Confocal and conventional immunofluorescent and immunogold electron microscopic localization of collagen types III and IV in human placenta

Confocal and conventional indirect immunofluorescence and immunogold electron microscopic methods were applied to examine the distribution of extracellular matrix constituents (collagens types III and IV) in the villi of immature and term human placentae. The immunofluorescence study revealed that collagen type III is more distinct in the villous stroma of term placenta as compared with that of the first trimester. Collagen type IV was detected mainly in endothelial and epithelial basement membranes and interestingly also to a certain extent in the stroma. Results obtained using immunoelectron microscopy support the proposal that collagen types III and IV are characteristic of stromal and basement membranes, respectively. Stromal collagen type IV is apparently localized in association with the interstitial types of collagen (I and III), in the villous stroma of term placenta.     

Placental extracellular matrix: gene expression,deposition by placental fibroblasts and the effect of oxygen

Database mining revealed 102 extracellular matrix (ECM) genes amongst about 10000 mRNA species expressed in human placenta, and these were classified into collagens (23), non-collagenous glycoproteins (59) and proteoglycans (23). A panel of antibodies to selected collagens and glycoproteins was used to examine ECM distribution in the placental villous stroma. Collagens I and IV, fibronectin and fibrillin I were abundant in first trimester and term tissue. Some areas lacked collagen I, while collagen IV was clearly evident in interstitial locations. At term, laminin was present in the stroma as well as in trophoblastic and vascular basement membranes. Thrombospondin I, tenascin C and elastin showed more restricted distributions. Fibrosis has been reported in association with ischaemia, so ECM production by cultured term and first trimester placental fibroblasts was evaluated at three different oxygen concentrations. Fibronectin and collagen IV were more strongly expressed than collagen I, fibrillin I or thrombospondin I, while the production of laminin and elastin was very low. Reducing the oxygen tension led to a selective increase in fibronectin and collagen IV production. Thus both quantitative and qualitative alterations in ECM composition may be expected to accompany prolonged hypoxia.     

The granzyme B inhibitor, PI-9, is differentially expressed during placental development and up-regulated in hydatidiform moles

The intracellular serpin Proteinase Inhibitor-9 (PI-9) is a potent inhibitor of the cytotoxic lymphocyte (CL) proteinase granzyme B, a major effector molecule used by CLs to induce target cell apoptosis. PI-9 is produced by CLs to protect against mis-directed granzyme B. However, PI-9 expression has also been reported in immune privileged tissues. In the present study, cell-specific expression of PI-9 in placental tissue of various gestational ages was examined by immunohistochemistry. PI-9 is highly expressed by the extravillous trophoblasts that have invaded the decidua, and this high expression is maintained throughout pregnancy. Similar levels were also observed in proliferative villous cytotrophoblasts. Syncytial trophoblasts generally do not produce PI-9 to a significant level until the last few weeks of pregnancy. The villous stroma contains mixed populations of PI-9 positive and negative cells throughout pregnancy, with highest expression during the second trimester. Compared to first trimester placentas, syncytial trophoblasts of partial and complete hydatidiform moles showed marked up-regulation of PI-9. Examination of choriocarcinoma cell lines also demonstrated a very high level of PI-9 is produced by these cells, which may provide protection from granzyme B-mediated apoptosis. The cell-specific expression of PI-9 in the placenta is consistent with a function in the maintenance of immune privilege, and it is proposed that up-regulated expression of PI-9 in gestational trophoblastic diseases contributes to disease pathogenesis via immune evasion.     

