Localization and purification of SBU-4--a pregnancy specific protein of the ovine uterus

In order to elucidate the function of molecules of the ovine maternal-fetal interface a monoclonal antibody was produced to intact interplacentomal trophoblast membranes. Extensive immunohistological studies revealed that the monoclonal antibody recognizes a protein designated SBU-4 which originates in the intercaruncular regions of the gravid sheep uterus at about the time of implantation and increases in concentration throughout gestation. The data suggest that SBU-4 is produced by endometrial epithelial cells and that adjacent uninucleate cells of the trophoblast acquire the antigen by endocytosis. Initial biochemical analysis of the purified SBU-4 molecule prepared by monoclonal antibody immunoaffinity chromatography indicates that SBU-4 is high molecular weight glycoprotein complex comprising several sub-units.     

Detection of fetal-specific DNA after enrichment for trophoblasts using the monoclonal antibody LK26 in model systems but failure to demonstrate fetal DNA in maternal peripheral blood

Trophoblast cells can be detected in maternal blood during normal human pregnancy and DNA from these cells may be used for non-invasive prenatal diagnosis of inherited diseases. The possibility of enriching trophoblast cells from maternal blood samples using a monoclonal antibody (LK26) against a folate-binding protein, which recognizes trophoblast in normal tissues, in conjunction with immunomagnetic cell sorting was investigated. Verification of the presence of fetal DNA in the sorted samples was done by detection of fetal/paternal-specific short tandem repeat (STR) alleles using polymerase chain reaction (PCR) and automated fluorescence-based genotyping. After successful initial experiments using retroplacental blood samples with a high number of trophoblast cells or an artificial mixture of trophoblast cells and blood, several versions of the enrichment method were attempted on peripheral maternal blood samples. However, it was not possible to detect fetal DNA sequences in these samples, most probably due to the extremely low number of trophoblast cells. Positive identification and retrieval of trophoblast cells in suspension or trophoblast nuclear material prepared on microscope slides after cell sorting procedures can be a solution to this problem.     

Ultrastructural immunogold investigation of the function and diversity of binucleate cells in the ovine placenta using a monoclonal antibody

Ultrastructural immunogold labelling of ovine placentomes demonstrated that the molecule recognized by the monoclonal antibody SBU-3 is restricted to the fetal binucleate cell granules and Golgi body, and granules of similar size in the syncytium. Quantitative examination shows that the percentage of placentomal mature binucleate cells that are SBU-3-positive increases rapidly from a low level at 29 days of pregnancy to a plateau at virtually 100 per cent from 41 days to term, whereas interplacentomal binucleate cells rarely show label at any stage. There were no detectable differences in ultrastructure between SBU-3-positive or -negative binucleate cells. These results corroborate the hypothesis of syncytium formation by migration of binucleate cells and indicate local control of SBU-3 production and its possible role in villus formation.     

Cytokeratin antibodies as differential markers of trophoblast and fetomaternal syncytial plaques in the sheep placentome

The sheep placenta is an often used model in placental research. Uterine epithelium and trophoblast of this synepitheliochorial placenta form a complex, intensely interdigitating epithelial barrier separating maternal and fetal organisms. The close topographical relation and additionally the presence of hybrid syncytia formed by focal fusion of both epithelia hamper identification of the various cellular constituents. Therefore we aimed to find a specific immunohistochemical marker differentiating between the fetomaternal syncytial plaques and trophoblast. A monoclonal antibody directed against type II cytokeratins strongly stained unicellular trophoblast. The syncytial plaques were only weakly stained while binucleate trophoblast remained unstained. This antibody proved to be a useful tool for easy histological orientation in the sheep placenta. In combination with other antibodies in double immunohistochemistry it facilitates exact localization of antigens.     

A trophoblast specific antigen, recognized by monoclonal antibody MA21, locates a unique trophoblast cell population in the murine placenta

Monoclonal antibody MA21 recognized a 44kDa plasma membrane protein on F9 teratocarcinoma cells, trophectoderm of mouse peri-implantation-stage blastocyst and ectoplacental cone cells of 5 day postcoitum implanted blastocyst (Vernon, Linnemeyer and Hamilton, 1989). We show here that this antigen is expressed by trophoblast cells of the maturing placenta. Immunohistochemical assays of early and mature placental tissue sections, indirect immunofluorescence labelling of placental cultures and blastocyst outgrowths in vitro, and immunoprecipitation of 35S-labelled NP-40 extracts of placental cultures indicate the presence of a plasma membrane-associated antigen with the same characteristics as MA21 antigen of peri-implantation embryos and F9 teratocarcinoma cells. In sections of placentae, antigen-positive cells are always situated in a thin layer between trophoblastic giant cells and maternal tissue. In cultures of postimplantation stage embryos, attached trophoblast cells express MA21 antigen initially, but following transformation to the giant cell state, antigen is no longer expressed. These results indicate the presence of a plasma membrane protein antigen associated with a distinct population of cells believed to be trophoblast. We believe that these cells are the foremost trophoblast cells opposing maternal decidua and that they may give rise to secondary trophoblastic giant cells.     

