Aberrations of early trophoblast differentiation predispose to pregnancy failure: lessons from the anti-phospholipid syndrome

OBJECTIVES: The epithelium of the human placenta comprises an inner cytotrophoblast (CT) which proliferates and fuses with the outer differentiated syncytiotrophoblast (ST). Turnover has been studied focussing on second and third trimester placentas but with a paucity of data describing the normal first trimester trophoblast. The aim of this study was to compare the nuclear CT:ST ratio in normal and pathological pregnancy and thus establish the relationship between cytotrophoblast and syncytiotrophoblast nuclear number during early gestation. METHODS: Archival first trimester material from placentas from healthy pregnancy and recurrent miscarriage (anti-phospholipid syndrome) was stained with H&E, cytokeratin-7 and Mib-1. The area of trophoblast as a fraction of total villous area was calculated and the number of sectioned cytotrophoblast and syncytiotrophoblast nuclei as well as the number of proliferating cytotrophoblast was evaluated. RESULTS: Normal features of trophoblast development during the first trimester (rise in trophoblast area, increase in number of syncytiotrophoblast nuclei, increase in number of proliferating cytotrophoblast, decrease in the nuclear CT:ST ratio) are absent/reversed in tissues from recurrent miscarriage (decreasing trophoblast area, constant number of syncytiotrophoblast nuclei, decreasing number of proliferating trophoblast, constant nuclear CT:ST ratio). CONCLUSIONS: Proliferation of cytotrophoblast in early gestation provides a pool of trophoblast stem cells critical for ongoing placental development. Premature cytotrophoblast differentiation in favour of syncytial fusion results in deficiencies of cytotrophoblast and rarification of villous trophoblast. Abnormal trophoblast differentiation in early gestation may be due to a premature onset of maternal perfusion of the placenta and may be a likely antecedent for conditions associated with failure of placentation such as recurrent miscarriage.     

The product of the imprinted gene IPL marks human villous cytotrophoblast and is lost in complete hydatidiform mole

The IPL/TSSC3 gene is expressed nearly exclusively from the maternal allele, and its protein product acts to limit placental growth in mice. This protein specifically marks Type II trophoblast in the labyrinthine layer of the mouse placenta. To investigate mouse-human homologies, we carried out immunohistochemistry with antibodies against human IPL. There was strong expression of IPL in villous cytotrophoblast of the human placenta, contrasting with complete lack of expression in syncytiotrophoblast. Staining for IPL was weak in cells of the villous mesenchyme and extravillous trophoblast, including the cytotrophoblast columns in the basal plate and the intervillous trophoblast islands. The IPL and p57(KIP2)/CDKN1C genes are closely linked and coordinately imprinted, and immunostaining showed that their protein products are co-expressed in villous cytotrophoblast. However, other cell types, including extravillous cytotrophoblast and cells in various non-placental tissues, expressed p57(KIP2), but not IPL. IPL protein was absent in both of two cases of androgenetic complete hydatidiform mole examined by immunostaining, and IPL mRNA was absent in an additional three cases of this neoplasm examined by northern blotting. In the mouse, Ipl-expressing cells disappear at mid- to late-gestation when placental growth ceases, but persistent IPL mRNA and protein expression was observed throughout human gestation, correlating with the continuous growth of the human placenta. These findings highlight dosage regulation of human IPL by imprinting and, more generally, suggest homology between Type II labyrinthine trophoblast in the mouse and villous cytotrophoblast in humans, both of which are proliferative stem cell-like compartments.     

