Placental Villi Research Articles

MUC1 Expression Is Increased During Human Placental Development and Suppresses Trophoblast-Like Cell Invasion In Vitro1

Mucin (MUC)1 is a multifunctional mucin expressed by a variety of reproductive tract epithelia. Trophoblast invasion is essential for normal placental development. However, MUC1 expression in the human placenta throughout pregnancy and the role of MUC1 in trophoblast-like cell invasion are still unclear. In the present study, results from quantitative RT-PCR and Western blot demonstrated that MUC1 mRNA and MUC1 protein expression, respectively, increased with gestational age of the human placenta. Immunohistochemistry revealed that MUC1 in placental villi was mainly expressed by syncytiotrophoblasts throughout pregnancy and increased with gestational age. Interestingly, we found two populations of extravillous trophoblasts, MUC1-positive and MUC1-negative cells, in decidua. The numbers of MUC1-positive extravillous trophoblasts were increased during placental development. Furthermore, MUC1 overexpression significantly (P < 0.01) suppressed matrigel invasion of trophoblast-like JAR cells by 34.6% +/- 4.5% compared with control, which was associated with a decrease in MMP9 activity assessed by gelatin zymography. Our results suggest that MUC1 expression in the human placenta is increased during placental development, and its overexpression suppresses trophoblast-like cell invasion in vitro.

Maintaining the integrity of trophoblast during growth of the placenta. Focus on “Insulin-like growth factor I and II regulate the life cycle of trophoblast in the developing human placenta”

maternal-fetal exchange of nutrients and respiratory gases occurs across the brush border of the trophoblast lining the intervillous space of the placenta. The trophoblast is syncytial in nature and has a high rate of turnover. Therefore syncytiotrophoblast is continually replenished from an

Insulin-like growth factor I and II regulate the life cycle of trophoblast in the developing human placenta

The main disorders of human pregnancy are rooted in defective placentation. Normal placental development depends on proliferation, differentiation, and fusion of cytotrophoblasts to form and maintain an overlying syncytiotrophoblast. There is indirect evidence that the insulin-like growth factors (IGFs), which are aberrant in pregnancy disorders, are involved in regulating trophoblast turnover, but the processes that control human placental growth are poorly understood. Using an explant model of human first-trimester placental villus in which the spatial and ontological relationships between cell populations are maintained, we demonstrate that cytotrophoblast proliferation is enhanced by IGF-I/IGF-II and that both factors can rescue cytotrophoblast from apoptosis. Baseline cytotrophoblast proliferation ceases in the absence of syncytiotrophoblast, although denuded cytotrophoblasts can proliferate when exposed to IGF and the rate of cytotrophoblast differentiation/fusion and, consequently, syncytial regeneration, increases. Use of signaling inhibitors suggests that IGFs mediate their effect on cytotrophoblast proliferation/syncytial formation through the MAPK pathway, whereas effects on survival are regulated by the phosphoinositide 3-kinase pathway. These results show that directional contact between cytotrophoblast and syncytium is important in regulating the relative amounts of the two cell populations. However, IGFs can exert an exogenous regulatory influence on placental growth/development, suggesting that manipulation of the placental IGF axis may offer a potential therapeutic route to the correction of inadequate placental growth.

Placental Matrix Metalloproteinase—1 Expression Is Increased in Labor

Matrix metalloproteinases (MMPs) are now known to process a broad spectrum of cell surface molecules and to function in several important biological processes. Testing for differences in gene expression in human placental chorionic villi in the absence or presence of labor, using cDNA microarray analysis, revealed that labor was associated with increased expression of MMP-1 gene expression in 5 placentas collected after term normal spontaneous deliveries compared with 5 placentas collected after term nonlaboring cesarean deliveries. Fibronectin 1 and collagen XVII, 2 other proteins involved in the homeostasis of the extracellular matrix, were also found to be upregulated in labor. MMP-1 was further tested in individual samples and found to be consistently overexpressed in labor. While previous microarray analyses have focused on either uterine tissue or the fetal membranes, the data presented here indicate for the first time that placental chorionic villus genes are likely to affect the initiation of parturition through altered processing of cell surface molecules by MMP-1.

