Expression pattern of the CCAAT/enhancer-binding protein C/EBP-beta in gestational trophoblastic disease.

The CCAAT/enhancer-binding protein (C/EBP) family consists of several factors that are important regulators of intracellular processes and hormone action. C/EBP-beta, the most important member of the C/EBP family, was shown recently to be expressed in the normal human placenta where it is localized in villous syncytiotrophoblast and in the extravillous (intermediate) trophoblast but not the villous cytotrophoblast. The purpose of this study was to investigate the expression pattern of C/EBP-beta in gestational trophoblastic disease (GTD) which has not been studied so far. We used immunohistochemistry on a total of 15 cases of GTD including nine complete hydatidiform moles, one placental site nodule (PSN), one placental site trophoblastic tumor (PSTT), and four choriocarcinomas. All our tested specimens showed positivity for C/EBP-beta. The strongest C/EBP-beta expression could be observed in villous syncytiotrophoblast and in the trophoblast proliferations on the villous surface of hydatidiform moles; villous cytotrophoblast was negative. The PSN also showed positive nuclear staining but the expression was not as strong as it was in the hydatidiform moles and the total amount of stained cells was the lowest of all GTD. The PSTT also showed immunoreactivity but with a weaker and more heterogeneous staining than in the choriocarcinomas. The specific expression pattern of C/EBP-beta in GTD indicate that C/EBP-beta could potentially be an additional marker of such lesions.     

Expression pattern of the adhesion molecule CEACAM1 (C-CAM, CD66a, BGP) in gestational trophoblastic lesions.

CEACAM1 (CD66a, BGP, C-CAM) is an adhesion molecule of the carcinoembryonic antigen (CEA) family which has been shown to be normally expressed at the apical pole of epithelial cells and to show a dysregulated expression pattern in tumors derived from the latter. The purpose of the present study was to investigate the expression pattern of CEACAM1 in gestational trophoblastic lesions and to compare this expression with the one observed in the normal trophoblast. For this purpose, we performed immunohistochemistry using the 4D1/C2 monoclonal antibody which specifically recognizes CEACAM1 and does not interact with other members of the CEA family. Immunohistochemistry was performed on a total of 20 cases of gestational trophoblastic lesions including complete hydatidiform moles, one placental site trophoblastic nodule (PSN), one placental site trophoblastic tumor (PSTT), and three choriocarcinomas. Immunostaining for cytokeratin, hPL, hCG, and Ki-67 was also performed. Normal placental samples served as a control. CEACAM1 was absent from villous cyto- and syncytiotrophoblast in both normal placenta and hydatidiform molar samples. It was present in the benign extravillous trophoblast, with stronger expression in the proximal extravillous trophoblast of anchoring villi, but was also observed in interstitial and endovascular intermediate trophoblast and chorionic intermediate-like trophoblast. Partial expression was observed in the trophoblast proliferating from the surface of molar villi. In choriocarcinomas, areas of weak expression could be observed along with large areas without CEACAM1 expression. In the PSN and especially in the PSTT, CEACAM1 expression was stronger and more diffuse. The specific localization to extravillous trophoblast and its expression pattern in gestational trophoblastic lesions indicate that CEACAM1 can potentially be a helpful additional diagnostic marker in the differential diagnosis of such lesions.     

Matrix metalloproteinases and their inhibitors in gestational trophoblastic diseases and normal placenta.

