Placental infection with human papillomavirus is associated with spontaneous preterm delivery

BACKGROUND: We sought to determine if human papillomavirus (HPV) infection of extravillous trophoblast cells reduces cell invasion and if placental infection is associated with adverse reproductive outcomes attributed to placental dysfunction. METHODS: We conducted apoptosis and invasion assays using extravillous trophoblast (HTR-8/SVneo) cells that were transfected with a plasmid (pAT-HPV-16) containing the entire HPV-16 genome. In order to associate HPV infection with reproductive outcomes, we conducted a case-control study to detect HPV DNA in the extravillous trophoblast region of placentas from cases of spontaneous preterm delivery, severe pre-eclampsia requiring delivery at <37 weeks and controls who delivered at term. RESULTS: Rates of apoptosis were 3- to 6-fold greater in transfected cells than in non-transfected cells or cells transfected with an empty plasmid. Invasion of transfected cells through extracellular matrices was 25-58% lower than that of the controls. HPV was detected more frequently in placentas from spontaneous preterm deliveries than in placentas from controls (P = 0.03). Identification of HPV in placentas from cases of pre-eclampsia was not significantly different to controls. CONCLUSIONS: HPV infection of extravillous trophoblast induces cell death and may reduce placental invasion into the uterine wall. Thus, HPV infection may cause placental dysfunction and is associated with adverse pregnancy outcomes, including spontaneous preterm delivery.     

Altered Expression of Human Leukocyte Antigen G (HLA-G) on Extravillous Trophoblasts in Preeclampsia: Immunohistological Demonstration With Anti-HLA-G Specific Antibody “87G” and Anti-cytokeratin Antibody “CAM5.2”

PROBLEM: Human leukocyte antigen-G (HLA-G) is suggested to be at play in the materno-fetal immune relationship during pregnancy. In the light of current concept that disruption of the materno-fetal immune relationship could account for several complications of pregnancy, including preeclampsia, we asked whether the expression of HLA-G protein on the trophoblasts is altered in preeclampsia. METHOD: The presence of HLA-G protein in the extravillous trophoblasts in placenta obtained from five preeclamptic patients and seven uncomplicated pregnant women was determined by means of an immunohistochemical technique. RESULTS: All of the extravillous trophoblasts, which were stained for cytokeratin, were stained for HLA-G protein in every woman with an uncomplicated pregnancy. In contrast, clusters of extravillous trophoblasts were insularly devoid of the staining for HLA-G in all the preeclamptic patients. CONCLUSION: The attenuated expression of HLA-G protein on the extravillous trophoblasts could be at play in the pathophysiology of preeclampsia.     

First trimester human endovascular trophoblast cells express both HLA-C and HLA-G.

PROBLEM: In human pregnancies, trophoblasts, in contrast to placental connective tissue and the fetus itself, come into direct contact with the maternal allorecognizing system at special sites. Villous syncytiotrophoblasts washed around by maternal blood lack HLA class I proteins, whereas extravillous trophoblasts, which deeply invade maternal uterine tissues, express high amounts of HLA-G and also HLA-C, the latter to a lesser degree, however. A subpopulation of extravillous trophoblasts, the endovascular trophoblast, enters maternal spiral artery lumen and, like syncytiotrophoblast, comes into direct contact with maternal blood. Less is known about HLA class I distribution on this endovascular trophoblast subpopulation. METHOD OF STUDY: A comparative immununohistochemical analysis was done on decidual cryo-sections containing trophoblast-invaded spiral arteries using different anti-HLA class I monoclonal antibodies (mAbs) and a peroxidase-labeled streptavidinbiotin detection system. RESULTS: MAbs W6/32 (anti-HLA-A, -B, -C, -G), HCA2 (anti-HLA-A, -G) G233 and 87G (both anti-HLA-G) resulted in strong positivity on endovascular trophoblasts. L31 (anti-HLA-C) and HC10 (anti-HLA-B, -C) revealed clear positivity, whereas TU149 (anti-HLA-B, -C, some -A) produced a heterogeneous staining pattern, faintly positive on some endovascular trophoblastic cells and negative on others. MAb LA45 (anti-HLA-A, -B) did not bind to any endovascular trophoblast, neither did BFL.1 (anti-HLA-G) nor 16G1 (anti-HLA-G, soluble). CONCLUSION: This study shows that trophoblastic cells belonging to the endovascular subpopulation express considerable amounts of HLA-G and slightly less HLA-C.     

