Expression of intercellular adhesion molecule 1 (ICAM-1) in Plasmodium falciparum-infected placenta.

We investigated the expression of intercellular adhesion molecule 1 (ICAM-1) in malarial placenta and related histological changes. Thirty-two malarial and 40 control term placentae were collected at Tanga, Tanzania and examined histologically and immunohistochemically. Malaria infected placentae were further divided into acute (15) and chronic (17) cases according to the presence of malarial pigment. The expression of ICAM-1 on monocyte, syncytio- and cytotrophoblasts, endothelial and stromal cells was assessed. Birthweight was lower and leukocyte counts higher in placentae with chronic infection. Many monocytes were present within the intervillous spaces, especially in placentae with chronic infection, and aggregated with parasitized erythrocytes. Some monocytes were adhesive to the surface of fibrinoid deposits. ICAM-1 expression on monocytes of malarial placentae was significantly conspicuous and correlated to the degree of intervillous leukocyte infiltration. Syncytiotrophoblasts often did not show ICAM-1, even though ICAM-1 was expressed by endothelium and weakly by cytotrophoblasts and stromal cells in both infected and control placentae. These results suggest that the expression of ICAM-1 on monocytes contributes to sequestration of infected erythrocytes within the intervillous spaces and their adhesion to fibrin masses and that ICAM-1 is unlikely to be associated with the direct adhesion of infected erythrocytes to the syncytiotrophoblasts.     

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.     

Placental hormones: II. Immunofluorescence studies of the localization of prolactin/placental lactogen- and human chorionic gonadotrophin-receptors in human and rat placenta

Human term placentae and rat placentae of gestation day 15 and 17 were examined for the localization of prolactin (PRL)/placental lactogen (PL) and human chorionic gonadotrophin (hCG) receptors by means of an immunofluorescence technique in order to determine possible target cells for PRL/PL and hCG and to obtain indirect evidence for the localization of steroid producing cells. In the human placenta, PRL/PL- and hCG-receptors were observed in the syncytiotrophoblast of the chorionic villi and in the giant cells of placental septa and cytotrophoblastic islands in the intervillous space. In the rat placenta, PRL/PL- and hCG-binding was localized mainly to the giant cells and vacuolated glycogen cells of the basal zone.     

Release of oncofetal fibronectin from human placenta

Oncofetal fibronectin (onfFN) is an extracellular matrix (ECM) protein synthesized by tumours and fetal tissue including placenta. The appearance of onfFN and other cellular FNs in cervico-vaginal secretions and maternal blood is associated with adverse pregnancy outcomes. In the current study, we used dual (maternal and fetal) perfusion of human term placentae and primary cultures of syncytiotrophoblasts (SCTs) to determine whether the human placenta releases onfFN. ELISA and Western blotting revealed that onfFN is preferentially released to the maternal perfusate in a time-dependent manner. Immunodetection with FDC-6, extra domain A (EDA) and cell binding domain-specific antibodies revealed onfFN in maternal perfusate to be a high molecular weight (>450 kDa) protein dimer, similar to that found in amniotic fluid. This suggested that onfFN was released intact from placenta and not cleaved from an ECM. In addition, a similar high molecular weight dimeric onfFN species was noted in conditioned media from cultures of SCTs. Since SCTs directly release proteins to the intervillous space, this suggests that SCTs may be a source of onfFN detected in maternal perfusate. These results indicate that onfFN is released from human placenta and thus levels in maternal sera may provide insight into placental pathophysiology.     

