Chorion laeve trophoblasts of preeclamptic fetal membranes: histochemically detectable enzyme activities do not change at a subcellular level.

We examined the subcellular localization of ADP-degrading activity and cytochrome c oxidase (CCO) activity in chorion laeve trophoblasts from term and near term human fetal membranes, and compared them with those from severe preeclamptic fetal membranes. The methods used for the detection of enzyme activities were the lead nitrate method for ADP-degrading activity and the diaminobenzidine method for CCO. Precipitates indicative of ADP-degrading activity were visible on surface microvillous plasma membranes of chorion laeve trophoblasts both from normal and preeclamptic fetal membranes. The intensity and distribution patterns were the same in the normal and preeclamptic subjects. CCO labeling was visible in almost all laeve trophoblastic mitochondria both in normal and preeclamptic cases. Previously, we demonstrated that in preeclamptic villous trophoblasts there were decreases in ADP-degrading activity and the presence of CCO-negative mitochondria, which were proposed to lead to dysfunction of each villous trophoblast, and finally to placental insufficiency in preeclampsia. Reductions or changes in enzyme intensities/distribution patterns, which are characteristic features of preeclamptic villous trophoblasts, were absent in chorion laeve trophoblasts in preeclampsia. These results suggest that in preeclampsia there are no, or at least less severe, abnormalities in the enzyme activities of chorion laeve trophoblasts, compared with villous trophoblasts, as far as enzyme-histochemically detectable enzymes are concerned.     

Human cytomegalovirus infection of placental cytotrophoblasts in vitro and in utero: implications for transmission and pathogenesis

Human cytomegalovirus (CMV) is the leading cause of prenatal viral infection. Affected infants may suffer intrauterine growth retardation and serious neurologic impairment. Analysis of spontaneously aborted conceptuses shows that CMV infects the placenta before the embryo or fetus. In the human hemochorial placenta, maternal blood directly contacts syncytiotrophoblasts that cover chorionic villi and cytotrophoblasts that invade uterine vessels, suggesting possible routes for CMV transmission. To test this hypothesis, we exposed first-trimester chorionic villi and isolated cytotrophoblasts to CMV in vitro. In chorionic villi, syncytiotrophoblasts did not become infected, although clusters of underlying cytotrophoblasts expressed viral proteins. In chorionic villi that were infected with CMV in utero, syncytiotrophoblasts were often spared, whereas cytotrophoblasts and other cells of the villous core expressed viral proteins. Isolated cytotrophoblasts were also permissive for CMV replication in vitro; significantly, infection subsequently impaired the cytotrophoblasts' ability to differentiate and invade. These results suggest two possible routes of CMV transmission to the fetus: (i) across syncytiotrophoblasts with subsequent infection of the underlying cytotrophoblasts and (ii) via invasive cytotrophoblasts within the uterine wall. Furthermore, the observation that CMV infection impairs critical aspects of cytotrophoblast function offers testable hypotheses for explaining the deleterious effects of this virus on pregnancy outcome.     

Death receptor Fas/Apo-1/CD95 expressed by human placental cytotrophoblasts does not mediate apoptosis

Trophoblasts, the fetal cells that line the villous placenta and separate maternal blood from fetal tissue, express both Fas antigen and the tumor necrosis factor (TNF) receptor p55 (TNFRp55), two members of the TNF receptor family that contain a cytoplasmic "death domain" that mediates apoptotic signals. We show that Fas mRNA expressed by cultured villous cytotrophoblasts isolated from term placentas encodes transmembrane sequences and that the protein is full-length (approximately 45 kDa), suggesting that the product is an active plasma membrane-anchored receptor. Its location on the cell surface was confirmed by cellular ELISA analysis of live cells. Although cytotrophoblast apoptosis was induced by TNFalpha, and both anti-Fas antibody (CH11) and FasL-expressing T lymphocyte hybridoma (activated A1.1) cells induced HeLa cell apoptosis, neither CH11 antibody nor activated A1.1 cells stimulated apoptosis in term or first-trimester cytotrophoblasts or in term syncytiotrophoblasts. We conclude that Fas- but not TNFRp55-mediated apoptosis is blocked in primary villous trophoblasts. These data suggest that the Fas response is specifically inactivated by unknown mechanisms to avoid autocrine or paracrine killing by Fas ligand constitutively expressed on neighboring cyto- or syncytiotrophoblasts.     