OPINION: Immunologic Abnormality of Intrahepatic Cholestasis of Pregnancy

Citation Yayi H, Danqing W, Shuyun L, Jicheng L. Immunologic Abnormality of Intrahepatic Cholestasis of Pregnancy. Am J Reprod Immunol 2010; 63: 267–273 Problem Intrahepatic cholestasis of pregnancy (ICP) is a pregnancy risk because of the possibility of pre‐term delivery and sudden intrauterine fetal death. Its pathogenesis is still under discussion. Method of study The analysis of the recent findings on the complex immunologic events that occur in ICP were performed. Results In ICP, an increase of type 1 cytokine (TNF‐α, IFN‐γ) associated with a decrease of type 2 cytokine (IL‐4). The decreased production of the suppressor cytokine TGF‐β2 may increase the type 1 cytokine. Fas appeared to be increased and FasL appeared to be decreased in syncytiotrophoblasts of ICP. The human leukocyte antigen gene (HLA‐G, E) in extravillous trophoblasts of ICP were significantly decreased. Conclusion Th1/Th2 cytokine balance and HLA play important roles in the tolerance and maintenance of pregnancy. ICP may be resulting from breach of the maternal fetal immune tolerance during pregnancy.     

Lack of human leukocyte antigen-G expression in extravillous trophoblasts is associated with pre-eclampsia

Pre-eclampsia, a common complication of first pregnancies, is thought to result from a poorly perfused placenta and may reflect an abnormal maternal immune reaction to the hemiallogenic fetus. Human leukocyte antigen (HLA)-G, a major histocompatibility tissue-specific antigen expressed in extravillous trophoblast cells (fetal-derived), may protect trophoblasts from maternal-fetal immune intolerance and allow these cells to invade the uterus. Through RNA in-situ hybridization analysis, we studied the expression pattern of HLA-G in normal placentae and placentae from pregnancies complicated by severe pre-eclampsia. In normal placenta we found HLA-G expression in the anchoring extravillous trophoblasts with an increasing gradient of expression in the more invasive cells. However, in nine out of 10 pre-eclamptic placentae HLA-G expression was absent or reduced. We conclude that HLA-G is normally expressed in invasive trophoblasts and HLA-G expression is defective in most pre-eclamptic placentae. We propose that trophoblasts lacking HLA-G are vulnerable to attack by the maternal immune system. These defective trophoblasts will be unable to invade the maternal spiral arteries effectively, thereby developing vessels which cannot adequately nourish the developing placenta. This poorly perfused placenta may initiate the systemic cascade of events associated with pre-eclampsia.     

Human trophoblast invasion and spiral artery transformation: the role of nitric oxide

During early human pregnancy extravillous cytotrophoblasts invade the uterus and also migrate up the spiral arteries, transforming them into large vessels of low resistance. Failure of transformation has been described in pre-eclampsia, fetal growth restriction, and miscarriage. Recent evidence suggests that some maternal vessels undergo structural changes without interaction with cytotrophoblasts. The possibility arises that local vasoactive mediators such as nitric oxide result in spiral artery dilatation before their invasion. In support of this, a recent histological study in the guinea pig suggested that cytotrophoblasts expressed nitric oxide synthase (NOS) as they surrounded vessels. This study tested the hypothesis that invading cytotrophoblasts express NOS and therefore have the potential to induce vasodilatation by releasing nitric oxide. The expression of NOS on extravillous cytotrophoblasts was studied in placental bed biopsies, obtained, using a transcervical sampling technique, from normal human pregnancies between 8 to 19 weeks of gestation and in the third trimester. Whereas eNOS was expressed by syncytiotrophoblast, neither eNOS or iNOS was expressed by extravillous cytotrophoblasts at any time during invasion. The mechanisms controlling spiral artery transformation are pivotal to understanding normal and abnormal placentation. These results suggest that trophoblast-derived nitric oxide is unlikely to contribute to spiral artery dilatation.     