Placental leptin receptor isoforms in normal and pathological pregnancies

Alternate mRNA splicing of human leptin receptor generates four membrane isoforms with different C-terminal sequences. They differ by the length of their intracellular domain which include specific motifs crucial for the specificity of leptin signalling. As a step towards functional studies, we have characterized leptin receptors in human placenta from normal pregnancies and pregnancies associated with diabetes and pre-eclampsia. Leptin and leptin receptors were visualized by immunohistochemistry of placentas obtained from first and third trimester pregnancies. Antibodies against N and C-terminal epitopes showed signals in the apical membrane of the syncytiotrophoblast in early and term placental villi as well as in JAr and BeWo derived trophoblast cells. In addition, a distinct isoform recognized by its extracellular juxtamembrane epitope was exclusively localized in cytotrophoblast cells and likely stains the soluble receptor. At contrast with the transmembrane receptors, the expression of this isoform is increased in placentas of pre-eclamptic and diabetic women which synthesize more leptin than placenta from uncomplicated pregnancy.These data demonstrate that short and long transmembrane leptin receptors are expressed in the trophoblast and indicate that leptin synthetized within the placenta can act locally through both receptor isoforms. Being also accessible to leptin from maternal origin, these transmembrane receptors may signal differently in pregnancy with normal and increased leptin production. The co-localization of leptin and the soluble receptor isoform suggests that this isoform serves for modulating maternal free leptin levels through modification of leptin binding capacities.     

Histochemical localization of acidic glycosaminoglycans in normal human placentae

First-trimester and term placentae were studied histochemically with alcian blue stain before and after specific enzyme treatments. A specific deposition of glycosaminoglycans was detected in the villous stroma, fetal blood vessels and on the surface of the syncytiotrophoblast, the latter being discontinuous and of variable thickness. Treatments of sections with hyaluronidases from Streptomyces and from bovine testes and with chondroitinase ABC indicated the presence of (1) chondroitin sulphates and hyaluronic acid mainly in the stroma; (2) heparan sulphate and dermatan sulphate associated with villous fetal blood vessels and the intervillous surface of the syncytiotrophoblast. It is suggested that the location of individual glycosaminoglycans could be related to their functions with regard to the maintenance of the structural integrity of the placenta by preventing its compression and to their involvement in blood anticoagulation and in lipid metabolism.     

The expression and localization of the human placental prorenin/renin-angiotensin system throughout pregnancy: roles in trophoblast invasion and angiogenesis?

The renin-angiotensin system (RAS) is thought to regulate placentation, however, the expression and localization of RAS pathways in early gestation human placenta is not known. Here we describe the expression of prorenin (REN), (pro)renin receptor (ATP6AP2), angiotensinogen (AGT), angiotensin-converting enzyme 1 and 2 (ACE; ACE2), angiotensin II type 1 and 2 receptors (AGTR1; AGTR2) and angiotensin 1-7 receptor (MAS1), as well as the angiogenic factor, vascular endothelial growth factor (VEGF), and transforming growth factor-β1 (TGF-β1), in early gestation (6-16 weeks) and term (>37 weeks) human placentae. We also describe the location of all of the key RAS proteins in the early gestation placentae. The highest levels of REN, ATP6AP2, AGT, AGTR1 and ACE2 mRNAs were found in early gestation, whereas ACE1 mRNA was highest at term. AGTR2 and MAS1 mRNA expression were low to undetectable in all samples. REN, ATP6AP2 and AGTR1 mRNA levels were correlated with VEGF expression, but not with TGF-β1 mRNA. In early gestation placentae, prorenin, (pro)renin receptor and the angiotensin II type 1 receptor (AT₁R) were localized to extravillous trophoblast cells, suggesting they play a key role in trophoblast migration. ACE2 in syncytiotrophoblasts could regulate release of Ang 1-7 into the maternal circulation contributing to the vasodilation of the maternal vasculature. ACE was only found in fetal vascular endothelium and may specifically target the growing fetal placental vessels. Because REN, ATP6AP2 and AGTR1 show strong correlations with expression of VEGF this pathway is likely to be important in placental angiogenesis.     