Oxygen tension directs the differentiation pathway of human cytotrophoblast cells

During placental development, human cytotrophoblast cells can differentiate to either villous syncytiotrophoblast cells or invasive extravillous trophoblast cells. We hypothesize that oxygen tension plays a critical role in determining the pathway of cytotrophoblast differentiation. A highly purified preparation of cytotrophoblast cells from human third trimester placenta was cultured for 5 days in either 20% or 1% oxygen tension. The cells incubated at 20% oxygen formed a syncytium as determined by immunohistochemistry using an anti-desmosomal protein antibody that identifies cell membranes. In addition, the mRNA was markedly induced for syncytin, a glycoprotein shown to be essential for syncytiotrophoblast formation, and for human placental lactogen (hPL), which is a specific marker for syncytiotrophoblast cells. In contrast, the cell incubated at 1% oxygen tension did not fuse by morphologic analysis and did not express syncytin or hPL mRNA. However, these cells expressed abundant amounts of HLA-G, a specific marker for extravillous trophoblast cells, which was not seen in cells incubated at 20% oxygen tension. These results suggest that low oxygen tension directs differentiation along the extravillous trophoblast cell pathway while greater oxygen tension directs differentiation along the villous trophoblast cell pathway.     

A positive immunoselection method to isolate villous cytotrophoblast cells from first trimester and term placenta to high purity

We developed a method for isolating highly pure villous cytotrophoblast cells from first trimester and term placenta that excludes extravillous trophoblast and syncytiotrophoblast fragments. The method is based on positive immunoselection using an antibody (mAb C76/18) reacting with hepatocyte growth factor activator inhibitor 1, HAI-1, a membrane antigen on villous cytotrophoblast. As a comparison, we also immunopurified cells using an antibody against CD105, present on syncytiotrophoblast and some extravillous trophoblast cells. The isolates were characterized by flow cytometry. HAI-1-positive cells from first trimester and term placentae were highly pure (>98 per cent cytokeratin 7-positive) mononuclear trophoblast cells. These isolations were contaminated with only very small percentages of vimentin and CD45-positive cells. HAI-1-positive trophoblast cells lacked CD105 and also HLA class I, a marker for extravillous trophoblast. In culture HAI-1-positive cells adhered, displayed an epithelial morphology, and survived for more than three days. In contrast, CD105-positive cell fractions from first trimester placenta were a heterogeneous mixture of mononuclear and multinuclear elements consisting of syncytiotrophoblast fragments, extravillous trophoblast cells, as well as around 5 per cent non-trophoblastic contaminants. In conclusion, the positive immunoselection method using antibody C76/18 yielded highly pure villous cytotrophoblast cells devoid of elements derived from syncytiotrophoblast or extravillous trophoblast.     

Expression of Fas-ligand in first trimester and term human placental villi

The expression of Fas-ligand (FasL) on trophoblast cells is thought to play a role in immune regulation during human pregnancy. However, there are some discrepancies in the published data concerning the cell types expressing FasL in the placental villi. Therefore, we examined the expression of FasL on cryosections of first trimester and term placental tissue with three different anti-sera against FasL, which are in common use. By immunohistochemistry, all three anti-sera principally gave the same staining result. In the first trimester of pregnancy, villous cytotrophoblast cells underlying the syncytium, as well as all extravillous trophoblast cells of cell columns and cell islands, gave a clear, mainly membrane-located staining, whereas the syncytiotrophoblast, which forms the borderline to the maternal blood flow, only gave a spot-like reaction in distinct areas. The same result was obtained with term placental villi; however, in this tissue, the staining of the villous cytotrophoblast cells was less pronounced. From our results, we suggest that in placental villi, an important role of FasL in immune regulation is not very conclusive because this molecule is mainly expressed on trophoblast with no access to maternal blood or tissue. This is in contrast to the uterine part of the placenta, where FasL expressing trophoblast cells are in close contact with apoptotic maternal leukocytes.     