OBJECTIVE: Our purpose was to investigate the expression of matrix metalloproteinases (MMPs) in gestational trophoblastic diseases and normal first-trimester placenta. METHODS: Paraffin sections of 16 partial moles, 25 complete moles, 10 gestational choriocarcinomas, and 11 normal first-trimester placentas were studied immunohistochemically for expression of MMP-1, MMP-2, MMP-3, MMP-9, MMP-13, and tissue inhibitor of metalloproteinase-1 (TIMP-1). RESULTS: Nine (90.0%) of the choriocarcinoma cases showed strong intensity of staining for MMP-1. Choriocarcinoma exhibited significantly stronger staining for MMP-1 than syncytiotrophoblast in normal placenta (P < 0.01), partial mole (P < 0.01), and complete mole (P < 0.01). Choriocarcinoma also showed significantly stronger staining for MMP-1 than the extravillous trophoblast in placenta (P < 0.05). MMP-2 was expressed only in syncytio- and extravillous trophoblasts in normal placenta, partial mole, and complete mole. Choriocarcinoma and the extravillous trophoblast in partial mole and complete mole had significantly stronger staining for MMP-2 than the extravillous trophoblast in placenta (P < 0.05, P < 0.01, P < 0.01, respectively). Choriocarcinoma also exhibited significantly stronger staining for MMP-2 than syncytiotrophoblasts in placenta (P < 0.01), partial mole (P = 0.05), and complete mole (P < 0.01). The expression of MMP-3, MMP-9, and MMP-13 was similar in all four tissues with the predominance of syncytiotrophoblast for MMP-3 and MMP-13 and cytotrophoblast for MMP-9. While 8 (73.0%) placentas, 14 (87.5%) partial moles, and 19 (76.0%) complete moles showed strong immunoreactivity for TIMP-1 in syncytiotrophoblasts, no strong staining was found in choriocarcinomas (P < 0.01, P < 0.01, P < 0.01, respectively). CONCLUSION: The extravillous trophoblast of first-trimester placenta has significantly less expression of MMP-1 than choriocarcinoma and significantly less expression of MMP-2 than choriocarcinoma and extravillous trophoblast of partial and complete mole. The expression of TIMP-1 was significantly less in choriocarcinoma than the syncytiotrophoblast of normal placenta, partial mole, and complete mole. MMPs and their inhibitors may play a role in the pathogenesis of gestational trophoblastic diseases.     

Expression pattern of the cell cycle promoter cyclin e in benign extravillous trophoblast and gestational trophoblastic lesions: correlation with expression of Ki-67.

In this study, a specific monoclonal antibody was used to immunohistochemically investigate correlated expression of the cell cycle promoter cyclin E and the proliferation marker Ki-67 in benign extravillous trophoblast and gestational trophoblastic lesions. Our data show that cyclin E is expressed in the normal extravillous trophoblast, with strongest levels of expression in the cell columns of anchoring villi. Differences could be observed in expression of Ki-67 in both normal extravillous trophoblast and gestational trophoblastic lesions. In the extravillous trophoblast of the cell columns, expression of cyclin E started more distal compared with Ki-67 and was maintained (with less intensity) into the deeper layers of interstitial trophoblast. In the benign trophoblastic lesions (exaggerated placental site [EPS] and placental site nodule [PSN]) and in the trophoblast proliferations on the surface of hydropic villi of hydatidiform moles (HM), the percentage of cells expressing cyclin E was higher than of those expressing Ki-67. The same observation could be made for a case of placental site trophoblastic tumor (PSTT). In contrast, choriocarcinomas (N=8), which are definitely malignant tumors, showed an opposite pattern, with a much higher percentage of strongly Ki-67-positive cells compared with cyclin E-positive cells. We conclude that cyclin E is expressed in benign extravillous trophoblast and gestational trophoblastic lesions, where a ratio cyclin E/Ki-67<1 characterizes choriocarcinomas, whereas PSTT and the benign lesions (HM, EPS, PSN) show expression of cyclin E in a higher percentage of cells than Ki-67 (cyclin E/Ki-67 ratio >1).     

c-mos immunoreactivity aids in the diagnosis of gestational trophoblastic lesions.