Perfusion with lipopolysaccharide differently affects the secretion of interleukin-1 beta and interleukin-1 receptor antagonist by term and preterm human placentae

The aim of the present study was to examine the effect of lipopolysaccharide (LPS) on the secretion of the pro-inflammatory cytokine interleukin-1beta (IL-1beta) and of its natural inhibitor interleukin-1 receptor antagonist (IL-1Ra), by perfused human term and preterm placental tissue. Eight term and eight preterm placentae were collected immediately after delivery; four term and four preterm placentae were perfused with control medium (without LPS) and the other four term and four preterm placentae were perfused with medium containing LPS. The release of IL-1beta into the maternal compartment by term placenta was significantly higher than the release by preterm placenta (p<0.001). However, there were no significant differences between IL-1beta levels released into the fetal compartments of term and preterm placentae. No significant differences were observed in the release of IL-1Ra into the maternal and fetal compartments of term placenta, when compared to preterm placenta. Exposure to LPS significantly decreased the capacity of term placenta to release IL-1beta into the maternal compartment (p<0.001) and increased the capacity of term placenta to release IL-1Ra into the maternal and fetal compartments (p<0.001 and p=0.017, respectively). However, the capacity of preterm placentae to release IL-1beta and IL-Ra into the maternal and fetal compartments was not affected by LPS. IL-1beta was expressed by both term and preterm placentae before and after perfusion (+/- LPS), by epithelial cells of the amnion, chorion, by syncytiotrophoblast and stromal cells of villous tissue and by the decidua. IL-1Ra in term and preterm placentae was expressed before perfusion mainly in epithelial cells of the amnion. After perfusion of term placentae (+/- LPS), additional IL-1Ra expression was seen in epithelial cells of the amnion and in syncytiotrophoblast and stromal cells of villous tissue and by the decidua. However, perfusion of preterm placentae (+/- LPS) did not affect IL-1Ra expression. The localization of IL-1beta and IL-1Ra in both term and preterm human placental tissue suggests a their physiologic role. The data presented indicates that the IL-1 system in term and preterm placentae seems to be differently affected by LPS. Down-regulation in the release of the pro-inflammatory cytokine IL-1beta and the up-regulation of its antagonist (IL-1Ra) may be a part of the inflammatory response to infection in human term, but not preterm, placentae. The IL-1 system in term and preterm placentae seems to be differently affected by LPS.     

The immunocytochemical localization of new soluble placental tissue proteins (PP14, 16, 17, 19, 20 and PP21) in human and cynomolgus monkey placentae

Apparently Placenta-specific placental tissue proteins (PP14 and PP17) and solitary tissue proteins (PP16, 19, 20 and PP21) were investigated by avidin-biotin immunoperoxidase technique in the human and cynomolgus monkey placentae, membranes, decidua and umbilical cords. In human early placentae, PP14, 16, 17, 19 and PP21 were localized mainly in the cytoplasm of villous syncytiotrophoblast. PP20 was localized in the cytoplasm of basal chorionic trophoblasts. In human term placentae, positive stainings for PP16, 19 and PP21 were observed mainly in all kinds of trophoblastic cells, while positive stainings for PP14, 17 and PP20 were weakened in the trophoblastic cells. PP20 was clearly localized in the cytoplasm of Hofbauer-like cells in the villous stroma. The membrane of villous syncytiotrophoblast showed strongly positive stainings for PP21. PP21 was also localized in the membrane of amniotic and umbilical epithelium. The umbilical epithelium was cytoplasmically positive for PP14, 16 and PP20. Clear positive stainings for PP14 and PP21 were found in the cytoplasm of fetal polymorphonuclear neutrophils. All of the placental proteins were immunocytochemically positive in the decidual large cells. In the cynomolgus monkey placentae, similar immunostaining results were obtained. The monkey could, thus, serve as a model for the investigation of the placental proteins.     

