Immunohistochemical study of tissue polypeptide antigen (TPA) and cancer antigen 125 (CA125) in the human and cynomolgus monkey placenta,umbilical cord and decidua

Summary Tissue polypeptide antigen (TPA) and cancer antigen 125 (CA125) were studied immunohistochemically by the avidin-biotin immunoperoxidase technique in human and cynomolgus monkey placentae, membranes, umbilical cords and decidua. In early human placentae, TPA was localized mainly in the cell membranes of villous syncytio- and cyto-trophoblast. The cytoplasm of those trophoblastic cells were weakly stained with TPA. The membrane of basal chorionic trophoblast cells was strongly stained with TPA and the cytoplasm stained weakly. In early cynomolgus placentae, similar immunostaining results were obtained. However, the positive stainings for TPA was more marked in the cytoplasm of villous syncytiotrophoblast and basal chorionic trophoblast, and less marked in the cell membrane of villous cytotrophoblast. In early human and cynomolgus placentae, CA125 was not demonstrated immunohistochemically in the villi and basal chorion. In human and cynomolgus term placentae, the villous syncytiotrophoblast and basal and reflected chorionic trophoblast showed similar immunostaining as the early placentae. In addition, TPA was found in the amniotic epithelium in both sorts of placentae. TPA was not detected immunohistochemically in the umbilical cord and decidual cells. While weakly positive stains for CA125 were observed in decidual cells, CA125 was localized mainly in the membrane and cytoplasm of amniotic epithelium in both human and cynomolgus term placentae. TPA and CA125 are thus oncoplacental antigens and the monkey could serve as a model for their investigation.     

Characteristic differences in immunohistochemical localization of new placental proteins (PP1, PP19, PP21) in the human placenta

The immunohistochemical localization in the human placenta of new placental proteins PP1, PP19, and PP21 was clarified using modified indirect enzyme-labeled antibody method and compared with that of pregnancy-specific beta 1-glycoprotein (SP1). The major results are as follows: positive staining for PP1 was seen at the nucleus and cytoplasm of villous cytotrophoblasts, the X cells at the basal plate, and of chorionic trophoblasts, while the decidua cells and amnion were not stained. PP19 was characteristically seen in the nucleus and cytoplasm of syncytiotrophoblasts. X cells in basal plate, chorionic trophoblasts, and maternal leukocytes. The villous cytotrophoblasts, decidua cells, and amnion were not stained. PP21 localization was found at the microvilli and basal membrane of syncytiotrophoblasts and at the cytotrophoblast plasma membrane of the chorionic villus in early gestation. In late gestation, increased staining was seen at the syncytiotrophoblast microvilli and the villous basement membrane, and moderate staining at plasma membrane of the amniotic epithelium and chorionic trophoblasts. SP1 was found only at the syncytiotrophoblast cytoplasm of chorionic villi. Studies using these four placental proteins simultaneously may therefore provide a new key learning about unknown metabolic functions of trophoblasts.     

Immunohistochemical expression analysis of inhibin-alpha and -beta subunits in partial and complete moles, trophoblastic tumors, and endometrial decidua.

The expression of inhibin-alpha subunit has been described in normal placentas, hydatidiform moles, and trophoblastic tumors. We performed a double immunohistochemical expression analysis of inhibin-alpha and inhibin-beta subunits in a cytogenetically well characterized series of 21 complete and 22 partial hydatidiform moles, 2 placental site trophoblastic tumors, and one choriocarcinoma. Syncytiotrophoblastic cells were consistently inhibin-alpha and inhibin-beta positive in all hydatidiform moles and in the one choriocarcinoma. Cytotrophoblast was negative for both subunits in all trophoblastic lesions studied. While villous intermediate trophoblastic cells were consistently inhibin-alpha negative in all hydatidiform moles, focal inhibin-beta immunoreactivity was detected in villous intermediate trophoblast in approximately one third of complete and partial hydatidiform moles. Decidual stromal cells in 40 hydatidiform moles were inhibin-alpha and inhibin-beta positive in approximately one third of cases. Both placental site trophoblastic tumors were inhibin-alpha positive but inhibin-beta negative. Our findings indicate that inhibin-alpha and -beta subunits are consistently coexpressed in syncytiotrophoblast in complete and partial moles. Immunohistochemical detection of inhibin subunits may be useful in the differential diagnosis of trophoblastic lesions.     

Localization and distribution of unconjugated steroid hormones in normal placenta at term

The localization and distribution of unconjugated oestrone, oestradiol-17 beta, oestriol and progesterone were studied in normal term placentae, using subcellular fractionation and indirect immunofluorescence techniques. Radioimmunoassay of these steroids in each subcellular fraction showed that they were detectable mainly in the cytosol fraction. The efficiency of fixatives for retaining steroids in placental tissue during immunohistochemical procedures was studied in order to validate the present experimental techniques. As compared with other fixatives, glutaraldehyde solution produced minimal leakage of steroid hormones from placental tissue. Using an indirect immunofluorescence technique oestrone, oestriol and progesterone were detectable in the cytoplasm of villous syncytiotrophoblast of fixed term placentae.     

