytotrophoblast or chorionic villous stroma. Approximately 2% of pregnancies studied by CVS show confined placental mosaicism (CPM) involving either cytotrophoblast, stroma or both. We present the results of a cytogenetic study of nine term placentae from pregnancies with prenatally diagnosed CPM. The aneuploid cell lines involved trisomies for chromosomes 7,9,16, and X. The cytotrophoblast and villous stroma from multiple biopsies of these placentae were examined using a combination of interphase and metaphase cytogenetic analysis. CPM was detected in all nine of the term placentae and both tissue-specific and site-specific patterns of mosaicism could be discerned. These results indicate that the analysis of villous stroma and cytotrophoblast from multiple placental biopsies is necessary to improve our understanding of the evolution of CPM during pregnancy and its effect on the fetus. Received: 1 May 1995 / Revised: 11 August 1995