Placental expression of erythropoietin mRNA, protein and receptor in sheep

In ovine placentae at 100 days gestation, we localized the expression of erythropoietin (EPO) mRNA by in situ hybridization and determined the cellular localization of EPO protein and EPO receptor protein by fluorescence immunohistochemistry. Erythropoietin mRNA was observed in maternal tissue at the apical tips of the fetal cytotrophoblastic villi at their interface with the maternal caruncle and was absent from both the centrally located fetal-maternal tissue and the more peripherally located maternal caruncle. An EPO-protein-associated fluorescent signal was observed in the same interface region as the EPO mRNA hybridization signal. An intense fluorescent signal associated with EPO receptor protein was observed in the apical fetal-maternal interface region with a lower density signal dispersed throughout the remainder of the interdigitating fetal-maternal tissue. The predominant cells in the apical fetal-maternal interface were identified as binucleate cells by immunohistochemistry. Thus the localization of the binucleate cells was the same as that for the EPO mRNA and the EPO protein, whereas the EPO receptor had a more generalized distribution. Since the binucleate cells are hormone producing cells, we speculate that the binucleate cells are the source of the EPO that is present in ovine placenta.     

Placental vessel adaptation during gestation and to high altitude: changes in diameter and perivascular cell coverage

The objective of this study was to determine how human placental vascular structures change during gestation and whether this would be altered by external factors such as reduced ambient oxygen. To achieve this, several experiments were carried out: Vessel profile diameter was measured and the presence of perivascular cells (pericytes or smooth muscle cells) noted. This was carried out in normal human first trimester and term placentae, and in term placentae obtained from high altitude and an ethnically matched lowland population. In addition, to characterize endothelial cells in human placenta a panel of endothelial markers anti-CD 105, CD31, CD34, Von Willebrand factor (vWF), Ulex europaeus agglutinin 1 (UEA I), Peanut agglutinin (PNA), Dolichos biflorus agglutinin (DBA) and Bandeieraea simplicifolia agglutinin 1 (BS 1) was used. The proportion of vessels associated with perivascular cells rises during gestation from 37 per cent in the first trimester to 63 per cent at term (P<0.0001) and vessels with perivascular cells have a larger median diameter at term. In placentae obtained at high altitude, the vessels are dilated and are less frequently associated with perivascular cells. The absence of perivascular cells may allow remodelling of capillaries and this is likely to be physiological important in the first trimester but also under physiological or pathological stress.     

Placental pathology in midtrimester pregnancies interrupted by intra-amniotic injection of hypertonic urea.

The histological appearances of 52 placentae from patients who had a urea-induced second trimester abortion were studied. Extensive subchorionic intervillous fibrin deposition and swollen degenerated stromal cells (probably Hofbauer cells), were found both in the placenta and membranes. Between urea injection and abortion, serial vaginal smears showed signs of progesterone deficiency. The urea-induced placental and fetal damage was similar to that seen with hypertonic saline.     

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.     

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.     

Identification and localization of divalent metal transporter-1 (DMT-1) in term human placenta

The mechanism by which iron is transported from mother to fetus is incompletely understood. Whereas transferrin receptor (TfR) is responsible for iron uptake from maternal serum by the syncytiotrophoblast, the proteins responsible for intracytoplasmic transport and for delivery to the fetal serum remain unknown. The aim of this study was to determine whether the recently characterized endosomal membrane iron transporter, divalent metal ion transporter-1 (DMT-1), is expressed in human syncytiotrophoblast, and whether its cellular localization would support roles for cytoplasmic and placental-fetal iron transport. Six micron sections of frozen, term human placenta were assessed immunohistochemically using a polyclonal antibody to rat DMT-1 and a monoclonal antibody to human TfR. DMT-1 was found both in the cytoplasm and at the junction of the fetal (basal) membrane and fetal vessels, while TfR was localized predominantly to the maternal (apical) side of the syncytiotrophoblastic membrane. Double staining demonstrated no overlap between the two proteins on the apical membrane and minimal areas of overlap in the cytoplasm. We postulate that the syncytiotrophoblast takes up diferric transferrin from serum via TfR, subsequently incorporating the transferrin : TfR complex via endosomes. Subsequent transport of iron out of the endosome and across the basal membrane to the fetus may occur via DMT-1.     