Detection of class I MHC mRNA in subpopulations of first trimester cytotrophoblast cells by in situ hybridization

Class I MHC mRNA has been identified in both villous and extravillous cytotrophoblast cells in first trimester placentas by in situ hybridization. In this report, we expand those observations to additional morphologically and anatomically distinct subpopulations of trophoblast cells in early placentas using the same experimental approach. In the transition zone of first trimester placental villi, where cytotrophoblast cells are proliferating to form new villi or to migrate into adjacent tissue, both cytotrophoblast cells beneath the uninterrupted syncytium and the proliferating cytotrophoblast cells contained class I mRNA whereas a layer of cytotrophoblast cells proximal to the villus core did not contain class I mRNA. In the placental bed, migrating cytotrophoblast cells contained high levels of class I mRNA as determined by the intensity of staining. In contrast, multinucleated giant trophoblast cells contained little specific message. Alterations in levels of class I mRNA seem therefore to be associated with trophoblast proliferation, migration and differentiation.     

Placental glucose transporter 3 (GLUT3) is up-regulated in human pregnancies complicated by late-onset intrauterine growth restriction

Introduction

Transport of glucose from maternal blood across the placental trophoblastic tissue barrier is critical to sustain fetal growth. The mechanism by which GLUTs are regulated in trophoblasts in response to ischemic hypoxia encountered with intrauterine growth restriction (IUGR) has not been suitably investigated.

Objective

To investigate placental expression of GLUT1, GLUT3 and GLUT4 and possible mechanisms of GLUT regulation in idiopathic IUGR.

Methods

We analyzed clinical, biochemical and histological data from placentas collected from women affected by idiopathic full-term IUGR (n = 10) and gestational age-matched healthy controls (n = 10).

Results

We found increased GLUT3 protein expression in the trophoblast (cytotrophoblast greater than syncytiotrophoblast) on the maternal aspect of the placenta in IUGR compared to normal placenta, but no differences in GLUT1 or GLUT4 were found. No differential methylation of the GLUT3 promoter between normal and IUGR placentas was observed. Increased GLUT3 expression was associated with an increased nuclear concentration of HIF-1α, suggesting hypoxia may play a role in the up-regulation of GLUT3.

Discussion

Further studies are needed to elucidate whether increased GLUT3 expression in IUGR is a marker for defective villous maturation or an adaptive response of the trophoblast in response to chronic hypoxia.

Conclusions

Patients with IUGR have increased trophoblast expression of GLUT3, as found under the low-oxygen conditions of the first trimester.     

Human trophoblast cells express the immunomodulator progesterone-induced blocking factor

Progesterone-induced blocking factor (PIBF) is an immunomoduatory factor with anti-abortive properties. In this study, we present evidence that PIBF is synthesized in the human placenta and determine its cellular source. Expression of PIBF was analysed with polyclonal rabbit anti-human PIBF antibodies against recombinant N-terminal 48kDa PIBF in first trimester and term placental tissues and in the choriocarcinoma cell line JAR by means of immunohistochemistry, confocal laser scanning microscopy of double immunofluorescence labelling, and Western blotting; RT-PCR was performed for analysis of PIBF mRNA in isolated trophoblast cells. PIBF protein is present in human first trimester and term placenta. Double immunofluorescence labelling localised PIBF to the extravillous cytotrophoblast. PIBF is also expressed heterogeneously by syncytiotrophoblast and part of the villous cytotrophoblast. Full-length PIBF mRNA encoded by exons 1-18 is present in isolated first trimester and term villous trophoblast and in the choriocarcinoma cell line JAR. The corresponding 90kDa protein is expressed by JAR cells, first trimester and term villous trophoblast cells. In addition, these cells express PIBF proteins of 50 and 34kDa. Trophoblast is a source of PIBF; its tissue distribution suggests a role both in systemic and local (decidual) immunoregulation.     