Immunohistological characterization of endometrial gland epithelium and extravillous fetal trophoblast in third trimester human placental bed tissues

Summary. The distribution, morphology and antigen expression of endometrial glands, uterine vessels and fetal trophoblast have been studied in third trimester placental bed tissues with a panel of monoclonal antibodies in immunohistochemical techniques. Residual endometrial glands were numerous but often were attenuated or compressed and could only be identified clearly with epithelial cell markers. These glands must be clearly distinguished from vessels and trophoblast in immunological studies of cells in the placental bed. The changing pattern of antigen expression of both maternal glands and fetal trophoblast in placental bed tissues may indicate a form of local regulation of gene expression.     

Immunohistological characterization of endometrial gland epithelium and extravillous fetal trophoblast in third trimester human placental bed tissues

The distribution, morphology and antigen expression of endometrial glands, uterine vessels and fetal trophoblast have been studied in third trimester placental bed tissues with a panel of monoclonal antibodies in immunohistochemical techniques. Residual endometrial glands were numerous but often were attenuated or compressed and could only be identified clearly with epithelial cell markers. These glands must be clearly distinguished from vessels and trophoblast in immunological studies of cells in the placental bed. The changing pattern of antigen expression of both maternal glands and fetal trophoblast in placental bed tissues may indicate a form of local regulation of gene expression.     

Monoclonal antibody GB17 recognizes human syncytiotrophoblast

A monoclonal antibody, GB17, was obtained from a hybridoma produced following murine immunization with isolated human term syncytiotrophoblastic microvilli. Immunohistological studies demonstrated the GB17 antigen to be present on syncytiotrophoblast of human term, first- and second trimester placenta, but not on human extravillous trophoblast or baboon placental trophoblast. Other normal adult human tissues and transformed cell lines tested were non-reactive with this antibody. Radio-iodinated term syncytiotrophoblastic microvillous protein was immunoprecipitated by GB17, and shown by sodium dodecyl sulfate--polyacrylamide gel electrophoresis to migrate as a single protein band at 175 kDa. The restricted tissue specificity suggests that the GB17 antigen could be useful for the development of a contragestational vaccine.     

HLA-G expression in trophoblast cells circulating in maternal peripheral blood during early pregnancy

OBJECTIVE: The aim of this study was to assess the use of circulating trophoblast cells in maternal peripheral blood for noninvasive prenatal diagnosis of numeric chromosomal aberrations. STUDY DESIGN: A combined procedure for immunocytochemical identification and deoxyribonucleic acid fluorescence in situ hybridization was used after a single enrichment step consisting of density gradient centrifugation. A specific HLA-G monoclonal antibody was used in combination with X and Y chromosome specific probes in deoxyribonucleic acid fluorescence in situ hybridization to confirm fetal identity of cells bearing HLA-G in the case of a male fetus. RESULTS: We detected fetal trophoblast cells expressing HLA-G in maternal blood starting at 9 weeks' gestation. In addition to fetal sex prediction with X and Y chromosome-specific probes, fetal aneuploidy was confirmed in peripheral blood from a pregnancy complicated by trisomy 21. CONCLUSION: Although the numbers of fetal cells were extremely low, the proof of concept was demonstrated. Early noninvasive prenatal screening for numeric chromosomal abnormalities with fetal trophoblast cells is feasible.     