C-mos is an important proto-oncogene involved in the mitogen-activating protein kinase pathway. This study was designed to explore c-mos immunoreactivity in gestational trophoblastic lesions and compare it with immunoreactivity in normal placentas as well as other gynecological lesions and germ cell tumors using antibody P-19. The immunohistochemical distribution of c-mos in 159 cases of gynecological lesions and 26 germ cell tumors using formalin-fixed, paraffin-embedded tissues was evaluated. The lesions included 45 (32 complete and 13 partial) hydatidiform moles, 17 choriocarcinomas, 5 placental site trophoblastic tumors, 18 squamous cell carcinomas and 5 adenocarcinomas of the cervix, 11 endometrial carcinomas, 9 ovarian carcinomas, 4 primary peritoneal papillary serous carcinomas, 9 low-grade endometrial stromal sarcomas, 4 epithelioid leiomyomas, 6 leiomyosarcomas, and 26 gem cell tumors (3 embryonal carcinomas, 5 yolk sac tumors, 6 immature teratomas, and 3 mature teratomas from the ovary; 9 testicular seminomas). Twenty-six normal placentas also were included for comparison. Among cases of gestational trophoblastic diseases, c-mos immunoreactivity was found in all hydatidiform moles and choriocarcinomas, but in none of the placental site trophoblastic tumors. The c-mos staining pattern was similar in trophoblastic diseases and normal placentas with strong expression in syncytiotrophoblast, moderate expression in villous intermediate trophoblast, and predominantly negative expression in implantation site intermediate trophoblast, chorionic-type intermediate trophoblast, and villous cytotrophoblast. All the nontrophoblastic tumors, including carcinomas, sarcomas, and germ cell tumors, were negative for c-mos expression. Immunohistochemical detection of c-mos is useful in differentiating choriocarcinoma from placental site trophoblastic tumor and nontrophoblastic tumors of the female genital tract that may sometimes cause problems in differential diagnosis.     

Low Molecular Mass Polypeptide-2 in Human Trophoblast: Over-Expression in Hydatidiform Moles and Possible Role in Trophoblast Cell Invasion

Embryo implantation involves invasion of placental extravillous trophoblast cell (EVTs) into the uterus. Hyperactive EVT invasion occurs in hydatidiform moles and choriocarcinomas. We have previously demonstrated that the 20S proteasome is involved in mouse embryo implantation and its action is mediated via regulating the expression and activities of matrix metalloproteinase (MMP)-2 and MMP-9 in the EVTs. Our objective was to investigate whether low molecular mass polypeptide-2 (LMP2), a beta subunit of the 20S proteasome, is involved in the regulation of human trophoblast invasion. Normal human placentas or placentas from hydatidiform mole patients were collected and the expression of LMP2 in different cell types including trophoblastic column (TC), cytotrophoblast cells (CTB) and syncytiotrophoblast (STB) under different pathological states were studied by immunohistochemical analysis. Furthermore, the effect of LMP2 or proteasome on cell invasion was measured by using RNAi and inhibitors in a Matrigel invasion assay system in HTR-8/SVneo cells, a human invasive extravillous trophoblast cell line. Changes in the invasion-related molecules including MMP-2 and MMP-9 were also examined by using real time PCR and gelatin zymography. We demonstrated that the expression of LMP2 in TC of partial hydatidiform mole and complete hydatidiform mole, is higher than that in TC of normal human placentas. Besides, LMP2 knockdown significantly attenuated IL-1β-induced cell invasion in vitro, a response readily induced by proteasome inhibitors. In summary, over-expression of the 20S proteasome β-subunit LMP2 in trophoblast cells of hydatidiform moles may contribute to its highly invasive phenotype.     

HLA expression by trophoblast of invasive moles

HLA expression by the trophoblast in invasive hydatidiform mole was analysed by immunoperoxidase staining. In the invading villi of an invasive mole, villous trophoblast, both syncytiotrophoblast and cytotrophoblast, failed to show a positive reaction for HLA-A, -B and -C and HLA-DR. By contrast, extravillous trophoblast showed an intense reaction for HLA-A, -B and -C. The distribution of HLA antigens in the invading villi was the same as in the non-invading villi, and the antigens were also indistinguishable from those noted in non-invasive hydatidiform moles. The histopathology of invasive mole may suggest that it is a malignant neoplasm. This immunohistochemical study, however, lends support to the current view that invasive mole is a variant of a benign hydatidiform mole rather than a form of malignant trophoblastic disease.     