Regulation of expression of the 3β-hydroxysteroid dehydrogenases of human placenta and fetal adrenal

The appropriate expression of 3β-hydroxysteroid dehydrogenase/Δ^5→4-isomerase (3β-HSD) is vital for mammalian reproduction, fetal growth and life maintenance. Several isoforms of 3β-HSD, the products of separate genes, have been identified in various species including man. Current investigations are targeted toward defining the processes that regulate the levels of specific isoforms in various steroidogenic tissues of man. High levels of expression of 3β-HSD were observed in placental tissues. It has been generally considered that the multinucleated syncytiotrophoblastic cells are the principal sites of 3β-HSD expression and, moreover, that 3β-HSD expression is intimately associated with cyclic AMP-promoted formation of syncytia. Herein we report the presence of 3β-HSD immunoreactive and mRNA species in uninucleate cytotrophoblasts in the chorion laeve, similar to that in syncytia but not cytotrophoblast placenta. In vitro, 3β-HSD levels in chorion laeve cytotrophoblasts were not increased with time nor after treatment with adenylate cyclase activators, whereas villous cytotrophoblasts spontaneously demonstrated progressive, increased 3β-HSD expression. Moreover, 3β-HSD synthesis appeared to precede morphologic syncytial formation. Thus high steroidogenic enzyme expression in placenta is not necessarily closely linked to formation of syncytia. Both Western immunoblot and enzymic activity analyses also indicated that the 3β-HSD expressed in these cytotrophoblastic populations was the 3β-HSD type I gene product (M_r, 45K) and not 3β-HSD type II (M_r, 44K) expressed in fetal testis. In cultures of fetal zone and definitive zone cell of human fetal adrenal, 3β-HSD expression was not detected until ACTH was added. ACTH, likely acting in a cyclic AMP-dependent process, induced 3β-HSD type II activity and mRNA expression. The higher level of 3β-HSD mRNA in definitive zone compared with fetal zone cells was associated with parallel increases in cortisol secretion relative to dehydroepiandrosterone sulfate formation.     

Developmental regulation of human cytomegalovirus receptors in cytotrophoblasts correlates with distinct replication sites in the placenta

Cytomegalovirus (CMV), the major viral cause of congenital disease, infects the uterus and developing placenta and spreads to the fetus throughout gestation. Virus replicates in invasive cytotrophoblasts in the decidua, and maternal immunoglobulin G (IgG)-CMV virion complexes, which are transcytosed by the neonatal Fc receptor across syncytiotrophoblasts, infect underlying cytotrophoblasts in chorionic villi. Immunity is central to protection of the placenta-fetal unit: infection can occur when IgG has a low neutralizing titer. Here we used immunohistochemical and function-blocking methods to correlate infection in the placenta with expression of potential CMV receptors in situ and in vitro. In placental villi, syncytiotrophoblasts express the virion receptor epidermal growth factor receptor (EGFR) but lack integrin coreceptors, and virion uptake occurs without replication. Focal infection can occur when transcytosed virions reach EGFR-expressing cytotrophoblasts that selectively initiate expression of alphaV integrin. In cell columns, proximal cytotrophoblasts lack receptors and distal cells express integrins alpha1beta1 and alphaVbeta3, enabling virion attachment. In the decidua, invasive cytotrophoblasts expressing coreceptors upregulate EGFR, thereby dramatically increasing susceptibility to infection. Our findings indicate that virion interactions with cytotrophoblasts expressing receptors in the placenta (i) change as the cells differentiate and (ii) correlate with spatially distinct sites of CMV replication in maternal and fetal compartments.     