Morphometry of the microvillous membrane of the human placenta in maternal diabetes mellitus

The syncytiotrophoblast microvillous membrane of the human placenta has been investigated with quantitative analyses in a group of placentae from diabetic mothers, these placentae being compared to a group of normal placentae. This study has shown that, in the placentae of the diabetic mothers, there was a significant increase in the surface density of the microvilli and in the microvillous surface enlargement factor as compared to those of the controls. Consequently, it has demonstrated that the already large villous surface area found in the placentae of diabetic mothers can be enlarged very significantly by the microvilli, in terms of the total trophoblastic surface area which is in contact with the maternal blood. On a functional basis, these findings support the theory that placental function is probably not adversely affected in these placentae, and that these placentae can efficiently support the growth of large fetuses.     

Plasma levels of eosinophil granule major basic protein in pregnant cynomolgus monkeys.

The sera of all pregnant women contain increased amounts of a protein biochemically and immunologically similar to the eosinophil granule major basic protein (MBP). Immunofluorescence shows that the pregnancy-associated MBP is localized to placental trophoblastic cells. This information raises important questions about the function of pregnancy-associated MBP because of the potential biological functions attributed to its eosinophil counterpart (namely, its potent toxic and cytostimulatory activities). Previous studies demonstrated the presence of an immunologically cross-reactive protein in the placentae and plasma of pregnant non-human primates. Here, plasma MBP levels were measured throughout gestation in cynomolgus monkeys. In early pregnancy, only modest increases in MBP were found in contrast to the sharp rise observed in the first 20 weeks of human pregnancy. During the final one-third of gestation, striking increases in plasma MBP occurred in the monkeys. This parallels the late rise in MBP seen in humans in the third trimester. Thus, the cynomolgus monkey may serve as a model to clarify the role of the MBP in the biochemical events that culminate in parturition.     

Immunohistochemical localization of pregnancy-associated plasma protein A (PAPP-A) in placentae from normal and pre-eclamptic pregnancies

An immunohistochemical method was used to locate pregnancy-associated plasma protein A (PAPP-A) in the placenta and uterus. In addition to 10 placentae and basal plates from normal pregnancies, ranging in gestational age from 37 to 40 weeks, the following specimens were studied: three uteri obtained by hysterectomy during early pregnancy; and three placentae from patients with severe hypertensive pre-eclampsia. In early gestation, PAPP-A was localized in the villous cytotrophoblastic cell layer and the endometrial glands but was not found in the villous syncytiotrophoblast, the cytotrophoblastic cell columns or the decidual cells. On histochemical examination of placentae from cases of pre-eclampsia with hypertension, an increased number of villous cytotrophoblastic cells and so-called X-cells was observed. The monospecific antiserum to PAPP-A reacted strongly and evenly with the cytoplasm of these cells. The present study strongly suggests that the active production sites of PAPP-A are the villous cytotrophoblastic cells and the X-cells.     

Immunohistochemical localization of HLA antigens and placental proteins (alpha hCG, beta hCG CTP, hPL and SP1 in villous and extravillous trophoblast in normal human pregnancy: a distinctive pathway of differentiation of extravillous trophoblast

Immunohistochemical localization of HLA antigens and placental proteins (alpha hCG, beta hCG CTP, hPL and SP1) in villous and extravillous trophoblast at various stages of normal human gestation were studied, using hysterectomy specimens. In the chorionic villi, the capacity for synthesizing placental proteins seemed to develop in parallel with the morphological change from mononuclear cells to multinucleated syncytiotrophoblast and no villous trophoblast expressed HLA antigens. In contrast, extravillous trophoblast, including the multinucleated trophoblastic cells at the deciduomuscular junction, expressed HLA-A, -B, and -C, and their capacity for synthesizing placental proteins did not seem to correspond with the degree of morphological change: the location of alpha hCG, beta hCG CTP and SP1 was restricted to mononuclear trophoblast in the superficial decidua, while hPL was present extensively in extravillous trophoblast. These findings strongly suggest that extravillous trophoblast possesses many distinctive biological features and differentiates in an independent manner. Mononuclear trophoblast forming the cell columns was also positive for HLA-A, -B, and -C, and no placental protein was demonstrated in these cells; this, together with previous morphological observations, may indicate the germinative nature of these cells.     