Practical management of gestational trophoblastic disease

Gestational trophoblastic disease (GTD) represents a spectrum of tumors that arise from the fetal chorion during pregnancy, including benign partial and complete hydatidiform moles, persistent invasive or metastatic moles, placental-site trophoblastic tumors, and gestational choriocarcinomas. Gestational trophoblastic diseases all exhibit proliferation of both cytotrophoblast and syncytiotrophoblast cells, with the exception of placental-site tumor, which is derived from the intermediate trophoblast cells. Practical management of these tumors begins with preoperative evaluation and screening for metastatic disease followed by uterine evacuation or hysterectomy. Postmolar GTD includes local noninvasive proliferation of molar tissue, invasive mole, or gestational choriocarcinoma. The management of malignant GTD may be directed from three classification systems developed to predict patient prognosis and guide the decision for either single or multiagent chemotherapy. These systems assess clinical risk factors in addition to sites of metastatic disease. Treatment with aggressive multiagent chemotherapy and individualized multimodality therapy is warranted in these extremely high-risk patients. After remission, patients should be followed closely for 1 year and periodically thereafter. Pregnancy is deferred and contraception instituted for 1 year to prevent disruption of serum human chorionic gonadotropin surveillance.     

Immunohistochemical localization of inhibin-alpha in the placenta and gestational trophoblastic lesions.

The immunohistochemical distribution of inhibin-alpha in formalin-fixed, paraffin-embedded tissues was evaluated in placentas (2 to 40 weeks of gestation), implantation sites, and a variety of trophoblastic lesions. In the first trimester placenta, inhibin-alpha was strongly and diffusely expressed in syncytiotrophoblast. Implantation site intermediate trophoblast in normal and exaggerated placental sites was either negative or only weakly and focally positive for inhibin-alpha. With increasing gestational age, the staining intensity and distribution of inhibin-alpha decreased in syncytiotrophoblast but increased in the implantation site intermediate trophoblast. Chorionic-type intermediate trophoblast, present in the chorion laeve of the term placenta, was weakly but diffusely positive for inhibin-alpha. Cytotrophoblast remained negative for inhibin-alpha throughout gestation. In trophoblastic lesions, inhibin-alpha immunoreactivity was detected in all 17 hydatidiform moles (7 complete and 10 partial), 32 placental site nodules, 23 placental site trophoblastic tumors, 15 epithelioid trophoblastic tumors, and 16 choriocarcinomas. Inhibin-alpha immunoreactivity was confined to the syncytiotrophoblast in hydatidiform moles and choriocarcinoma. As with the normal placenta, inhibin-alpha was not detected in cytotrophoblast. To evaluate the utility of inhibin-alpha in the differential diagnosis of gestational trophoblastic lesions, we tested 32 squamous cell carcinoma of the cervix, 11 low-grade endometrial stromal sarcomas, 12 endometrial (7 well differentiated and 5 moderately differentiated) carcinomas, 7 epithelioid leiomyomas, and 10 leiomyosarcomas for the expression of inhibin-alpha. None of these lesions was positive. These data indicate that inhibin-alpha is expressed by all populations of trophoblast except cytotrophoblast and in all gestational trophoblastic lesions. Accordingly, immunohistochemical detection of inhibin-alpha is useful in the differential diagnosis of gestational trophoblastic lesions.     

Differential expression of the receptor tyrosine kinase EphB4 and its ligand Ephrin-B2 during human placental development