The human villous cytotrophoblast: Interactions with extracellular matrix proteins, endocrine function, and cytoplasmic differentiation in the absence of syncytium formation

Human syncytiotrophoblasts are derived from villous cytotrophoblasts by cell fusion. Coincident with this morphologic transformation, trophoblasts acquire specific endocrine functions, including elaboration of chorionic gonadotropin (hCG). We wondered if syncytia formation was a prerequisite for biochemical differentiation or simply was one part of the differentiation program. By growing purified human cytotrophoblasts under serum-free conditions and manipulating the culture surface, we were able to disassociate morphologic from biochemical differentiation. We have shown previously (Endocrinology 1986, 118:1567) that human cytotrophoblasts grown in the presence of fetal calf serum flatten out, aggregate, and form functional syncytiotrophoblasts in vitro over 24-96 hr. Here we demonstrate that when grown in the absence of serum, the cells do not undergo these morphologic changes, but remain as individual spherical cells. If the culture surface was precoated with fibronectin or a variety of collagens, but not albumin, the cells regained their ability to flatten, aggregate, and form syncytia. Attachment to and syncytia formation on fibronectin was blocked by the addition of the R-G-D-S-containing peptide, Gly-Arg-Gly-Asp-Ser-Pro. Attachment to and syncytia formation on type I collagen was blocked by anti-human fibronectin F(ab')2 fragments, while association with type IV collagen was not affected by this antibody, suggesting that fibronectin mediates trophoblast association with type I collagen, but not type IV. Although syncytia formation did not occur when cytotrophoblasts were cultured under serum-free conditions in the absence of ECM proteins, biochemical differentiation was not affected. These cells secreted hCG at the same rate under serum-free conditions whether they were plated on plastic only--which prevented syncytia formation--or fibronectin, laminin or, type IV collagen--which allowed syncytia formation to occur. Furthermore, cytoplasmic differentiation in the absence of syncytia formation was confirmed by performing transmission electron microscopy on cytotrophoblasts grown under serum-free conditions in the presence of 8-bromo-cAMP. We conclude that syncytia formation is not a prerequisite for biochemical differentiation, but simply part of the trophoblast differentiation program.     

Enzyme-histochemically detectable glucose-6-phosphatase is present in chorion laeve trophoblasts of human fetal membranes

The purpose of the present study was to demonstrate the presence of glucose-6-phosphatase (G6Pase) in fetal membranes from various gestational ages (20-40 weeks of gestation). Ultrastructural enzyme-histochemical analysis of G6Pase was performed using cerium and lead as capturing agents. Precipitates indicating G6Pase activity were present mainly in the endoplasmic reticulum and partly in the nuclear envelope of chorion laeve trophoblasts, but absent in amniotic epithelial cells. Stringent histochemical control experiments performed ensured specific detection of G6Pase activity. The results indicate that histochemically detectable G6Pase is present in the chorion laeve trophoblasts of human fetal membranes. This enzyme may have some physiological significance in carbohydrate metabolism in human fetal membranes and regulation of amniotic fluid glucose concentration.     

Placental alkaline phosphatase expression at the apical and basal plasma membrane in term villous trophoblasts.

Human placental alkaline phosphatase (PLAP) was localized at the apical and basal plasma membrane of syncytiotrophoblasts and at the surface of cytotrophoblasts in term chorionic villi using immunoelectron microscopy. Similarly, apical and basolateral PLAP expression was found in polarized trophoblast-derived BeWo cells. Trophoblasts isolated from term placentas exhibited mainly vesicular PLAP immunofluorescence staining immediately after isolation. After in vitro differentiation into syncytia, PLAP plasma membrane expression was upregulated and exceeded that observed in mononuclear trophoblasts. These data call for caution in using PLAP as a morphological marker to differentiate syncytiotrophoblasts from cytotrophoblasts or as a marker enzyme for placental brush-border membranes. (J Histochem Cytochem 49:1155-1164, 2001)     

Choriocarcinoma cells increase the number of differentiating human cytotrophoblasts through an in vitro interaction