Immunohistologic identification of trophoblast populations in early human pregnancy with the use of monoclonal antibodies

Three murine monoclonal antibodies (H315, H316, and NDOG1) have been used in a peroxidase-antiperoxidase technique on formalin-fixed paraffin-embedded tissues to identify populations of fetal trophoblast cells by their expression of membrane antigens in chorionic and decidual tissue from the first trimester of normal human pregnancy. H315 and H316 showed comparable staining of placental villous syncytiotrophoblast and cytotrophoblast and were also able to distinguish subpopulations of nonvillous trophoblast in the placental bed, including perivascular and endovascular trophoblastic cells as well as cytotrophoblastic elements within the decidua and myometrium. H315 and H316 also showed cytoplasmic staining of columnar epithelium of endometrial glands throughout the first trimester. In contrast, NDOG1 stained chorionic syncytiotrophoblast but not villous cytotrophoblast and also did not react with any cytotrophoblastic elements in the placental bed. NDOG1 distinguished these different subpopulations of trophoblast as early as 13 to 15 days after ovulation.     

An image analysis technique for the investigation of variations in placental morphology in pregnancies complicated by preeclampsia with and without intrauterine growth restriction

OBJECTIVE: The purpose of this study was to use visual image analysis to observe changes in the morphology and composition of placental villi in pregnancies complicated by preeclampsia (PE) and intrauterine growth restriction (IUGR). METHODS: Placental biopsies from nine normal pregnancies, five cases of PE, five cases of IUGR, and five cases of PE with IUGR (PE x IUGR) were randomly sampled. Formalin-fixed, wax-embedded sections were stained with hematoxylin and eosin (H&E) and subjected to image analysis. The placental areas occupied by villi, syncytiotrophoblast, and syncytial cytoplasm and nuclei were quantified. RESULTS: Significantly smaller placentas were obtained from growth-restricted pregnancies. PE, with and without IUGR, had no effect on the total area occupied by villi or intervillous space. IUGR alone showed a real and consistent reduction in villous area (56.0 +/- 2.4% vs 43.6 +/- 3.3%, P <.03). While the ratio of syncytial to villous areas were noticeably reduced in all cases of PE (0.38 +/- 0.03 vs 0.24 +/- 0.07, P <.05), this ratio remained unchanging in IUGR. Birth weight was positively correlated to both placental size and total villous area occupied. Moreover, increasingly positive relationships were recorded between both syncytiotrophoblast area and syncytiotrophoblast cytoplasm and birth weight (P <.01 and P <.001, respectively). CONCLUSION: These measurements point to impoverished villus development in idiopathic IUGR. The observed changes in PE with IUGR were more akin to PE without growth restriction than IUGR alone. This suggests that idiopathic IUGR and IUGR in PE have a separate etiology, idiopathic IUGR arising through a reduction in villous area alone, and IUGR in PE caused by changes in syncytiotrophoblast quantity, more specifically the amount of syncytiotrophoblast cytoplasm.     

Cullin 7 and Fbxw 8 expression in trophoblastic cells is regulated via oxygen tension: implications for intrauterine growth restriction?

Objective: The F-box protein Fbxw8 is a cofactor of Cullin 7 (Cul7), which regulates protein transfer to the proteasome and cell growth. Cul7 or Fbxw8 deficiency is associated with intrauterine growth restriction (IUGR) due to abnormal placental development leading to poor oxygen supply to the fetus. We studied the role of hypoxia for Fbxw8 and Cul7 expression in trophoblastic cells. Methods: Immunomagnetic bead-separated extravillous trophoblast (EVT) and villous trophoblast (VT) and trophoblast cell lines were incubated with 1 or 8% O₂. Fbxw8 and Cul7 expression was determined in IUGR versus matched control placentas. Results: Fbxw8 was expressed uniformly in trophoblasts, whereas Cul7 expression was most prominent in trophoblast cell lines. Hypoxia reduced expression of Cul7 and Fbxw8 in all trophoblastic cells, except for villous trophoblasts. In vivo, Cul7 and Fbxw8 were detected in syncytiotrophoblast cells, VT, and EVT cells. Although no significant changes in expression levels of Fbxw8 or Cul7 were noted in IUGR compared with control placentas, Fbxw8 expression correlated negatively with gestational age in the control, but not in the IUGR group. Conclusion: Fbxw8 and Cul7 expression reveals a complex regulation in trophoblastic cells. Our findings suggest that dysregulation of Cul7 and Fbxw8 expression might affect trophoblast turnover in IUGR.     