Normal placentation involves the development of an utero-placental circulation following the migration of the extravillous cytotrophoblasts into the decidua and invasion of the spiral arteries, which are thereby transformed into large vessels of low resistance. Given the documented role of the receptor tyrosine kinase EphB4 and its ligand ephrin-B2 in the establishment of the embryonal vascular network, we hypothesized that these molecules are also instrumental in the development of the human placenta. Monitoring the expression during placental development revealed that in first trimester and term placentae both molecules are equally expressed at the RNA level. In contrast, the protein levels were significantly reduced during gestation. Immunohistochemistry revealed a distinct localization of the EphB4 and ephrin-B2 proteins. EphB4 was predominantly expressed in the villous syncytial trophoblast layer and in a subset of intravillous capillaries. Prominent expression was also observed in the extravillous cytotrophoblast giant cells. In contrast, ephrin-B2 expression was detected in the villous cytotrophoblast and syncytial trophoblast cell layers, as well as initially in all intravillous capillaries. Strong expression was also observed in extravillous anchoring cytotrophoblast cells. Hypoxia is a major inducer of placental development. In vitro studies employing trophoblast-derived cell lines revealed that predominantly ephrin-B2 expression is induced by hypoxia, however, in an Hif-1alpha independent manner. These experiments suggest that EphB4 and ephrin-B2 are instrumental in the establishment of a functional placental structure and of the utero-placental circulation.     

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.     

Distribution of cells bearing the HNK-1 epitope in the human placenta

HNK-1 (Leu-7 antigen or CD57) is a unique carbohydrate moiety found in certain glycosphingolipids and several cell adhesion glycoproteins on the cell membrane. Previous studies have suggested that HNK-1 carbohydrates act as adhesive ligands in cell-cell interactions. Using a monoclonal antibody reactive to the HNK-1 moiety and an immunoperoxidase method on formalin-fixed paraffin-embedded tissue, the expression of the HNK-1 epitope in human placentae was confined to the intermediate trophoblast (IT) in trophoblastic columns. The number of HNK-1 immunoreactive IT cells increased from the proximal to the midportion of the trophoblastic column, and then disappeared at the junction of the column with the basal plate where IT infiltrates the endomyometrium and becomes extravillous IT. Neither cytotrophoblast nor syncytiotrophoblast reacted with the HNK-1 antibody. In hydatidiform moles, HNK-1 immunoreactivity was localized to areas that structurally resembled trophoblastic columns. In contrast, placental site trophoblastic tumours which do not contain structures analogous to trophoblastic columns did not express HNK-1 epitope. Expression of HNK-1 was only rarely observed in choriocarcinomas, being present in less than 5 per cent of trophoblastic cells in two of 13 cases. The murine placenta, which lacks trophoblastic columns, was negative for HNK-1. Thin-layer chromatography immunostaining demonstrated the HNK-1 reactive glycosphingolipids in placental lipid extracts, whereas Western blot analysis from placental protein extracts failed to reveal detectable glycoproteins that demonstrated HNK-1 immunoreacdvity. In conclusion, the specific localization of HNK-1 reactive glycosphingolipids in trophoblastic columns of the human placenta suggests that the HNK-1 moiety may play an important role in maintaining cohesion between intermediate trophoblastic cells in the trophoblastic columns thereby contributing to the structural integrity of the villi that anchor the placenta to the basal plate.     

Expression pattern of osteopontin in endometrial carcinoma: correlation with expression of the adhesion molecule CEACAM1.

Osteopontin (OPN) and CEACAM1 have diverse biological functions in the uterus and placenta throughout the estrous cycle and pregnancy and have been shown to interact with integrin beta3. OPN is a glycoprotein of the extracellular matrix, which has been shown to mediate cellular migration and invasion and to contribute to tumorigenesis in several types of cancers. Recently we showed the expression pattern of OPN in gestational trophoblastic tumors. CEACAM1 is an adhesion molecule of the carcinoembryonic antigen family that we have recently found to be expressed in endometrial cancer and that has been shown to be down-regulated in colorectal, prostate, and breast cancer. In this study, immunohistochemistry and immunofluorescence with specific antibodies were performed on a series of 20 normal endometrial samples, 17 endometrial hyperplasias, and 43 endometrial carcinomas (28 endometrioid, 10 serous, and 5 clear cell carcinomas) to investigate the expression pattern and cell-type specific localization of OPN and to correlate it with the expression of CEACAM1. In addition, Western blot was performed on normal human endometrium and endometrial neoplasia. Strong OPN expression with a consistent cytoplasmic localization in epithelial glandular cells was observed in the normal human endometrium in 80% of the samples of the proliferative and secretory phase (score 8-12). Similar results could be found in endometrial hyperplasias. Strong expression of OPN could be observed in 29 (67.4%) of the 43 analyzed endometrial carcinomas. Of the 43 analyzed tumors, 18 (41.8%) were in the high score (8-12) category with a strong OPN expression level; 11 of 43 (25.5%) showed a moderate score (4-7) category. In endometrioid carcinoma with increasing malignancy grade, increasing areas with low OPN expression level or complete loss of OPN expression could be observed. In contrast, serous tumors showed a strong OPN expression level. Similar results could be found in Western blot analysis. CEACAM1 showed similar results and could be found to be coexpressed with OPN in normal human endometrium and in endometrial neoplasia as we showed using immunofluorescence. In this study, the different expression patterns of OPN in endometrial tumors could additionally support the biological diversity of endometrioid and serous carcinomas together with other markers. We suggest that OPN might play a different role in the pathogenesis of endometrial cancer (possibly as a functional complex with CEACAM1) and could be relevant for invasive growth of such lesions.     