D2-40/podoplanin expression in the human placenta

Placental tissue expresses many lymphatic markers. The current study was undertaken to examine if D2-40/podoplanin, a lymphatic endothelial marker, was expressed in the human placenta, and how it is altered developmentally and pathologically. We examined D2-40/podoplanin and VEGFR-3 expressions in placentas from normotensive pregnancies at different gestational ages and in placentas from women with clinically defined preeclampsia. D2-40 expression in systemic lymphatic vessel endothelium served as a positive control. Protein expression for D2-40, VEGFR-3, and β-actin was determined by Western blot in placentas from normotensive (n = 6) and preeclamptic (n = 5) pregnancies. Our results show that D2-40/podoplanin was strongly expressed in the placenta, mainly as a network plexus pattern in the villous stroma throughout gestation. CD31 was limited to villous core fetal vessel endothelium and VEGFR-3 was found in both villous core fetal vessel endothelium and trophoblasts. D2-40/podoplanin expression was significantly decreased, and VEGFR-3 significantly increased in preeclamptic placental tissues compared to normotensive placental controls. Placental villous stroma is a reticular-like structure, and the localization of D2-40 to the stroma suggests that a lymphatic-like conductive network may exist in the human placenta. D2-40/podoplanin is an O-linked sialoglycoprotein. Although little is known regarding biological functions of sialylated glycoproteins within the placenta, placental D2-40/podoplanin may support fetal vessel angiogenesis during placenta development and reduced D2-40/podoplanin expression in preeclamptic placenta may contribute to altered interstitial fluid homeostasis and impaired angiogenesis in this pregnancy disorder.     

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.     

Abnormal expression of HLA-DR antigen in the placenta of a patient with pemphigoid gestationis

The villous stroma and fetal endothelium in chorionic villi adjacent to maternal decidua in a placenta of a woman suffering from pemphigoid gestationis were found to have abnormal expression of HLA-DR antigen. This aberrant DR expression may be a reflection of an immune attack on the placenta.     

Altered perlecan expression in placental development and gestational diabetes mellitus

The proteoglycan perlecan is involved in cell signaling, regulation of growth factor activity, and maintenance of basement membranes. This study aims to investigate the expression of perlecan during placental development and whether hyperglycemia of gestational diabetes mellitus induces the alteration of perlecan expression in placenta. Immunohistochemistry, immunoprecipitation/sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and quantitative real-time PCR were carried out to study the placental perlecan expression at different trimesters of pregnancies and in gestational diabetes mellitus. The perlecan protein was mainly immunolocalized in the trophoblast and vessel basement membranes with some staining in the villous stroma of placental villus. Perlecan was also found to co-localize with laminin and collagen IV in the basement membranes of placenta. The protein and mRNA levels of placental perlecan were significantly decreased as the gestational age increased. However, a significant increase in perlecan expression was observed in the third trimester placentas with gestational diabetes mellitus compared to the gestational age-matched controls. Furthermore, trophoblast cells cultured in a high glucose (30 mM) medium and a high osmotic pressure medium (5.6 mM glucose and 24.4 mM mannitol) showed increased perlecan expression compared to cells cultured in the low glucose (5.6 mM) regular medium. These alterations of perlecan expression may be associated with the structural changes of placenta during maturation. The metabolic effect of high glucose and high osmotic pressure of gestational diabetes mellitus may contribute to the increased perlecan expression of diabetic placentas.     

Does plasminogen activator inhibitor-1 (PAI-1) control trophoblast invasion? A study of fetal and maternal tissue in intrauterine, tubal and molar pregnancies