Capacity for hormone production of cultured trophoblast cells obtained from placentae at term and in early pregnancy

Problem: There is an increased doubt about the identity of isolated cytotrophoblast cells at term. Therefore, we compared pregnancy serum levels of three hormones [human placental lactogen (hPL), human chorionic gonadotropin (hCG), and leptin] with the capacity for hormone production of early placentae [EP; 8–13 weeks of gestation (WG)] and term placentae (TP; 38–42 WG).Methods: Serum levels of these hormones were determined in 15 paired maternal (7–41 WG) and fetal (37–41 WG) samples. Cytotrophoblast cells were isolated from term (TP; 38–42 weeks) and early (EP; 8–13 weeks) placentae by enzymatic digestion and subsequent purification on a Percoll gradient. These cells were cultured for 6 days. The production of the hormones hPL, hCG, and leptin was determined as release during culture + cell content after culture – cell content before culture.Results: Serum levels (mean ± SD; n ± 15) at 7–12 and 37–41 WG were 89,652 ± 21,431 and 13,620 ± 5854 mIU/ml for hCG, 400 ± 182 and 7088 ± 2030 ng/ml for hPL, and 12,675 ± 4266 and 32,236 ± 10,961 pg/ml for leptin, respectively. For cultured cells from EP and TP, hCG and hPL showed different patterns of release during the first 2–3 days. While the release of these two hormones by EP cytotrophoblast cells continued during 6 days in culture, their concentrations reached a plateau for TP cytotrophoblasts between 4 and 6 days. Leptin was undetectable (<15 pg/ml) in TP cell cultured media, while for EP all three hormones showed the same release profiles. Production calculated for 30,000 TP trophoblast cells cultured for 6 days (n = 8) was 2–31 mIU for hCG and 0.5–2 ng for hPL. For EP (n = 11), it was 50–1070 mIU for hCG, 15–323 ng for hPL, and 137–580 pg for leptin. Net synthesis of hCG and hPL for TP was >10-fold and <1-fold, respectively. In contrast, the production of all three hormones for EP was at least 100 times the initial cell content.Conclusions: These results demonstrate that trophoblasts from early pregnancy show much higher production rates of hCG, hPL, and leptin than at term. However, the in vitro findings are difficult to be reconciled with the different serum concentrations of the two hormones hPL and leptin observed during the course of pregnancy.     

Distribution of glutamate transporters in the human placenta

Glutamate metabolism is known to be important for growth and development of the human fetus. The glutamate transporters EAAT1, EAAT2 and EAAT3 are key components of the glutamate-glutamine cycle and responsible for active transport of glutamate over the cell membrane. The placenta is thought to regulate glutamate transport during fetal development. Glutamate transporters have been found in placentae of rats, but their distribution in the human placenta is unknown. Therefore, the distribution of glutamate transporters EAAT1, EAAT2 and EAAT3 were analysed in the human placenta during normal pregnancies ending between 8 and 40 weeks of gestation and in placentae of intrauterine growth restricted infants with gestational ages between 28 and 35 weeks of pregnancy. Using immunohistochemistry, EAAT1 expression was found in the syncytiotrophoblast layer, while EAAT2 was detected in the syncytiotrophoblast layer and in endothelial cells of about 5 per cent of all fetal blood vessels. EAAT3 was observed in the endothelium of the fetal blood vessels in all placentae examined. However, expression was also found in the syncytio- and the cytotrophoblast layer of the fetal villi at 8 weeks of gestational age. The expression patterns of EAAT1, EAAT2 and EAAT3 suggest involvement in active transport of glutamate between the fetal and maternal blood circulation. No differences were found in the distribution of the glutamate transporters between control and IUGR placentae. Our data show specific localization of EAAT1, EAAT2 and EAAT3 in the human placenta during development.