Spatial and temporal patterns of expression of messenger RNA for insulin-like growth factors and their binding proteins in the placenta of man and laboratory animals

To better understand the role of the insulin-like growth factors (IGF-I and -II) and their binding proteins (IGFBPs 1-6) in placental development and function, it is important to review similarities and differences between species in expression of the respective mRNAs. In human placenta, IGF-II mRNA is expressed in chorionic mesoderm and first trimester villous cytotrophoblast, but not in syncytiotrophoblast. In contrast, in rhesus monkey placenta, IGF-II mRNA is expressed in syncytiotrophoblast but not in chorionic mesoderm. IGFBP-3 mRNA is present in the chorionic mesoderm of placental villi from both these species and may modulate IGF-II action through a paracrine mechanism. In rodent placentae, IGF-II mRNA is expressed both in fetal mesoderm and in the trophoblast of the placental labyrinth. In guinea pig, where IGFBP-5 mRNA is expressed in the marginal and interlobular syncytium and IGF-II mRNA in the labyrinth, interaction between IGF-II and IGFBP-5 mRNA may be involved in vascularization of the placenta by fetal vessels. In sheep placenta, IGF-II mRNA is expressed, not in the trophoblast layer, but in the fetal mesoderm immediately adjacent to it. In the basal plate of human, rhesus monkey and baboon placentae, extravillous trophoblasts express IGF-II mRNA and uterine decidual cells IGFBP 1-6 mRNAs. The inference is that there is interaction between IGF-II and IGFBPs at the maternal-fetal interface of the primate placenta during trophoblast invasion and decidualization. IGFBP-1 expressed by the decidua may also interact with alpha(5)beta(1)integrin expressed by the extravillous trophoblast. The placentae of rodents are also of the invasive type. Glycogen cells of the mouse placenta are analogous with human extravillous trophoblast and express IGF-II mRNA. However, expression of IGFBP mRNAs in the mouse, as in the guinea pig, is confined to non-decidualized endometrium and myometrium. IGF-II mRNA is strongly expressed by trophoblasts invading uterine vessels in human and guinea pig placentae. Interactions probably occur between IGF-II expressed by these trophoblasts and IGFBPs expressed in the vessel walls. However, it is possible that IGFBPs expressed by maternal vessels are associated with processes that are independent of trophoblast invasion. Thus, IGFBP-3 mRNA is highly expressed in the maternal blood vessels of the non-deciduate sheep placenta. Findings to date highlight the diversity in the expression of the IGF system among placentae of man and different laboratory animals, and even between closely related species. Comparative studies will continue to be required to understand the functional role of IGFs and IGFBPs in each species.     

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.     