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 of pregnancy-associated plasma protein-A (PAPP-A) during human villous trophoblast differentiation in vitro

Pregnancy-associated placental protein-A (PAPP-A), first isolated from maternal serum, has been identified as a metalloprotease cleaving insulin-like growth factor binding protein-4 (IGFBP-4). The source of PAPP-A during pregnancy is unclear. We therefore investigated PAPP-A expression during in vitro human villous cytotrophoblast cell (CT) differentiation into syncytiotrophoblast (ST). CT were isolated from normal first trimester, second trimester and term placentae (n=10) and cultured to form ST. PAPP-A mRNA was quantified by real-time PCR, and PAPP-A protein expression was studied by immunocytochemistry and TRACE technology with specific monoclonal antibodies. PAPP-A mRNA expression in total placental extracts increased during the course of pregnancy. PAPP-A protein was detected in the cytoplasm of both CT and ST. ST formation in vitro was associated with a 19-fold increase in PAPP-A mRNA expression and an 8-fold increase in PAPP-A secretion into the culture medium. No significant difference in PAPP-A production was observed between cultured cells isolated from early and term placentae. In conclusion, PAPP-A production in vitro, is associated to the differentiation of villous cytotrophoblast cells into syncytiotrophoblast, independently of the age of gestation.     

Intrauterine growth retardation: morphometry of the microvillous membrane of the human placenta

The syncytiotrophoblast microvillous membrane of the human placenta has been investigated with quantitative analyses in cases of severe fetal growth retardation associated with a marked reduction in the surface area of exchange at the peripheral villous level. This study has shown that, in placentae of intrauterine growth-retarded infants of unknown origin, there were morphological changes in the microvillous membrane characterized by an increase in the microvillous surface density and surface enlargement factor, associated with a reduction of the intermicrovillous space. It is not possible to state whether these morphological changes represent a delayed maturation of the placental tissue, or compensatory mechanisms to improve the functional efficiency of the placenta. In pre-eclampsia, these placental changes were much less pronounced, possibly due to severe uteroplacental ischaemia in this complication of pregnancy. Despite these morphological changes, both groups of placentae showed significant reductions in absolute values for the microvillous and total trophoblastic surface areas, which can have major implications on the functional efficiency of the placenta.     

Immunohistochemical demonstration of epidermal growth factor (EGF) receptors in normal human placental villi

With an avidin-biotin-peroxidase (or glucose oxidase) complex method using anti-epidermal growth factor receptor monoclonal antibody (528 IgG), the tissue and cellular distribution of the receptors for epidermal growth factors (EGF) in normal human placental villi, from 6 to 42 weeks of gestation, were studied. EGF receptors were mainly localized on the free surface of the syncytiotrophoblast that directly faced to intervillous space of the maternal circulation. The cell surface of cytotrophoblasts, except for the region that was adjacent to the basal lamina, was also positive for EGF receptors. The receptors were in close contact to the fetal vessels in the villous stroma. The EGF receptors on the syncytiotrophoblast were thought to be involved in the production and secretion of human chorionic gonadotropin and placental lactogen, probably under the control of maternal EGF. The receptors on cytotrophoblasts may play a role in trophoblastic proliferation, possibly mediated by EGF in the fetal circulatory system.     

The distribution of activin and activin receptors in gestational tissues across human pregnancy and during labour

The aim of this study was to investigate localization and content of activin beta A-subunit and activin receptors in gestational tissues throughout pregnancy and in association with term labour. Placenta and fetal membranes were collected from women undergoing first and second trimester terminations and from women before and after term labour. Activin beta A-subunit and activin receptors IA, IB, IIA and IIB were studied by immunohistochemistry. Term tissues were analysed for activin A and follistatin content by ELISA and the presence of receptor proteins was assessed by Western hybridization. Activin beta A-subunit was localized to the syncytiotrophoblast and cytotrophoblast in placentae from all gestational ages, and to the amniotic epithelial and chorionic trophoblast layer at term. In placentae of first and second trimester, receptor proteins were localized to the syncytium, whereas at term, the distribution was confined predominantly to vascular endothelial cells of villous blood vessels. Receptor proteins in amnion were localized to some epithelial cells, mesenchyme and chorionic trophoblast. These findings suggest that activin A is secreted by and targets the placental syncytium and amniotic epithelium in early pregnancy, but at term targets the vascular endothelium of placenta and the fetal membranes. There were no differences with labour onset.     

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.     