Urokinase plasminogen activator, its receptor and the inhibitor PAI-1 are believed to control proteolysis and remodelling of maternal tissue during trophoblast invasion. This system appears to be strictly regulated in normal intrauterine pregnancies whereas tubal and molar pregnancies seem to be characterized by an uncontrolled excessive placental invasion. This study evaluates subcellular PAI-1 by immunohistochemistry in the villous placenta, in the basal plate and placental bed, and in the decidual compartments of normal, tubal and molar pregnancies. PAI-1 was present in villous syncytiotrophoblasts and co-localized focally with fibrin-type fibrinoid on the surface of the chorionic villi. Basal plate and placental bed extravillous interstitial trophoblasts, as well as vascular trophoblasts, were also PAI-1 positive. In the decidua parietalis, PAI-1 was observed in the cytoplasm of the non-invaded decidual cells. In the decidua basalis comprising the basal plate, PAI-1 was seen to be membrane-associated or confined to the extracellular matrix (ECM) facing the invasive front of anchoring villi. The ECM of decidua capsularis and chorion laeve displayed the most pronounced PAI-1 expression towards the maternal interface. In contrast, the majority of placental bed decidual cells adjacent to the interstitial and vascular trophoblasts were PAI-1 negative. Only a few stromal cells distant from the implantation site were PAI-1 positive in the tubal pregnancies and decidualization was not present. Likewise, excessive decidual necrosis and fibrinoid deposition devoid of PAI-1 was a common finding in complete molar pregnancies. These results suggest that PAI-1 defines specific extravillous invasive trophoblasts within the maternal decidua. Moreover, maternal cellular lack of PAI-1 in tubal pregnancies and excessive decidual necrosis in molar pregnancies indicate an uncontrolled placental invasion. The present data indicate that trophoblast invasion is primarily regulated by signals from decidual cells.     

Localization and distribution of adrenomedullin receptor in the human placenta: changes with gestational age

OBJECTIVE: To examine the distribution and localization of adrenomedullin (AM) receptor (AM-R) in human placenta and fetal membranes to assess any change during pregnancy or with labor. STUDY DESIGN: Immunohistochemistry was performed by the avidin/biotin immunoperoxidase method using an antibody specific to AM-R on intrauterine tissues collected from 7-41 weeks of gestation (n=73). RESULTS: AM-R was localized in the placenta and fetal membranes in all 3 trimesters. The distribution of AM-R in the villous and extravillous trophoblast cells of the placenta and in chorion and decidua cells of the fetal membranes changed with gestational age but not with labor. CONCLUSION: AM is secreted by decidua and trophoblast cells that also possess AM-R, suggesting that placental tissues function in both the synthesis and action of AM. Changes in AM-R in the placenta during pregnancy may reflect changes in AM function throughout gestation.     

Increased expression of interleukin-1 beta is associated with persistence of the disease and invasion in complete hydatidiform moles (CHM)

Complete hydatidiform moles (CHM), a post-conceptual pathologic condition of the placenta, have a high prevalence rate (12/1,000 deliveries) in Kerala, India. This study addresses the expression of IL-1 alpha and beta by immunohistochemistry in relation to persistence and invasion of the disease. Mild to moderate expression of IL-1 alpha in the villous cytotrophoblasts, syncytiotrophoblasts and decidua of the first trimester in the normal placenta and all gestational ages in the molar placenta were observed. IL-1 beta expression was observed in the extravillous trophoblasts, syncytiotrophoblasts and decidua in both the normal and molar placentae and also in the villous cytotrophoblasts and the stromal Haufbaur cells in molar placentae. Strong expression of IL-1 beta in the placenta suggests its involvement in placental physiology supporting earlier reports. Higher expression of IL-1 beta correlated well with the invasive and persistent nature of the tumour and holds potential as a marker of persistence and invasion in CHM.     

Expression and localisation of FoxO3 and FoxO4 in human placenta and fetal membranes

Forkhead box O (FoxO) proteins regulate inflammation, extracellular matrix (ECM) remodelling and apoptosis. We have previously identified FoxO1 proteins in human gestational tissues, and demonstrated a link between FoxO1 and rupture of fetal membranes. There is, however, no data available on the expression and localisation of FoxO3 and FoxO4 in human intrauterine tissues. Thus the aim of this study was to characterise the localisation and expression of FoxO3 and FoxO4 in (i) human placenta and fetal membranes before term spontaneous labour onset, and (ii) supracervical site (SCS) and distal site (DS) fetal membranes from non-labouring women. Immunohistochemistry, Western blotting and quantitative RT-PCR (qRT-PCR) was used to localise and quantitate FoxO3 and FoxO4 protein and mRNA expressions. Cytoplasmic and nuclear FoxO3 was localised in the syncytiotrophoblast layer, chorionic trophoblasts, amnion epithelium and decidua. Cytoplasmic FoxO4 was localised in the syncytiotrophoblasts and chorionic trophoblasts. No or very little FoxO4 protein and mRNA was present in amnion epithelium. The intensity and extent of staining of FoxO3 and FoxO4 was greater in fetal membranes obtained from the SCS compared to DS. Presence of FoxO3 and FoxO4 are expected to contribute to apoptosis and/or cell cycle regulation associated with fetal membrane rupture.     