Macroscopic and microscopic aspects of collared peccary and white-lipped peccary placenta

This study examines middle and late gestational placentae from 13 Tayassu tajacu (collared peccary) and 3 Tayassu pecari (white-lipped peccary), which are Artiodactyla belonging to the Family Tayassuidae. The chorionic sac of Tayassu species is diffuse and chorioallantoic. These epitheliochorial placentae show no trophoblast invasion into the uterine epithelium and there is interdigitation between fetal and maternal microvilli. Two distinct regions of the fetomaternal interface can be identified: the interareolar and the areolar regions. The uterine epithelium has eosinophilic cytoplasm with dispersed, basophilic and electron-dense granules. Trophoblast cells are irregularly cuboidal on top of the fetal ridges and columnar on troughs, where cells have cytoplasmic vesicles and large basal vacuoles, surrounded by whorls of smooth membranes. Capillaries indent the trophoblast cells forming a placental barrier 3 microm or less thick. The columnar uterine glandular epithelium has a subpopulation of granules staining with Perl's Prussian blue reaction, suggesting iron secretion. In areolar areas, the trophoblast cells show apical microvilli, a basophilic cytoplasm with electron-dense intracellular vacuoles and cisternae. The placenta can therefore be classified as non-deciduate. The ultrastructural aspects of this study reveal features that have not previously been described and extend our knowledge of functions relating to materno-fetal transport in these species.     

The rat placenta expresses paternal class I major histocompatibility antigens

The expression of paternally inherited class I MHC antigens on the placental trophoblast of the rat has been investigated using a mouse anti-rat monoclonal antibody (MN4-91-6) in an indirect immunoperoxidase labelling assay on cryostat sections. Strong specific staining was obtained on the spongy zone trophoblast of the mature placenta from DA male (RT1a) X PVG female (RT1c) matings. In marked contrast, no staining was observed on the labyrinthine trophoblast nor on the trophoblastic giant cells at any stage of gestation from 8 to 19 days post-coitum. None of the trophoblastic cell populations at any stage of gestation were reactive with an anti-class II monoclonal antibody. Class I positive endovascular cytotrophoblast cells were present in the maternal arterial sinusoids of the decidua. These findings imply that maternal immunoregulatory mechanisms must be essential for the survival of the placenta and fetus.     

HLA antigen on the trophoblast of normal pregnancy

The mechanism of fetal survival as a semiallograft in the uterus remains to be clarified. In this context, the expression of HLA antigen on the trophoblast which stands between the mother and the fetus is the main problem, because HLA antigen plays an important role in immunological reaction to an allograft. For this purpose, 41 pregnant uteri (6-18 weeks of gestation) were examined immunohistochemically (avidin-biotin-peroxidase complex method) using monoclonal antibodies to HLA antigens. Troma 1, a rat monoclonal antibody, was used as a trophoblast marker in immunohistochemical studies. The results were as follows: HLA-A,B,C is not expressed on syncytiotrophoblast and villous cytotrophoblast. HLA-A,B,C is expressed on nonvillous cytotrophoblast which exists in cytotrophoblastic cell column, cytotrophoblastic shell, and endometrium. HLA-DR is not expressed on any trophoblast. The absence of HLA antigen on syncytiotrophoblast and villous cytotrophoblast seems to be essential for the survival of the fetus. But it is proved that some trophoblasts express HLA-A,B,C on their cell surface and they are adjacent to endometrial cells or maternal blood. From these findings, it seems that fetal HLA antigen might be recognized by the mother and there might be an exquisite immune escape mechanism in decidua.     

Purification of human cytotrophoblast from term amniochorion by flow cytometry

Term cytotrophoblast do not express polymorphic MHC Class I antigens, unlike other fetal and maternal cells in the amniochorion/decidua. This allows cytotrophoblast to be isolated and purified from this tissue, utilizing 4E, a monoclonal antibody specific for HLA-B, which labels only non-trophoblast. We have developed a method using enzymic dispersion and Percoll gradient centrifugation, followed by flow cytometry, that yields, on average, a total of 5 X 10(6) term extravillous cytotrophoblast, 97 per cent pure. The availability of highly purified extravillous cytotrophoblast, for the first time, permits precise investigation of trophoblast function.     

Antigen expression by trophoblast populations in the human placenta and their possible immunobiological relevance

Antigen expression by villous and extravillous human trophoblast populations at discrete anatomical sites has been reviewed. The various different antigenic phenotypes have been highlighted using a panel of monoclonal antibodies reactive with characteristic trophoblast membrane antigens, a trophoblast-leucocyte common antigen, class I MHC antigens, epithelial cell cytokeratin and epithelial membrane markers. This approach has allowed three separate fetal trophoblast populations to be identified within term amniochorionic membranes, and also has facilitated further definition of trophoblast populations in maternal uterine tissues. Furthermore, antigenic alterations have been noted in the maternal uterine gland epithelium in pregnancy leading to the expression of a trophoblastic phenotype, thereby suggesting a mechanism of extrinsic regulation of gene expression in these tissues. The possible involvement in the immunoregulatory control of maternal responses in pregnancy of MHC-linked gene products expressed by trophoblast has been discussed.     