A single base-pair mutation in the 3'-untranslated region of HLA-G mRNA is associated with pre-eclampsia

Human leukocyte antigen-G (HLA-G) is a non-classical class I HLA molecule that is expressed by extravillous cytotrophoblast cells. This protein may play a critical role in the protection of cytotrophoblasts from maternal immune response, allowing these semi-allogeneic cells to invade the uterus unimpeded. We have demonstrated that diminished placental HLA-G expression is associated with pre-eclampsia. In order to explore fundamental mechanisms underlying this reduced HLA-G expression in pre-eclampsia, we looked for, and found by sequences analysis, a single base-pair mutation in the HLA-G gene 3'-untranslated region (3'UTR) adjacent to an AUUUA motif. This mutation is significantly associated with pre-eclampsia, the severe form being more strongly associated with homozygosity for this mutation than the mild form. Since the null allele was discovered in the HLA-G mRNA 3'UTR adjacent to an AUUUA motif, we also examined the effect of this mutation on HLA-G mRNA stability, and found that half-lives of HLA-G mRNA with the mutation were significantly shorter than without the mutation. These data provide evidence that this mutation could be one of the fundamental mechanisms for lower levels of placental HLA-G protein expression in patients with pre-eclampsia.     

The regulation of trophoblast differentiation by oxygen in the first trimester of pregnancy

In the first trimester of human pregnancy villous cytotrophoblasts are able to differentiate to form either the overlying syncytiotrophoblast layer or, in anchoring villi, extravillous trophoblasts which grow out from the villi and invade into the maternal decidua, acting to both physically attach the placenta to the decidua, and modify the maternal spiral arteries to sustain pregnancy. During the first 10-12 weeks of gestation, extravillous trophoblast plugs block the spiral arteries and prevent maternal blood flow entering the intervillous space, thereby creating an environment of physiological hypoxia in which placental and fetal development occur. As extravillous trophoblasts migrate away from the villus they differentiate from a proliferative to an invasive phenotype. The hypoxic environment of the first trimester is believed to play an important role in the regulation of trophoblast differentiation. However, there is currently a large body of conflicting experimental evidence concerning this topic. This review examines the experimental evidence to date on the role of oxygen in trophoblast differentiation.     

HLA-G expression in trophoblast cells is independent of embryonic development

HLA-G is a nonclassical major histocompatibility complex class I molecule that is selectively expressed on cytotrophoblasts at the feto-maternal interface where it may play a major role in maternal-fetal tolerance. In this study, we compared HLA-G expression in trophoblasts from normal and pathologic pregnancies by immunohistochemical analysis. First, we found a defective HLA-G expression in miscarriages associated with hypotrophic but normal eggs. Conversely, by studying molar pregnancies, we observed a high HLA-G expression in complete and partial hydatidiform moles. Finally, HLA-G expression could be visualized in extravillous trophoblasts that develop outside of their normal environment, as reported here in ectopic pregnancies. Taken together, these results suggest that HLA-G expression in extravillous trophoblasts is induced in an autonomous manner, independently of embryonic development, and may be an integral part of placental development allowing its tolerance from maternal immune system.     

Placental HLA-G protein expression in vivo: where and what for?

In contrast to HLA-A and -B class Ia genes that are down-regulated in human trophoblast cells, HLA-G class Ib molecules are expressed in the placenta throughout gestation. In addition to extravillous cytotrophoblast that invade the decidua basalis essentially, HLA-G was also observed in endothelial cells of fetal vessels in the chorionic villi as well as in amnion cells and amniotic fluid. Both membrane-bound and soluble HLA-G isoforms have been detected. In view of the recently published functional data showing that HLA-G: (i) has the capability to bind and present peptides; (ii) is recognized by at least three different killing inhibitory receptors; and (iii) is a regulator of HLA-E expression, we can predict that such functions are likely to be exerted by extravillous cytotrophoblast. Of particular importance will be the anti-viral function of HLA-G at this materno-fetal interface, knowing that HLA-G was shown to be expressed by thymic medullary epithelial cells. In addition to these immunological functions, due to its presence on chorionic fetal endothelial cells, we hypothesize that HLA-G could also be a regulator of chorionic villous angiogenesis. Finally, soluble HLA-G isoforms may act as specific immunosuppressors during pregnancy.     