The human placenta arises from the zygote through single cell intermediates called cytotrophoblasts that in turn give rise to a syncytium. In culture, mononucleated cytotrophoblasts exhibit little, if any, cell division but are converted to multinucleated cells. Choriocarcinoma, the malignant tumor of placenta trophoblast, comprises a mixed population of dividing cellular intermediates that resemble cytotrophoblasts but are less differentiated. Because the choriocarcinoma intermediates arise from dividing cells, the tumor may contain one or more cell types in abundance not present in the population of isolated placental cells. To study placental differentiation through cell-cell interaction, choriocarcinoma cell lines were co-cultured with placenta-derived cytotrophoblasts, and placental hormone biosynthesis, as a marker of differentiation was examined. We reasoned that intermediates formed by the tumor might interact with and complement those intermediates in the placenta-derived cytotrophoblast population. Co-culturing either the JAr or JEG choriocarcinoma cell lines with cytotrophoblasts elevated the synthesis of the chorionic gonadotropin alpha and beta subunits 10-20 fold, and human placental lactogen 5-fold. The effect was specific for these trophoblast-derived cells, since comparable quantities of Chinese hamster ovary or HeLa cells did not affect the placental cytotrophoblast culture. Further experiments suggested that the source of enhanced synthesis was the cytotrophoblasts. We propose that an interaction between cytotrophoblasts and choriocarcinoma cells occurs, which results in an increased number of differentiating cytotrophoblasts. Such co-cultures may represent a model system for examining choriocarcinoma cell interaction with normal cells, a process known to occur in vivo. The data are also consistent with the hypothesis that the regulated chorionic gonadotropin production in the placenta is determined by interaction among trophoblast cells at different stages of differentiation.     

Biochemical characterization and modulation of LH/CG-receptor during human trophoblast differentiation

Due to the key role of the human chorionic gonadotropin hormone (hCG) in placental development, the aim of this study was to characterize the human trophoblastic luteinizing hormone/chorionic gonadotropin receptor (LH/CG-R) and to investigate its expression using the in vitro model of human cytotrophoblast differentiation into syncytiotrophoblast. We confirmed by in situ immunochemistry and in cultured cells, that LH/CG-R is expressed in both villous cytotrophoblasts and syncytiotrophoblasts. However, LH/CG-R expression decreased during trophoblast fusion and differentiation, while the expression of hCG and hPL (specific markers of syncytiotrophoblast formation) increased. A decrease in LH/CG-R mRNA during trophoblast differentiation was observed by means of semi-quantitative RT-PCR with two sets of primers. A corresponding decrease ( approximately 60%) in LH/CG-R protein content was shown by Western-blot and immunoprecipitation experiments. The amount of the mature form of LH/CG-R, detected as a 90-kDa band specifically binding (125)I-hCG, was lower in syncytiotrophoblasts than in cytotrophoblasts. This was confirmed by Scatchard analysis of binding data on cultured cells. Maximum binding at the cell surface decreased from 3,511 to about 929 molecules/seeded cells with a kDa of 0.4-0.5 nM. Moreover, on stimulation by recombinant hCG, the syncytiotrophoblast produced less cyclic AMP than cytotrophoblasts, indicating that LH/CG-R expression is regulated during human villous trophoblast differentiation.     

Ultrastructural study on human placentae from women subjected to assisted reproductive technology treatments

This study compared the ultrastructural differences of term placentae from human pregnancies resulting from assisted reproductive technology (ART) with term placentae from spontaneous human pregnancies. Term placentae were taken from women who had undergone an ART procedure (n = 8) and matched with term placentae from women who had had a spontaneous pregnancy (controls, n = 15). Using light microscopy (LM) and transmission-electron microscopy (TEM), terminal villi were evaluated with respect to the placental blood barrier, fetal capillaries, villous stroma, as well as cytotrophoblasts and syncytiotrophoblasts (ST) along with their substructures. No obvious differences were found between the ART-derived and control placentae when LM was used. With TEM, however, differences in the ultrastructural features were seen in the ART-derived placentae, specifically degenerative alterations of the terminal villi, mainly in ST, including a thicker placental barrier, decreased apical microvilli, and increased multiple vacuoles. The results demonstrate that some ultrastructural differences exist between ART-derived and control placentae with respect to the placental blood barrier, which may suggest maternofetal traffic downregulation following ART treatment. Further studies are required to understand the ultrastructural changes and their potential functional aspects in ART pregnancies.     

Disorders in lipid composition and properties of plasma membrane in epithelial cells of the placental villous chorion in chlamydial infection

Lipid composition of plasma membranes of placental epithelial cells of villous chorion of healthy women and those with chlamydiosis has been studied. Lipid composition of plasma membranes of ill women differs from that of healthy women by reduction of total phospholipids quantity, by the increase of the level of free cholesterol and free fatty acids. A disturbance in the ratio between certain lipid fractions and increasing quantity of lysophospholipids is observed. Two-fold oppression of plasma membranes Na+, K+ -ATPase activity of placental epithelial cells of villous chorion in ill women has been detected but Mg2+, Ca2+ -ATPase activity has not changed. Thus chlamydial infection causes significant disorders in lipid composition and functioning of epithelial cells membranes of chorion.     