Expression of galectin-1, -3 (gal-1, gal-3) and the Thomsen-Friedenreich (TF) antigen in normal, IUGR, preeclamptic and HELLP placentas

BACKGROUND: Galectin-1 (gal-1) and galectin-3 (gal-3), which are members of the mammalian beta-galactoside-binding proteins, recognise preferentially (Galbeta1-4GlcNAc) sequences of several cell surface oligosaccharides. In addition, gal-1 also binds to the Thomsen-Friedenreich (TF) antigen (Galbeta1-3GalNAc-). MATERIALS AND METHODS: Slides of frozen and paraffin-embedded placental tissue of patients with fetal intrauterine growth retardation (IUGR), preeclampsia, haemolysis, elevated liver enzymes, low platelets (HELLP) and normal term placentas were incubated with monoclonal and polyclonal antibodies against gal-1, gal-3 and TF. Staining reaction was performed with the avidin-biotinylated peroxidase complex (ABC) reagent. The intensity of the immunohistochemical reaction on the slides was analysed using a semi-quantitative score. The identity of galectin-expressing cells was analysed by using a double immunofluorescence method. RESULTS: We demonstrated immunohistochemically that the expression of gal-1 and gal-3 on the extravillous trophoblast (EVT) is significantly up-regulated in preeclamptic and HELLP placentas and unchanged compared with normal controls in IUGR placentas. The expression of the TF antigen is significantly up-regulated in IUGR and preeclamptic extravillous trophoblast cells and unchanged in HELLP placentas compared with normal controls. In addition, the expression of gal-1 is significantly up-regulated in the decidual tissue of preeclamptic placentas and in the villous trophoblast tissue of HELLP placentas. CONCLUSION: Our data showed that gal-1, gal-3 and TF were up-regulated on the membrane of EVT in preeclamptic placentas. In addition, the expression of gal-1 is significantly up-regulated in decidual tissue of preeclamptic placentas and villous trophoblast tissue of HELLP placentas. Taking into consideration the results of this study, we speculate that expression of both galectins and TF on the membrane of preeclamptic EVT and up-regulation of gal-1 in preeclamptic decidual cells may at least in part compensate for the apoptotic effects of maternal immune cells.     

Antibody reaction patterns in first trimester placenta: implications for trophoblast isolation and purity screening

The aim of this immunohistochemical and cytochemical study was to select specific antibodies to establish an efficient purification protocol for first trimester trophoblast and for subsequent purity screening of isolated trophoblast cells. The reactivity of antibodies to various cytokeratin filaments, glycoprotein CD9, fibroblast specific antigen (FSA), common leukocyte antigen CD45RB and macrophage antigens CD163, CD68 and CD14 were studied on cryosections of placental tissue. Among the cytokeratins tested, cytokeratin 7 was the only keratin filament type, which was not expressed in placental mesenchymal cells, but in all trophoblast subpopulations. Since anti-CD9, in addition to mesenchymal cells, also strongly labels extravillous cytotrophoblast cells, whereas the antibody to FSA only reacts with mesenchymal cells, anti-FSA is suitable as a depletion antibody for mesenchymal cells. Among the macrophage markers anti-CD163 was the most specific for Hofbauer cells. CD45RB was expressed on maternal and fetal leukocytes as well as on Hofbauer cells. Isolated first trimester placental cell preparations that have been collected from a density gradient contained up to 45 per cent non-trophoblast cells. Immunocytochemistry using antibodies to CK7, FSA, vimentin, CD45RB and CD163 demonstrated that subsequent immunodepletion with antibodies to CD45RB and FSA increased the purity of the trophoblast preparation to greater than 98 per cent.According to this study trophoblasts from first trimester placentae should be identified by cytokeratin antibodies specific for the isoform 7. Purification of isolated trophoblasts by density gradient alone does not result in a sufficient degree of purity.     