The novel inflammatory cytokine high mobility group box protein 1 (HMGB1) is expressed by human term placenta

High mobility group box protein 1 (HMGB1) was previously considered a strict nuclear protein, but lately data are accumulating on its extranuclear functions. In addition to its potent proinflammatory capacities, HMGB1 has a prominent role in a number of processes of specific interest for the placenta. Our overall aim was to investigate the expression of HMGB1 in human term placenta and elucidate a potential difference in HMGB1 expression comparing vaginal deliveries with elective Caesarean sections. In addition, placentas from normal pregnancies were compared with placentas from pregnancies complicated by pre-eclampsia. Twenty-five placentas, 12 from normal term pregnancies and 13 from pregnancies complicated by pre-eclampsia were analysed with immunohistochemistry for HMGB1 and its putative receptors; receptor for advanced glycation end-products (RAGE), Toll-like receptor 2 (TLR2) and TLR4. We present the novel finding that in addition to a strong nuclear HMGB1 expression in almost all cells in investigated placentas, an individual variation of cytoplasmic HMGB1 expression was detected in the syncytiotrophoblast covering the peripheral chorionic villi, by cells in the decidua and in amnion. Production of HMGB1 was confirmed by in situ hybridization. Although labour can be described as a controlled inflammatory-like process no differences in HMGB1 expression could be observed comparing active labour and elective Caesarean sections. However, a tendency towards a higher expression of cytoplasmic HMGB1 in the decidua from women with pre-eclampsia was demonstrated. The abundant expression of the receptors RAGE, TLR2 and TLR4 implicates a local capability to respond to HMGB1, although the precise role in the placenta remains to be elucidated.     

Is the Expression of Classical HLA Class I Antigens on Trophoblast of Importance for Human Pregnancy?

PROBLEM: Human leukocyte antigen (HLA)-C and possibly also HLA-B seem to be expressed on the extravillous trophoblast. These antigens carry epitopes that function as ligands for natural killer (NK)-cell-inhibitory receptors. Antitrophoblast cytotoxicity mediated by decidual NK cells might be involved in miscarriage. We thus found it relevant to elucidate whether parental HLA-C and -Bw polymorphism play a role in recurrent miscarriage (RM). METHOD OF STUDY: HLA-C and -Bw investigations by DNA-based techniques were undertaken in 35 couples with unexplained RM and in 30 couples with normal fecundity. The number of HLA-C- and -Bw-related supertypic specificities that can bind NK-cell-inhibitory receptors was evaluated in selected couples. RESULTS: The proportions of couples with RM and control couples carrying four HLA-C alleles with the same NK-cell-inhibitory supertypic specificities were equal. In 46% of studied couples with RM, all four HLA-B alleles carried the HLA-Bw6 supertypic specificity, which was significantly higher than the corresponding frequency (17%) in the control couples (P < 0.02). CONCLUSIONS: The expression of polymorphic HLA-C on trophoblasts does not seem to play a role in RM. Assuming that HLA-B is expressed on trophoblasts, we may suggest that the revealed predominance of HLA-Bw6 expression (which excludes the presence of HLA-Bw4—protective antigens) may predispose a particular couple to the RM phenomenon.     

不同浓度Dlll4调节绒毛外滋养细胞的生物学功能

背景：早孕时期绒毛外滋养细胞功能的正常发挥对妊娠有着至关重要的影响,绒毛外滋养细胞功能异常时将导致子痫前期、胎儿生长受限及胎盘植入等多种妊娠期疾病。 目的：探讨Notch信号家族配体Dll4对绒毛外滋养细胞的生物学功能的影响。 方法：以不同质量浓度外源性Dll4(0,0.125,0.25,0.5,1.0 mg/L)处理人绒毛外滋养细胞系HTR-8/SVneo,以CCK-8试验检测细胞活性,Transwell实验检测细胞迁移、侵袭能力的改变。 结果与结论：Dll4可明显促进绒毛外滋养细胞迁移与侵袭能力,而对细胞活性不产生显著影响;Dlll4对绒毛外滋养细胞侵袭功能的影响具有浓度依赖性。结果提示Dll4可通过调节绒毛外滋养细胞生物学功能,在妊娠过程很中发挥重要作用。  中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程     