Villous trophoblast abnormalities in extremely preterm deliveries with elevated second trimester maternal serum hCG or inhibin-A

Elevated levels of the maternal prenatal screening markers hCG and inhibin-A, measured at 15-20 weeks gestation, increase the subsequent risk of severe pre-eclampsia and intra-uterine growth restriction (IUGR). Since both markers are produced by syncytiotrophoblast, we tested the hypothesis that these elevations were due to accelerated differentiation of the villous trophoblast compartment. We performed a retrospective study of 12 cases from our Placenta Clinic with total hCG and/or inhibin-A levels of ≥3.0 multiples of the median that subsequently delivered by 28 weeks gestation and compared their placental pathology findings with 24 gestational age-matched controls. Morphometric analysis demonstrated a 41% reduction in the volume ratio of Ki67 positive cytotrophoblast nuclei to total trophoblast in cases vs controls (Student’s T-test; p = 0.028). Distal villous hypoplasia (DVH) was significantly more common in cases (10/12) than controls (4/24); Fisher’s exact test, p = 0.002. Wave-like syncytial knot (WLSK) formation was significantly more common in cases (9/12) than controls (1/24); Fisher’s exact test, p < 0.0001. WLSK formation was associated with DVH and resulted from accumulation of senescent/apoptotic syncytiotrophoblast nuclei along inherent lines of syncytial nuclear organization. Our data support the hypothesis that elevated second trimester maternal serum levels of total hCG and/or inhibin-A may result from premature accelerated differentiation of the villous cytotrophoblasts. The subsequent pathologic findings in the syncytiotrophoblast could render the pregnancy at risk of severe pre-eclampsia and IUGR.     

The placenta in pre-eclampsia and intrauterine growth restriction: studies on exchange surface areas, diffusion distances and villous membrane diffusive conductances

We test the null hypothesis that the morphometric diffusive conductance of the placental villous membrane does not alter in pregnancies complicated by intrauterine growth restriction (IUGR) or pre-eclampsia (PE). Placentas were collected from cases of normotensive IUGR, pure PE, PE+IUGR and from control pregnancies. Microscopical fields on formalin-fixed, trichrome-stained histological sections were randomly sampled for location and orientation. Using stereological methods, the exchange surface areas of peripheral (terminal and intermediate) villi and their fetal capillaries and the arithmetic and harmonic mean thicknesses of the villous membrane (maternal aspect of trophoblast to luminal aspect of vascular endothelium) were estimated. An index of the variability in thickness of this membrane, and an estimate of its oxygen diffusive conductance, was derived secondarily. Group comparisons were drawn using two-way analysis of variance to identify main effects (of PE or IUGR) and interaction effects (between PE and IUGR). PE did not have significant effects on placental morphology and there were no significant effects of PE or IUGR on membrane thickness or its variability. In contrast, IUGR (with or without PE) was associated with reduced surface areas and this was the principal factor leading to a smaller membrane diffusive conductance in these placentas. When account was taken of fetal mass, specific conductance showed no effects of PE or IUGR despite the mass-specific conductance in pure IUGR placentas appearing to be smaller than that in controls. The decline in total conductances is indicative of perturbations operating at the levels of villous trophoblast and fetal vasculature and these may contribute to fetal hypoxic stress.     