Abnormal expression of class II MHC antigens in placentae from patients with pemphigoid gestationis: analysis of class II MHC subregion product expression

Abnormal expression of class II MHC antigens was consistently observed in an immunohistological study on placentae from patients with pemphigoid gestationis. The area affected in all the placentae was the chorionic villi adjacent to the maternal decidua. The villous stroma, and in some cases the chorionic fetal endothelium, had abnormal expression of class II MHC antigens. Monoclonal antibodies specific for the class II MHC subregion products (DR, DP and DQ) were used to analyse the class II MHC antigen expression. Differential expression of the class II MHC subregion products was observed on the villous stroma and chorionic fetal endothelium; DR and DP were always expressed but DQ in some cases was heterogeneous. This abnormal expression of class II MHC antigens may reflect an immunological attack on the placenta in pemphigoid gestationis.     

The regional expression of insulin-like growth factor II (IGF-II) and insulin-like growth factor binding protein-1 (IGFBP-1) in the placentae of women with pre-eclampsia

Insulin-like growth factors and their binding proteins regulate cellular proliferation, differentiation and function, and play an important role in placental development. IGF-II and IGFBP-1 are abundantly expressed by cells at the maternal-fetal interface and mediate cell-to-cell communication between trophoblasts and decidua. Placentae of pre-eclamptic pregnancies show villous cytotrophoblast proliferation, increased syncytial sprout formation and impaired trophoblast invasion. We hypothesized that the expression of IGF-II and IGFBP-1 by cells at the maternal-fetal interface is altered in pre-eclampsia. We determined the regional abundance and cellular localization of IGF-II mRNA and IGFBP-1 mRNA and protein in placentae from normotensive control and pre-eclamptic pregnancies. IGF-II mRNA was expressed in both the chorionic villi and basal plate decidua regions. Increased IGF-II mRNA abundance was observed in the intermediate trophoblasts of peri-infarct regions. IGFBP-1 expression was present only in the decidua of the basal plate and membranes, and this expression was decreased significantly in pre-eclamptic placentae. The increased IGF-II expression in the intermediate trophoblast surrounding placental infarcts suggests a role for IGF-II in placental repair or remodelling. Decreased IGFBP-1 mRNA expression in the basal plate decidua suggests that the increased concentrations of IGFBP-1 the circulation of pre-eclamptic women is not of decidual origin. The altered IGF-II and IGFBP-1 expression at the fetomaternal interface may be important in the pathophysiology of pre-eclampsia.     

Expression,Regulation and Functional Characterization of Matrix Metalloproteinase-3 of Human Trophoblast