Interaction of the LILRB1 inhibitory receptor with HLA class Ia dimers

Leukocyte immunoglobulin‐like receptor subfamily B member 1 (LILRB1) has been reported to interact with a wide spectrum of HLA class I (HLA‐I) molecules, albeit with different affinities determined by allelic polymorphisms and conformational features. HLA‐G dimerization and the presence of intracellular Cys residues in HLA‐B7 have been shown to be critical for their recognition by LILRB1. We hypothesized that dimerization of classical HLA class Ia molecules, previously detected in exosomes, might enhance their interaction with LILRB1. A soluble LILRB1‐Fc fusion protein and a sensitive cellular reporter system expressing a LILRB1‐ζ chimera were employed to assess receptor interaction with different HLA class Ia molecules transfected in the human lymphoblastoid 721.221 cell line. Under these conditions, intracellular Cys residues and HLA‐I dimerization appeared associated with increased LILRB1 recognition. On the other hand, a marginal interaction of LILRB1 with primary monocytic cells, irrespective of their high HLA‐I expression, was enhanced by type I interferon (IFN). This effect appeared disproportionate to the cytokine‐induced increase of surface HLA‐I expression and was accompanied by detection of HLA class Ia dimers. Altogether, the results support that a regulated assembly of these noncanonical HLA‐I conformers during the immune response may enhance the avidity of their interaction with LILRB1.     

Human cytotrophoblast differentiation/invasion is abnormal in pre-eclampsia.

During human placental development, cytotrophoblast stem cells differentiate and invade the uterus. Simultaneously, the cells modulate their expression of several classes of stage-specific antigens that mark transitions in the differentiation process and play a role in either uterine invasion (integrin cell-extracellular matrix receptors and matrix metalloproteinase-9) or immune interactions (HLA-G). The pregnancy disease pre-eclampsia is associated with shallow cytotrophoblast invasion. Previously we showed, by immunofluorescence localization on placental tissue, that in pre-eclampsia invasive cytotrophoblasts fail to properly modulate their integrin repertoire. This finding suggests possible abnormalities in the differentiation pathway that leads to uterine invasion. Here we used a culture system that supports this differentiation process to compare the differentiative and invasive potential of cytotrophoblasts obtained from control (n = 8, 22 to 38 weeks) and pre-eclamptic (n = 9, 24 to 38 weeks) placentas. In culture, the cells from pre-eclamptic placentas failed to properly modulate alpha1 integrin and matrix metalloproteinase-9 expression at the protein and mRNA levels. Their invasive potential was also greatly reduced. Likewise, the cells failed to up-regulate HLA-G protein and mRNA expression. These results suggest that defective cytotrophoblast differentiation/invasion can have significant consequences to the outcome of human pregnancy (ie, development of pre-eclampsia) and that, by the time delivery becomes necessary, the defect is not reversed by removing the cells from the maternal environment.     

Human trophoblast invasion and spiral artery transformation: the role of PECAM-1 in normal pregnancy, preeclampsia, and fetal growth restriction

During early human pregnancy extravillous cytotrophoblasts invade the uterus and spiral arteries transforming them into large vessels of low resistance. Failure of trophoblast invasion and spiral artery transformation occurs in preeclampsia and fetal growth restriction (FGR); these processes are not well understood. Recent studies have suggested that cytotrophoblasts that invade spiral arteries mimic the endothelial cells they replace and express PECAM-1. It was also reported that in preeclampsia, cytotrophoblasts fail to express PECAM-1 and that failure to express endothelial cell adhesion molecules may account for failed trophoblast invasion. Despite the possible importance of adhesion molecules in trophoblast invasion, no study has systematically investigated the expression of PECAM-1 in the placental bed throughout the period of invasion, particularly in the myometrial segments where the key failure occurs. There are no studies on PECAM-1 expression in the placental bed in FGR. We have examined the expression of PECAM-1 in placental bed biopsies and placentas from 8 to 19 weeks of gestation and in the placenta and placental bed in the third trimester in cases of preeclampsia, FGR, and control pregnancies. PECAM-1 was expressed on endothelium of vessels in the placenta and placental bed but not by villous or extravillous trophoblasts in normal or pathological samples. These findings do not support a role for PECAM-1 in normal invasion or in the pathophysiology of preeclampsia or FGR.     

Trophoblastic tumors: ultrastructural comparison of choriocarcinoma and placental-site trophoblastic tumor