Increased endothelial thrombomodulin (TM) expression in pregnancies complicated by IUGR

Thrombomodulin (TM) is a cell surface receptor playing an important role in endothelial cell anticoagulant activity. TM is also known as a factor of angiogenesis; low TM activity correlates with impaired angiogenesis. Since vascular lesions with disorders of the placental coagulation and inadequate angiogenesis can be associated with IUGR, we hypothesized that thrombomodulin expression in the villous vasculature and syncytiotrophoblast of placentae complicated by IUGR might differ from those of normal pregnancies. Representative tissue samples of normal, IUGR as well as 1st and 2nd trimester (n = 12) placentae were collected. Immunohistochemistry (APAAP) of paraffin tissue sections was performed using monoclonal antibodies specific for TM and PECAM. The percentage of immunopositive vessels and the intensity of immunoreactivity was assessed. Vascular endothelium and syncytiotrophoblast stained positive for TM. Immunoreactivity for TM in villous vasculature rose significantly with gestational age. Villous vessels of IUGR placentae, showed a higher expression of TM, compared to placentae of appropriately grown fetus (p < 0.05). The number of terminal villi and the number of blood vessels per intermediate villi was significantly reduced in IUGR placentae (p < 0.05). These differences reflect inadequate vascularisation and impaired angiogenesis in IUGR.     

Paradoxical discrepancy between the serum level and the placental intensity of PP5/TFPI-2 in preeclampsia and/or intrauterine growth restriction: possible interaction and correlation with glypican-3 hold the key

There have been controversies whether maternal serum placental protein 5 (PP5)/tissue factor pathway inhibitor (TFPI)-2 is increased in the patients with preeclampsia and/or intrauterine growth restriction (IUGR). Here, we have estimated the serum PP5/TFPI-2 in these patients by a sandwich enzyme-linked immunosorbent assay with a newly developed monoclonal antibody, coupled with placental immunohistochemical studies of their placentae with semiquantitative scoring. Serum PP5/TFPI-2 level was significantly elevated only in the patients with preeclampsia alone (p=0.033), while PP5/TFPI-2 was detected significantly less intensely in the placentae of the same patients (p=0.035) in immunohistochemistry, as compared to Controls. A proteoglycan present on the placental villous surface, glypican-3, showed the same pattern of staining as PP5/TFPI-2, and there was a positive correlation (C.I.=0.506, p=0.004) between the immunohistochemical scores for these. Further experiments using HepG2 cells transfected with PP5/TFPI-2 suggested that glypican-3 could anchor PP5/TFPI-2 on the placental villi. A possibility that a decrease in glypican-3 in the placenta increases the outflow of PP5/TFPI-2, which in turn increases its serum level, was proposed. Preeclampsia and IUGR, often regarded to have the same pathological basis in common, showed distinct distributions of PP5/TFPI-2, which could be a clue to elucidate the pathogenesis of preeclampsia and IUGR.     

Immunofluorescent localization of placental lactogen, chorionic gonadotrophin and its alpha and beta subunits in organ cultures of human placenta

Localization of human placental lactogen (HPL), chorionic gonadotrophin (HCG) and its free alpha and beta subunits in mature and immature placental villi before and during organ culture was examined with an 'indirect' immunofluorescent technique using highly specific antisera. HPL fluorescence was strictly localized to the syncytiotrophoblast and the intensity of this fluorescence increased with gestational age and decreased with the time of culture. Undissociated HCG and HCG beta immunofluorescence was localized to the syncytiotrophoblast. Maximum intensity was observed in immature placentae and was not significantly affected by the duration of culture. However, irregular and patchy HCG and HCG beta immunofluorescence was seen in the cytotrophoblasts under conditions of extensive syncytiotrophoblastic damage. HCG alpha immunofluorescence was localized in the syncytiotrophoblast of immature placentae and was more intense in mature placentae. Beginning the third day of culture, HCG alpha fluorescence increased and was also present in the cytotrophoblast. On the basis of these observations and additional data, the possibility is discussed that cytotrophoblastic cells, better preserved than the syncytiotrophoblast in case of restricted energy and oxygen supply, may actively synthesize free HCG alpha, in addition to syncytiotrophoblastic production of this subunit. By contrast, HPL, undissociated HCG, and HCG beta are mainly or exclusively eleborated in the syncytiotrophoblastic layer.     

Placental proteins (PP 5, PP 12 and PP 14) in ovarian tumors. A preliminary report

Using a specific radio-immunoassay, the placental proteins PP 12, PP 14 and PP 5 were studied in serum for 2 patients with ovarian cancer and 7 with benign ovarian tumor. Elevated levels of PP 14, found in one endometroid ovarian cancer, decreased during successful treatment. PP 14 could also be found in the blood of nude mice grafted with this tumor. One patient with serous kystadenocarcinoma had a slightly elevated level of PP 14 at diagnosis, increasing as the tumor progressed. PP 14 was demonstrated histochemically in both the original tumor and the grafted. Ascitic fluid was rich in PP 14. Elevated levels of the proteins PP 12 and PP 5 were not found in these patients, however. In seven benign ovarian tumours, low PP 14 serum levels were found. The conclusion drawn from this study is that PP 14 can constitute a tumor marker in ovarian cancer.