MMP-3 has been detected in human placenta and reduced expression of the enzyme was observed in invasive trophoblasts of patients with severe preeclampsia. However, detailed expression pattern, regulation and biological properties of the placental protease have not been elucidated so far. RT-PCR analyses, Western blotting and enzyme activity assays revealed that pro- and active form of MMP-3 were predominantly expressed in purified first trimester villous trophoblasts, in invasive cytotrophoblasts of differentiating explant cultures and in trophoblastic SGHPL-4 cells. Accordingly, immunofluorescene of first trimester placental tissues detected MMP-3 mainly in villous and extravillous cytotrophoblasts. IL-1β, an inducer of MMP-3 in decidual cells, increased secretion and activity of the protease in trophoblast supernatants in a dose- and time-dependent manner. IL-1β-stimulated production of the enzyme was suppressed in the presence of inhibitors of MAPK and AKT signalling. Similar to recombinant MMP-3, MMP-3 in supernatants of IL-1β-stimulated decidual stromal or SGHPL-4 cells degraded IGFBP-1 in vitro resulting in the appearance of cleavage products at approximately 25, 22, 17, 14 and 11 kD. However, cleavage assays using recombinant MMP-2 suggested that the gelatinase may contribute to IGFBP-1 degradation in trophoblast supernatants. Despite its effects on MMP-3 expression IL-1β failed to significantly alter invasion of SGHPL-4 cells through Matrigel-coated transwells. In conclusion, the data suggest that invasive trophoblast cell models secrete bioactive MMP-3. Inducible expression of the protease involves MAPK and AKT signalling. In addition to the decidua, MMP-3 of trophoblasts may contribute to the regulation of the IGF system by degrading IGFBP-1.     

Tumour necrosis factor-alpha converting enzyme in human gestational tissues from pregnancies complicated by chorioamnionitis

Chorioamnionitis increases the risk of preterm labour and is associated with adverse neonatal outcomes including cerebral palsy. Tumour necrosis factor-alpha (TNF-alpha) derived from the gestational tissues (placenta, fetal membranes and maternal decidua) is thought to play a pivotal role in the induction of cytokine response in chorioamnionitis. Tumour necrosis factor-alpha converting enzyme (TACE) is essential for the release of TNF-alpha. Our aim was to determine whether the expression of TACE is increased in human gestational tissues from pregnancies complicated by chorioamnionitis, and whether lipopolysaccharide (LPS) causes increased expression of TACE in the human gestational tissues in vitro. The immunostaining of TACE was generally more intense, in particular in the syncytiotrophoblast and stromal cells, in villous samples from pregnancies complicated by chorioamnionitis than those from normal pregnancies. Increased immunoreactivity of TACE was also noted in the amnion and choriodecidua. In parallel, there was an increased infiltration of monocytes/macrophages within the villous stroma and choriodecidua. As a complement to our in vivo findings, LPS significantly increased the levels of mRNA and protein of TACE in a dose-dependent response in villous and fetal membrane explant cultures. Together, our results imply a potential role of TACE in the pathogenesis of chorioamnionitis.     

Basement membranes (amniotic,trophoblastic, capillary) and adjacent tissue in term placenta: An electron microscopic study

Electron microscopic studies of normal human term placenta show that the amniotic epithelium is attached by semidesmosomes to a basement membrane. The latter consists of a 300 to 350 Å thick lamina lucida which is in continuity with the interepithelial space, and an outer 400 to 450 Å thick lamina densa. The thick (1,000 to 3,000 Å) trophoblastic basement membrane is composed of a lamina lucida, 300 to 350 Å in width, and the outer lamina densa, measuring 650 to 2,700 Å in width. The basement membrane of the villous capillary consists of a lamina lucida measuring 300 to 350 Å; the outer lamina densa is usually ill defined. Microfibril-like structures are present at the level of all basement membranes. Typical microfibrils are found in the extracellular space adjacent to all basement membranes examined and throughout the amniotic connective tissue stroma. They are probably responsible for the elastic-like properties of the amnion.     