Ten trophoblastic tumors, including seven classical choriocarcinomas, two choriocarcinomas with atypical histology, and one placental-site trophoblastic tumor (PSTT), were studied to compare their fine structural features. Ultrastructurally, the classical choriocarcinomas showed well-defined cytotrophoblasts and syncytiotrophoblasts. The cytotrophoblasts were primitive epithelial cells, while the syncytiotrophoblasts were complex cells with multiple nuclei and dense cytoplasm containing dilated endoplasmic reticulum, lysosomes, vesicles, and tonofilaments. The syncytiotrophoblast cell membranes often contained numerous microvilli. In the choriocarcinomas, scattered intermediate trophoblasts showed features transitional between the cytotrophoblasts and the syncytiotrophoblasts, with moderately complex cytoplasm containing some of the organelles found in the syncytiotrophoblasts. Histologically, the atypical choriocarcinomas showed a predominance of mononucleate and binucleate cells and indistinct syncytiotrophoblasts. Ultrastructurally, these atypical tumors were composed largely of intermediate trophoblasts, yet contained scattered syncytiotrophoblasts with microvilli in compressed aggregates. The PSTT was composed primarily of intermediate trophoblasts that contained prominent paranuclear filaments not seen in the intermediate trophoblasts of the choriocarcinomas. Rare cells resembling syncytiotrophoblasts were found in the PSTT, but no cytotrophoblasts were observed. Immunoreactivity for human chorionic gonadotropin and human placental lactogen was found in the intermediate trophoblasts and syncytiotrophoblasts of both the choriocarcinomas and the PSTT, demonstrating functional homology between these tumors despite some ultrastructural differences. These results demonstrate ultrastructural features of trophoblastic cells that correlate with the morphologic diversity seen in these tumors by light microscopy. Furthermore, the comparisons suggest that the PSTT is composed of a distinct form of intermediate trophoblast that appears to reflect its origin from the extravillous trophoblast.     

Vascular endothelial growth factor, epidermal growth factor and fibroblast growth factor-4 and -10 stimulate trophoblast plasminogen activator system and metalloproteinase-9

Trophoblast invasion, accompanied by degradation of extracellular matrix, is crucial to normal pregnancy development, whereas shallow placental invasion and implantation likely plays a role in the subsequent development of pre-eclampsia. The growth factors vascular endothelial growth factor (VEGF), epidermal growth factor (EGF) and fibroblast growth factor (FGF) are placental growth factors that activate degradation of extracellular matrix. We determined the effect of VEGF, EGF, FGF-2, FGF-4 and FGF-10 on the plasminogen activator system of first trimester cytotrophoblasts cultured in vitro. We studied the activity of urokinase plasminogen activator (uPA), its inhibitor plasminogen activator inhibitor-1 (PAI-1), and 92 kDa gelatinase-B (matrix metalloproteinase-9, MMP-9), using protein gel and reversed gel zymography. The expression pattern of FGF-4 and FGF-10 in human placental sections was determined by immunohistochemistry. FGF-4 was expressed in first trimester villi stroma, primarily in endothelial cells. FGF-10 expression was localized to first trimester extravillous trophoblasts. VEGF, EGF, FGF-4 and FGF-10, but not FGF-2, stimulate the activity of trophoblast uPA, PAI-1 and MMP-9. These results support the hypothesis that specific growth factors modulate the invasive potential of trophoblasts, and therefore may play an important role in early placental development. Our findings may contribute to the understanding of the pathophysiology of diseases associated with shallow placentation, such as pre-eclampsia.     

Endothelial cells in chorionic fetal vessels of first trimester placenta express HLA-G

Using four different HLA-G-recognizing monoclonal antibodies (mAb), we investigated whether this nonclassical HLA class I molecule could be expressed in placental cell types other than extravillous cytotrophoblasts (evct) in which HLA-G has already been detected. Immunohistochemical analysis was performed on serial cryosections of first trimester placenta as well as on maternal decidual tissue. In addition to some proliferative evct, the recently described BFL.1 mAb also slightly stained some villous cytotrophoblast (vct) stem cells located near cell columns and cell islands, which until now have been considered as HLA-G negative. The same staining pattern was obtained with the 16G1 mAb raised against the soluble HLA-G isoform, whereas neither 87G nor HCA2 reacted with vct but did strongly label the invasive populations of evct, including interstitial and endovascular trophoblasts. Surprisingly, BFL.1 strongly and reproducibly stained endothelial cells in the fetal capillaries present in the mesenchymal core of the chorionic villi, whereas none of the other surrounding cellular components were stained. The same specific labeling was obtained, although with less intensity, with the three other HLA-G-recognizing mAb. In contrast, maternal endothelial cells present in spiral arteries of the decidua parietalis remained unstained. This unexpected cellular localization suggests that HLA-G may be present as a soluble form during the whole period of fetal vascularization and/or exert a nonimmunological function related to the endothelial cell type, in particular in the angiogenesis process which is highly active, until term, in chorionic villi.