Immunohistochemistry of carbonic anhydrase in human placenta and fetal membranes

The localization of human carbonic anhydrase (CA) isoenzymes HCA I, HCA II, and rat CA II have been studied in human umbilical cord, chorion laeve including amnion and placenta from first and second trimester and also from term pregnancies. Detection techniques of immunofluorescence and immunoperoxidase were used in cryostat and paraffin sections. Both isoenzymes were found in the villous syncytiotrophoblast throughout pregnancy. HCA I staining patterns in the villous endothelium were highly variable whereas increasing immunoreactivity levels of endothelial HCA II were detected as pregnancy advances. The extravillous cytotrophoblast showed generally weaker levels of immunoreactivity. In amnionic epithelium of membranes, chorionic plate and umbilical cord, higher activities for HCA I, HCA II and rat CA II were found than in all other localizations. Our findings emphasize the importance of enzyme mediated bicarbonate/CO₂ removal from the feto-placental unit as opposed to simple bicarbonate diffusion or carrier mediated transport. As effective transfer routes should be considered not only umbilical cord — placental villi — intervillous space, but also fetal kidney — amnionic fluid — amnion — uterine vessels.     

Hypoxia alters the epigenetic profile in cultured human placental trophoblasts

The mechanisms by which the placenta adapts to exogenous stimuli to create a stable and healthy environment for the growing fetus are not well known. Low oxygen tension influences placental function, and is associated with preeclampsia, a condition displaying altered development of placental trophoblast. We hypothesized that oxygen tension affects villous trophoblast by modulation of gene expression through DNA methylation. We used the Infinium HumanMethylation450 BeadChip array to compare the DNA methylation profile of primary cultures of human cytotrophoblasts and syncytiotrophoblasts under < 1%, 8% and 20% oxygen levels. We found no effect of oxygen tension on average DNA methylation for either cell phenotype, but a set of loci became hypermethylated in cytotrophoblasts exposed for 24 h to < 1% oxygen, as compared with those exposed to 8% or 20% oxygen. Hypermethylation with low oxygen tension was independently confirmed by bisulfite-pyrosequencing in a subset of functionally relevant genes including CD59, CFB, GRAM3 and ZNF217. Intriguingly, 70 out of the 147 CpGs that became hypermethylated in < 1% oxygen overlapped with CpG sites that became hypomethylated upon differentiation of cytotrophoblasts into syncytiotrophoblasts. Furthermore, the preponderance of altered sites was located at AP-1 binding sites. We suggest that AP-1 expression is triggered by hypoxia and interacts with DNA methyltransferases (DNMTs) to target methylation at specific sites in the genome, thus causing suppression of the associated genes that are responsible for differentiation of villous cytotrophoblast to syncytiotrophoblast.     

Altered Bcl-2 and Bax expression is associated with cultured first trimester human cytotrophoblasts apoptosis induced by hypoxia

Preeclampsia is associated with placental hypoxia at early gestation. We therefore investigated the effect of hypoxia on the apoptosis of cultured first trimester human cytotrophoblasts and the expression of apoptosis relevant proteins, Bcl-2 and Bax. First trimester human cytotrophoblasts were isolated and cultured under either standard or hypoxic conditions. Cellular apoptosis was monitored by TUNEL and Annexin V binding, and the expression of Bcl-2 and Bax was determined by Western blot analysis. Apoptosis increased significantly in cytotrophoblasts cultured for 24 h under hypoxic conditions in contrast with those cultured under standard conditions, meanwhile expression of Bcl-2 reduced, and that of Bax increased. These changes suggested that hypoxia induced apoptosis in cultured first trimester cytotrophoblasts with altered Bcl-2 and Bax expression. Further study is needed to explore the role of cytotrophoblasts apoptosis in hypoxia-induced maternal and fetal diseases.     