Double Immuno-labelling of Proliferating Villous Cytotrophoblasts in Thick Paraffin Sections: Integrating Immuno-histochemistry and Stereology in the Human Placenta

In order to understand the pathological basis of abnormal villous trophoblast development in diseased placentas, the organ must be sampled by non-biased methods and subject to analysis by stereological tools. This approach permits quantification of cytotrophoblast density and syncytiotrophoblast structure including evidence of apoptotic shedding via syncytial knots. The stereological quantification of cells (or their) nuclei requires that each should be unambiguously identified and counted within a defined volume of tissue. A major limitation of such studies at present is the inability to accurately identify and phenotype subsets of villous cytotrophoblasts that either proliferate or are destined to fuse into the overlying syncytiotrophoblast.We describe the development of a novel double immuno-labelling protocol to selectively identify proliferating villous cytotrophoblast cells in human placental villi using thick (25 μm) paraffin sections suitable for stereological quantification. Cytotrophoblast cells were selectively stained using a monoclonal anti-cytokeratin 7 (CK 7) antibody without antigen retrieval, followed by nuclear Ki-67 co-localisation. Both antibodies displayed full depth penetration with sharp, clearly defined staining precipitates and no cross-reactivity. This double immuno-labelling protocol is reproducible, cost effective and time efficient (8 h). Use of a variety of antibodies following antigen retrieval will be a significant advancement in the ability to accurately quantify sub-populations of villous cytotrophoblast in normal and pathological placentas.     

Carcinoembryonic antigen and related molecules in normal and transformed trophoblast

Carcinoembryonic antigen (CEA, CD66e) and CEA-related cell adhesion molecules (CEACAMs) are important mediators in remodeling of diverse human tissues, and modulators of cell proliferation and differentiation. Expression by normal and transformed trophoblast of gestational trophoblastic diseases (GTDs), isolated cytotrophoblast and choriocarcinoma cell lines is presented here. Immunocyto/histochemistry of normal placenta (n=9), invasive mole (n=8), choriocarcinoma (n=7), a placental site trophoblastic tumor, cytotrophoblast in primary culture and JAr and JEG-3 cells was performed using polyclonal anti-CEA and specific monoclonal anti-CEA antibodies. Data were analyzed and scored using Mann-Whitney Test. CEA and CEA-related molecules were identified by Western blot and immunoaffinity chromatography in JAr and JEG-3 cells and extracts of 1st and 3rd trimester of pregnancy tissue and cytotrophoblast cell lysates. CEA is expressed throughout pregnancy, in first trimester predominantly in syncytiotrophoblast, but also in villous cytotrophoblast and extravillous trophoblast. Data presented here demonstrate that CEA is significantly increased in transformed trophoblast of GTDs (p<0.05). Both cytotrophoblast in primary culture and choriocarcinoma cell lines express CEA, with staining of granular deposits in JAr and cell membrane in JEG-3. The results suggest that CEA (CD66e) and other CEA-related protein(s) could be involved in trophoblast differentiation.     

Fas and FasL expression in placentas complicated with intrauterine growth retardation with and without preeclampsia

Objective: To compare the level of Fas and FasL immunohistochemical expression in villous trophoblast (VT), extravillous trophoblast (EVT) cells, decidual cells (DC), endothelial cells (EC) of villous blood vessels and spiral arteries between the study groups of intrauterine growth retardation (IUGR) placentas with and without preeclampsia (PE).

Methods: The study included 17 placentas from pregnancies complicated by IUGR?+?PE and 17 placentas from pregnancies complicated by idiopathic IUGR (I-IUGR). Seventeen placentas from normal pregnancies served as a control group. CD31 was used to detect endothelial cells (EC). Immunohistochemical expression of Fas and FasL was assessed in all examined parts of placenta using the semi-quantitative HSCORE method.

Results: FasL expression was significantly higher in all examined parts of placenta in I-IUGR as compared to IUGR?+?PE and control group. Placentas with IUGR?+?PE had the significantly lowest expression of FasL in VT and EC of villi vessels. Expression of Fas did not differ significantly between the study groups.

Conclusion: Different expression of FasL in placentas from I-IUGR and IUGR?+?PE suggests that FasL probably has a different role in the etiology of these two syndromes.     