Proteinase and proteinase inhibitor localization in the human placenta

Standard immunoperoxidase techniques were used to investigate the distribution of the intracellular proteinase cathepsin D, two serine proteinase inhibitors--alpha 1-antitrypsin (alpha 1-AT) and alpha 1-antichymotrypsin (alpha 1-AChy)--and plasma fibrin stabilizing factor XIII (FXIII) in paraffin-embedded tissues from early and late intrauterine pregnancy and ectopic pregnancy. Localization of cathepsin D, alpha 1-AT, and alpha 1-AChy was identical in ectopic and intrauterine gestation: there was labeling of villous syncytiotrophoblast and a proportion of Hofbauer cells but no labeling of villous cytotrophoblast. The majority of interstitial extravillous trophoblast yielded negative results, but alpha 1-AT and alpha 1-AChy were consistently demonstrated in endovascular trophoblast. FXIII was not found in any trophoblast population but was demonstrated in Hofbauer cells, stromal fibroblasts, and interstitial dendritic cells. Granular, extracellular FXIII reactivity was present among sheets of infiltrating extravillous trophoblast in ectopic pregnancy but only occasionally in early intrauterine pregnancy. The results document further the heterogeneity of trophoblast, with the endovascular trophoblast forming an immunophenotypically distinct population. Furthermore, the pattern of extravillous trophoblastic invasion of maternal tissues in ectopic pregnancy appears to differ from intrauterine pregnancy; the poor decidualization of tubal mucosa in ectopic pregnancy may play a role in this variation.     

Involvement of PPARγ in Human Trophoblast Invasion

The peroxisome proliferator-activated receptor-γ (PPARγ) is a member of the nuclear receptor superfamily that controls the expression of a large array of genes in a ligand-dependent manner. In the human placenta, PPARγ is specifically expressed in the villous cytotrophoblast and syncytiotrophoblast as well as in the extravillous cytotrophoblastic cells (EVCT) along their invasive pathway. The present study used two cellular models, primary cultures of trophoblastic cells differentiated in vitro in extravillous trophoblastic cells and a cell line (HIPEC65), which was established from a primary culture of EVCT transformed by T-SV40. We observed that natural (15d-PGJ₂) or synthetic ligands of PPARγ (rosiglitazone) inhibit cell invasion in a concentration-dependent manner, with no effect on cell proliferation. This is associated with a modulation of the expression of trophoblastic genes described to be directly involved in the control of EVCT invasiveness, such as GH-V (−20%), TGFβ2 (−30%), PAPP-A (−60%) and IL1β (+300%.). In order to identify PPARγ potential ligands at the fetomaternal interface, we purified LDL (low density lipoprotein) from human sera and oxidized them in vitro in the presence of copper. OxLDL inhibit in vitro extravillous trophoblast cell invasion, whereas native LDL have no effect. In situ OxLDL and their LOX-1 receptor, as well as PPARγ are immunodetected in trophoblasts at the maternofetal interface.     

Osteopontin is down-regulated in hydatidiform mole

OBJECTIVE: Osteopontin (OPN) is a glycoprotein of the extracellular matrix that can bind to different types of receptors including integrins and CD44 receptors. Multiple binding affinity enables OPN to play a role in many physiological and pathological processes. OPN contributes to tumorigenesis in several types of cancers. OPN is also expressed by the endometrium and by trophoblast cells of the chorionic villus in human placenta, where OPN may regulate implantation and placentation in early pregnancies by promoting cell-cell interactions, adhesion, spreading, and migration of trophoblast. Our purpose was to determine the expression of OPN mRNA and protein in hydatidiform mole and in normal placenta of comparable gestational age. METHODS: A total of 13 fresh tissues from complete hydatidiform moles, 2 from partial hydatidiform moles, and 9 from normal placentas were analyzed by performing quantitative real-time PCR on microdissected trophoblast cells and immunohistochemistry on frozen sections of tissue. RESULTS: Our results showed significantly lower expression of OPN mRNA and protein in hydatidiform mole, and in particular complete mole (P = 0.001 by real-time PCR and P < 0.001 by immunohistochemistry) as compared to nermal placenta. CONCLUSION: Although precise molecular mechanisms of gestational trophoblastic diseases have not yet been determined, down-regulation of osteopontin may play an important role in the pathogenesis of molar pregnancy.