Regulation of placental villous angiopoietin-1 and -2 expression by estrogen during baboon pregnancy

We recently showed an increase in vascular endothelial growth factor (VEGF), decrease in angiopoietin-1 (Ang-1) and unaltered Ang-2 expression by the villous placenta with advancing baboon pregnancy. Moreover, placental VEGF expression was increased by estrogen in early pregnancy. In the present study, we determined whether placental Ang-1 and Ang-2 are regulated by estrogen. Ang-1 and Ang-2 mRNA and protein were determined by RT-PCR and immunocytochemistry in the placenta of baboons on Day 60 of gestation (term is 184 days) after administration of estrogen precursor androstenedione on Days 25-59 or on Day 54 after acute estradiol administration. Chronic androstenedione treatment increased serum estradiol levels three-fold (P < 0.001) and decreased (P < 0.05) villous cytotrophoblast Ang-1 mRNA to a level (0.36 +/- 0.08 relative to 18S rRNA) that was one-third of that in untreated animals (0.98 +/- 0.26). Within 2 hr of estradiol administration, cytotrophoblast Ang-1 mRNA was decreased to a level (0.24 +/- 0.05) one-fifth (P < 0.05) of that in untreated animals (1.14 +/- 0.23). However, Ang-2 mRNA levels were unaltered. Ang-1, Ang-2 and estrogen receptors alpha and beta protein were localized within villous cytotrophoblasts providing a mechanism for estrogen action at this site. In summary, estrogen increased VEGF, decreased Ang-1, and had no effect on Ang-2 expression within placental cytotrophoblasts during early baboon pregnancy. We propose that the estrogen-dependent differential regulation of these angioregulatory factors underpins the unique pattern of neovascularization established within the villous placenta during primate pregnancy.     

Endogenous heparin-binding lectin activity in human placenta: purification and developmental expression

Human placental extracts contain a herapin-inhibitable lectin activity. The lectin, which closely resembles those from chicken and rat tissues, was purified by heparin-affinity chromatography. It shares many properties with the previously reported lectins, including hapten specificity, molecular weight of monomers, and immunological cross-reactivity. Sections from different stages of placental development, stained by immunohistochemistry procedures using lectin-specific antibody, showed that the lectin was initially present only in cytotrophoblasts of early first trimester villi. Later in the first trimester, both cytotrophoblasts and syncytiotrophoblasts were stained positively for lectin. From second trimester to term, the lectin was seen only in syncytiotrophoblasts.     

Impaired cytotrophoblast cell-cell fusion is associated with reduced Syncytin and increased apoptosis in patients with placental dysfunction

Preeclampsia (PE), Hemolysis Elevated Liver Enzymes and Low Platelets (HELLP)-syndrome, and intrauterine growth restriction (IUGR) are associated with abnormal placentation. In early pregnancy, placental cytotrophoblasts fuse and form multinuclear syncytiotrophoblasts. The envelope gene of the human endogenous retrovirus-W, Syncytin, is a key factor for mediating cell-cell fusion of cytotrophoblasts. This study investigated clinical parameters of PE and HELLP-associated IUGR and analyzed the cell-cell fusion index and beta-human chorionic gonadotropin (beta-hCG) secretion of cytotrophoblasts isolated and cultured from placentas of these patients. In addition, we performed absolute quantitation of Syncytin and determined the apoptosis rate in both cultured cytotrophoblasts and placental tissues. Cultured cytotrophoblasts from PE and HELLP-associated IUGR correlated with a pronounced lower cell-cell fusion index, 1.8- and 3.6-fold; less nuclei per syncytiotrophoblast, 1.4- and 2.0-fold; a significantly decreased beta-hCG secretion, 4.3- and 17.2-fold and a reduction of Syncytin gene expression, 8.1 (P = 0.019) and 222.7-fold (P = 0.011) compared with controls, respectively. In contrast, a significantly 2.3-fold higher apoptosis rate was observed in cultured PE/IUGR cytotrophoblasts (P = 0.043). Importantly, Syncytin gene expression in primary placental tissues of PE/IUGR was 5.4-fold lower (P = 0.047) and in HELLP/IUGR 10.6-fold lower (P = 0.019) along with a 1.8- and 1.9-fold significant increase in the apoptosis rate compared with controls, respectively. Low Syncytin expression in both cultured cytotrophoblasts and primary tissues from pathological placentas supports an intrinsic placenta-specific deregulation of cell-cell fusion in the formation of syncytiotrophoblasts leading to increased apoptosis. These processes could contribute to the development and severity of PE and HELLP-associated IUGR.