Circulating and placental endoglin concentrations in pregnancies complicated by intrauterine growth restriction and preeclampsia

Inadequate trophoblast invasion and spiral artery remodeling leading to poor placental perfusion and hypoxia are believed to underlie preeclampsia (PE) and intrauterine growth restriction (IUGR). Recent studies implicate increased circulating endoglin as a contributor to the pathogenesis of PE. The objective of this study was to determine whether placental and circulating endoglin concentrations are altered in pregnancies complicated by intrauterine growth restricted (IUGR) infants and to address the role of hypoxia on the regulation of placental endoglin. We analyzed 10 placentas each from normal pregnant (NP), PE, and IUGR subjects. Endoglin levels were 2.5-fold higher in preeclamptic placentas compared to NP (15.4+/-2.6 versus 5.7+/-1.0, p<0.01). In contrast, endoglin levels were similar in NP and IUGR placentas (5.7+/-1.0 vs 5.9+/-1.1, p=NS). Placentas from pregnancies with both PE and IUGR exhibited endoglin levels comparable to the PE group and significantly different from normotensive pregnancies with and without IUGR pregnancies (mean 14.9+/-4.0, n=9, p=0.013). Soluble endoglin concentrations in maternal plasma were comparable in NP and IUGR, but higher in women with PE (n=10 per group, p<0.05). Despite a 2-fold increase in hypoxia inducible factor, HIF-1alpha, we did not observe endoglin upregulation in NP, PE, or IUGR placental villous explants exposed to hypoxia (2% oxygen). In contrast to PE, placental or circulating endoglin is not increased in normotensive women delivering small, asymmetrically grown (IUGR) infants at term. The placentas of women with IUGR appear to be fundamentally different from PE women with respect to endoglin, despite the proposed common pathology of deficient trophoblast invasion/spiral artery remodeling and poor placental perfusion.     

Expression and cellular localisation of chloride intracellular channel 3 in human placenta and fetal membranes

Chloride channels regulate the movement of a major cellular anion and are involved in fundamental processes that are critical for cell viability. Regulation of intracellular chloride is achieved by multiple classes of channel proteins. One class of putative channels are the chloride intracellular channel (CLIC) family. Evidence suggests that several CLICs are expressed in human placenta, although their roles in this tissue are not certain. Northern blot analysis has shown that CLIC3 is highly expressed in placenta relative to other human tissues; however, its cellular distribution is not known. This study used microarray expression profiling to clarify which CLICs are expressed in human placenta and RT-PCR, Western blot and immunohistochemistry to determine the expression pattern of CLIC3 in human placenta and fetal membranes. Placentas and fetal membranes were obtained from term pregnancies after delivery and placental tissue was obtained from first trimester following either chorionic villous sampling or elective pregnancy termination. Trophoblast cells were isolated from first trimester and term placentas and placental endothelial cells were isolated from term placentas. Microarray expression profiling identified high expression of mRNA for CLICs 1, 3 and 4 in the isolated first trimester and term trophoblast cells. High mRNA expression in the isolated endothelial cells was also found for CLICs 1 and 4, but not CLIC3. Low expression was found for CLIC5 in all three types of isolated cells. RT-PCR confirmed that CLIC3 mRNA was expressed in trophoblast cells at both gestational ages, but was not present in endothelial cells. CLIC3 mRNA was also identified in whole placental extracts at both gestational ages and in term amnion and choriodecidua. Immunohistochemistry using a chicken anti-human CLIC3 antibody localised strong CLIC3-specific staining to the syncytiotrophoblast and villous cytotrophoblast cells in both first trimester and term placentas, and weaker staining in extravillous trophoblast cells in first trimester. In fetal membranes at term strong CLIC3-specific staining was localised to chorionic trophoblast cells, with weaker staining in amniotic epithelial and decidual cells. It was previously shown that chloride uptake was increased into cells that had been transfected with CLIC3. CLIC3 may facilitate chloride ion movement and the regulation of cellular processes associated with the movement of chloride in the placental and fetal membrane cells in which it is expressed.