Effects of corticotropin-releasing hormone and stresscopin on vascular endothelial growth factor mRNA expression in cultured early human extravillous trophoblasts

Corticotropin-releasing hormone (CRH) takes a role in the regulation of the onset of parturition. Stresscopin (SCP) is a high affinity ligand for CRH receptor (CRHR)-2. CRHR-2 inhibits VEGF-induced neovascularization. In the present study, we investigated the effects of CRH and SCP on VEGF expression in early placental extravillous trophoblasts (EVTs). Isolation and culture of trophoblasts differentiating into EVTs were performed by the enzymatic digestion of anchoring early placental villi. The presence of CRH, SCP, CRHR-1, and CRHR-2 in cultured EVTs was examined by RT-PCR and immunocytochemistry. The effects of CRH and SCP on VEGF mRNA levels in cultured EVTs were assessed by real-time RT-PCR. CRH, SCP, CRHR-1, and CRHR-2 were expressed in cultured EVTs at mRNA and protein levels. Treatment with either 100 nM CRH or 100 nM SCP for 24 h decreased VEGF mRNA levels in cultured EVTs. The CRH- and SCP-induced decrease in VEGF mRNA levels was counteracted by the concomitant treatment with CRHR-2 antagonist antisauvagine-30, but not with CRHR-1 antagonist antalarmin. We demonstrated that CRH and SCP inhibited VEGF mRNA expression in cultured EVTs through the interaction with CRHR-2, suggesting that CRH and SCP may inhibit angiogenesis during early placentation.

Villous explant culture using early gestation tissue from ongoing pregnancies with known normal outcomes: the effect of oxygen on trophoblast outgrowth and migration

Early placental and embryo development occur in a physiologically low oxygen environment, with a rise in oxygen tension within the placenta towards the end of the first trimester. Oxygen is implicated in the regulation of trophoblast differentiation and invasion. This study examined the effects of oxygen tension on extravillous trophoblast outgrowth and migration from normal pregnancies free of significant pathology. Early gestation villous tissue (11-14 weeks gestation), obtained by chorionic villus sampling, was cultured in 3 or 20% oxygen. Maternal and fetal outcomes were ascertained for all samples. The frequency and amount of trophoblast outgrowth and migration from villi were measured for up to 192 h. Significantly fewer explants produced outgrowths in 3% compared with 20% oxygen. The number of sites of trophoblast outgrowth and the extent of migration were also significantly less in 3% compared with 20% oxygen. In vitro hypoxia/reoxygenation further reduced trophoblast growth compared with 3% oxygen alone. HLA-G expression in extravillous trophoblasts was not affected by oxygen tension, with HLA-G positive extravillous trophoblasts being universally Ki67 negative. Human placental villi and extravillous trophoblasts in the late first trimester of pregnancy are sensitive to oxygen tension, with low oxygen inhibiting extravillous trophoblast outgrowth and migration.

In vitro Zidovudine-Induced Apoptosis Demonstrated by a Model of Placental Histoculture Useful for the Study of the Apoptotic Effects of Antiretroviral Drugs

This study aims to evaluate the use of a placenta histoculture system based on placental villi culture on collagen sponge gels at the liquid/air interface, for the detection of zidovudine-induced apoptosis. Explant cultures from full term placentas were exposed to different concentrations of zidovudine (1, 10, 100 uM) up to 7 days and samples were analysed for apoptosis with different methods after 3, 24, 48 hours and 7 days of exposure. Apoptotic phenomena were demonstrated at 10 and 100 uM zidovudine, more delayed at the lowest concentration: this demonstrates that zidovudine-induced placenta apoptosis in vitro is both time- and dose-dependent and that also concentrations near to those achievable in vivo might cause placenta apoptosis. This has to be taken into account when considering possible consequences of antiretroviral treatments during pregnancy. Placental histocultures on collagen sponge cells are a reliable tool for the study of antiretroviral-induced toxicity in placenta: in fact, explants cultured for more prolonged periods (up to 7–14 days) in a “physiological milieu” allow the detection of biological effects otherwise not evident in the commonly used, short-term, placental cultures.

Human type 2 17beta-hydroxysteroid dehydrogenase mRNA and protein distribution in placental villi at mid and term pregnancy