Lysyl oxidases: expression in the fetal membranes and placenta

The cross-linking of the connective tissues in the fetal membranes and placenta is important for their tensile strength and elasticity. We have studied the expression of lysyl oxidase (LOX) because it is the classical enzyme responsible for the cross-linking of collagen and elastin. We have also studied the two recently described, genetically distinct lysyl oxidase-like genes and proteins, lysyl oxidase-like (LOXL) and lysyl oxidase-like 2 (LOXL2), of unknown functions.Specific antisera have been used for immunolocalization in fetal membranes and placentae from early pregnancy terminations and after caesarean section at both preterm and term, prior to labour. In addition, the steady state mRNA levels of the three genes has been quantitated in separated amnion, chorion, decidua and placentae collected at term before labour. The immunocytochemistry shows that the spatial expression of the three lysyl oxidases is similar in early pregnancy in both the fetal membranes and placentae. However, by preterm this pattern had diverged and becomes greatest at term. The expression of the genes found at term was similar to the results of protein expression obtained by immunocytochemistry, with the exception of LOXL which had high placental gene expression, but low levels of immunolocalized protein. Thus by term, LOX was expressed predominantly in the amniotic epithelium, with little expression in the placenta, while LOXL showed highest gene expression in the placenta and lowest expression in the amnion. LOXL2 expression was again different and was expressed predominantly in the chorionic cytotrophoblast of the membranes with low expression in both the amnion and placentae. These results suggest that these three members of the lysyl oxidase family may have similar roles in early pregnancy during the development of the placenta and fetal membranes, but their divergence as pregnancy advances to term, may reflect changes in substrate specificity and connective tissue composition.     

Pre-eclampsia does not change the adhesion molecule status in the placental bed

During normal placentation trophoblast cells invade maternal tissues and remodel the uterine arteries into low-resistance channels. In pre-eclampsia, trophoblast invasion is impaired and this, along with endothelial dysfunction, has been suggested to play a role in the pathogenesis of pre-eclampsia. We studied the expression of adhesion molecules important for leukocyte extravasation in the placental bed with immunohistochemistry and compared the expression in pre-eclampsia to that in normal pregnancy. Our major finding was that only invasive trophoblasts expressed cutaneous lymphocyte antigen-1 (CLA-1) in the third trimester of pregnancy, whereas villous trophoblasts did not. In the first trimester both villous trophoblasts and invasive trophoblast cells in decidua remained negative for CLA-1. Pre-eclampsia did not change the expression of leukocyte-endothelium adhesion or lymphocyte homing-associated antigens, ICAM-1, ICAM-2, VCAM, P-selectin, E-selectin, L-selectin, CLA-1, CD73, VAP-1 and alphaEbeta7 in the placental bed. Furthermore, pre-eclampsia was not associated with an aberrant accumulation of lymphocytes carrying antigens of any particular known organ-specific homing systems. The results on the unchanged pattern of adhesion molecule expression in pre-eclampsia suggests that there is no major change in the adhesive properties of the endothelium of the placental bed in pre-eclampsia.     

Eph and ephrin expression in normal placental development and preeclampsia

Eph receptors and their ephrin ligands play a fundamental role in embryogenesis. Their functions include cell targeting and angiogenesis. In placental development, trophoblasts migrate and invade maternal tissue and spiral arteries, where they play a role in both anchoring the placenta to the uterus and increasing blood flow to the developing fetus (interstitial and endovascular invasions). We investigated the cellular distribution and expression patterns of representative Eph and ephrin RNA and protein in an effort to identify the molecules involved in trophoblast migration during normal placental development and placental pathologies. We found ephrin-A1 expressed exclusively in the invasive extravillous trophoblast (EVT) cell lineage. We therefore proceeded to investigate ephrin-A1 in placental pathologies with defects in EVT invasion. In preeclampsia, where trophoblast invasion is shallow, we observed ephrin-A1 expression similar to normal placenta. Furthermore, in initial experiments on the deeply invading trophoblasts of placenta accreta, which lacks decidua, ephrin-A1 is found to be expressed highly in extravillous trophoblasts that have invaded the myometrium. In addition, we found the prototype ephrin-A1 receptor, EphA2, localized in several placental cell types. EphB4 and ephrin-B2 molecules, which have specific expression patterns during artery and vein development, respectively, were also expressed in the placenta. The cell specific distribution of ephrin-A1 suggests that it may play a role in targeting and migration of trophoblasts, and in the vascular remodeling induced by the invading extravillous trophoblasts. Failure of ephrin-A1 expression is unlikely to be the primary cause in defective migration of trophoblasts observed in preeclampsia. Specific roles for other Eph and ephrin proteins remain to be investigated.