Studies on the interaction between trophoblastic invasion and maternal immune cell infiltration at the implantation site in early human pregnancy by means of double immunoperoxidase technic using monoclonal antibodies

Interaction between trophoblastic invasion and maternal immune cell infiltration at the implantation sites in early human pregnancy was analyzed by means of a double immunoperoxidase technique using Troma-1, a rat monoclonal antibody, which recognizes trophoblastic cells and a set of mouse monoclonal antibodies to react with various immune cells. The results were as follows. The most prominent immune cells in the implantation sites were monocytes/macrophages, which were positive for HLA-DR. These cells were adjacent to trophoblastic cells which were infiltrating into the decidua basalis. It therefore appeared that these cells function as "antigen presenting cells" which recognize and present the processed fetal information to maternal T cells. A small number of cells with mature T cell markers were found to be infiltrating around the anchoring villi and the extra-villous trophoblastic cells in the decidua compacta. But a larger number of T cells were adjacent to the villi in the decidua spongiosa and the extra-villous trophoblastic cells invading the decidua spongiosa and the myometrium. These cells may therefore play a role in preventing trophoblastic cells from invading the myometrium in the implantation sites. A relatively large number of cells with E rosette receptors but without mature T cell markers were observed in the decidua basalis, but few were found in the myometrium, into which a larger number of mature T cells were infiltrating. The distribution of particular cells was similar to that of endometrial granulocytes studied in our laboratory. There were thus likely to be immune cells in humans equivalent to non-T granulated suppressor cells in mice, which have been shown to suppress the generation of cytotoxic T cells (Clark et al.).     

Enrichment and identification of fetal trophoblast cells from first trimester maternal cervical lavage and uterine blood specimens

First trimester prenatal diagnosis of fetal aneuploidies is an active area of research despite years of disappointing data employing maternal peripheral blood samples. To remedy this situation we have investigated other first trimester maternal specimens attempting to find a consistent fetal cell source. Using our previously established positive enrichment procedure along with a commercially available depletion method, fetal trophoblast cells were identified employing immunocytochemistry using an antibody cocktail or by using mRNA in-situ hybridization employing a cocktail of trophoblast specific probes. Fetal origin of positively identified cells was verified using interphase fluorescent in-situ hybridization (FISH) for X and Y-chromosomes. Artificial model systems were established that indicated yields of trophoblast cells and allowed the enrichment procedure to be optimized for minimal losses from maternal specimens.We demonstrate herein that blood drawn from maternal vessels near the placental implantation site to be the most consistent source of fetal cells from any first trimester maternal specimen described to date. In addition, a high yield of multinucleated syncytiotrophoblast cells was obtained using a cell depletion strategy to enrich the target cells. The safety of the procedure or even the clinical utility of blood drawn from maternal vessels near the placental implantation site is yet to be demonstrated.     

Cross-reactivity of monoclonal antibodies directed against lymphocyte markers with trophoblast cells of normal placenta, hydatidiform mole, and gestational choriocarcinoma

The current study was undertaken to characterize the expression of trophoblast-lymphocyte cross-reactive antigens on normal, molar, and malignant trophoblast. A panel of monoclonal antibodies directed against lymphoid cell markers were tested in immunofluorescence assay on cryostat sections of placenta and mole and on monolayers of choriocarcinoma cells. NKH-1, a monoclonal antibody to natural killer cells, reacted with both molar and placental villous trophoblast and with two choriocarcinoma cell lines. NKH-2, a monoclonal antibody reactive with a subset of natural killer cells, did not react with placental villous trophoblast but reacted with molar villous trophoblast in three of five moles tested and with both choriocarcinoma cell lines. B5, a monoclonal antibody which reacts with activated B cells, reacted with both choriocarcinoma cell lines but did not react with normal placental or molar trophoblast. MY7, a monoclonal antibody to myeloid colony-forming cells, reacted with only one of the choriocarcinoma cell lines. Trophoblast-lymphocyte cross-reactive antigens may be important in the immunobiology of gestational trophoblastic disease by modulating interactions between the trophoblast and the maternal immune system.     

Antigen presenting capacity of human decidua: no evidence for human decidual antigen presenting cell mediated immunoregulation.

Decidual antigen presenting cell (APC) mediated maternal immunoregulation has been reported. In the present study the ability of villous chorion as well as fetal cell pulsed early human pregnancy decidual APC to generate selectively antigen non-specific and MHC class II unrestricted CD8 positive T suppressor cells was reassessed in view of the fact that placental trophoblast, unlike the fetus, constitutes the fetal tissue of major contact at the maternal-fetal interface. Neither fetal cell nor villous chorion pulsed decidual APC generated maternal T cells with the ability to immunosuppress PHA-, Con A- and PWM-induced autologous or allogeneic lymphoproliferation. In only 2 out of 45 assays with villous chorion pulsed decidual APC was significant inhibition of mitogen induced lymphoproliferation detected and on no occasion with fetal cell pulsed decidual APC. No change in CD4/CD8 ratio of the maternal putative regulatory cells was detected by FACS analysis compared with control cultures. These findings suggest that decidual APC mediated immunoregulation plays no role in directing the maternal immune response.