BackgroundDuring human pregnancy, the placental villi produces high amounts of estradiol. This steroid is secreted by the syncytium, which is directly in contact with maternal blood. Estradiol has to cross placental foetal vessels to reach foetal circulation. The enzyme 17beta-hydroxysteroid dehydrogenase type 2 (17beta-HSD2) was detected in placental endothelial cells of foetal vessels inside the villi. This enzyme catalyzes the conversion of estradiol to estrone, and of testosterone to androstenedione. It was proposed that estradiol level into foetal circulation could be regulated by 17beta-HSD2.MethodsWe obtained placentas from 10 to 26 6/7 weeks of pregnancy from women undergoing voluntary termination of pregnancy, term placentas were collected after normal spontaneous vaginal deliveries. We quantified 17beta-HSD2 mRNA levels in mid-gestation and term human placenta by RT-QPCR. We produced a new anti-17beta-HSD2 antibody to study its spatio-temporal expression by immunohistochemistry. We also compared steroid levels (testosterone, estrone and estradiol) and 17beta-HSD2 mRNA and protein levels between term placenta and endometrium.ResultsHigh 17beta-HSD2 mRNA and protein levels were found in both mid-gestation and term placentas. However, we showed that 17beta-HSD2 mRNA levels increase by 2.27 fold between mid-gestation and term. This period coincides with a transitional phase in the development of the villous vasculature. In mid-gestation placenta, high levels of 17beta-HSD2 were found in mesenchymal villi and immature intermediate villi, more precisely in endothelial cells of the stromal channel. At term, high levels of 17beta-HSD2 were found in the numerous sinusoidal capillaries of terminal villi. 17beta-HSD2 mRNA and protein levels in term placentas were respectively 25.4 fold and 30 to 60 fold higher than in the endometrium. Steroid levels were also significantly higher in term placenta than in the endometrium.ConclusionThe spatial and temporal expression of 17beta-HSD2 in the placenta during pregnancy and the comparison of 17beta-HSD2 expression and steroid levels between placental villi and endometrium are compatible with a role in the modulation of active and inactive forms of estrogens. Our observations strongly support the hypothesis that 17beta-HSD2 acts as a barrier decreasing estradiol secretion rates in the foetal circulation.

Transfer of folic acid inside the first-trimester gestational sac and the effect of maternal smoking

The objective of the study was to investigate the transfer pathways of folic acid inside the first-trimester gestational sac and to evaluate the impact of maternal smoking. Folate and cotinine levels were evaluated in maternal serum (n = 125) and coelomic fluid (n = 42), and immunostaining was used to identify folate receptors in placental villi, decidual tissue, and secondary yolk sacs from normal pregnancies at 6-12 weeks' gestation. Folate receptors-alpha were found on the apical surface of syncytiotrophoblast, uterine glandular epithelial cells, and mesothelial layer of the yolk sac. Significantly higher (P < .05) median folate levels were found in maternal serum than in coelomic fluid. The median folate maternal serum and coelomic levels were significantly (P < .0001 and P < .005) lower in smokers, compared with nonsmokers. Uterine glands and the secondary yolk sac play key roles in supplying folic acid to the developing fetus before the placental circulations are established, and maternal smoking does not seem to impair directly its transfer mechanism in early pregnancy.

A Light and Electron Microscopical Study of the Tragulid (Mouse Deer) Placenta

The Tragulidae are the living relics of the basal ruminant stock. They have a diffuse placenta, with no aggregations of the placental villi into localised placentomes characteristic of all other ruminants. Despite this difference, this ultrastructural and immunocytochemical investigation demonstrates that in Tragulus the trophoblast binucleate cell (BNC) plays the same central role in development and structure as in all other ruminants. It shows an identical development and ultrastructure, produces granules reactive with bovine placental lactogen and pregnancy associated glycoprotein antibodies, and migrates when mature through the trophoblast tight junction to fuse into a mosaic of syncytial plaques from which the granules are released to the mother and which have replaced the uterine epithelium. Unlike the persistent plaques in the sheep and goat placenta, in Tragulus they are transient, dying by apoptosis with the fragments phagocytosed by the trophoblast. This brings the trophoblast into direct endotheliochorial apposition to maternal tissue until BNC migration and fusion replace the dead plaque. This intimate fetomaternal confrontation has not been shown in any other ruminant, and could be a relic of the evolutionary development of the synepitheliochorial from the original basic eutherian endo- or hemo-chorial placenta.

Matrix metalloproteinases-2, -3 and -9 in human term placenta

Matrix metalloproteinases (MMPs) are zinc-dependent enzymes that degrade the components of the extracellular matrix (ECM) and are known to be the main mediators of human placentation and parturition. Although there are many studies on the roles and distribution of MMPs in human term placenta, so far none of the studies has investigated the distribution of MMP-2, -3 and -9 in different cells of various placental sites. In this study, we aimed to determine the distribution and enzymatic activities of MMP-2, -3 and -9 with regard to different regions of term human placenta, such as amnion, basal plate, chorionic plate, decidua, chorion laeve, Nitabuch's stria, umbilical cord and placental villi. Eighteen normal human term placentas were obtained after vaginal deliveries. Immunohistochemistry and zymography were performed for MMP-2, -3 and -9 on placental tissue sections and protein extracts, respectively. Nearly all tissues showed immunoreactivity for MMPs. The strongest enzymatic activity for MMP-2 was seen in areas where invasive trophoblast cells invaded maternal tissues. MMP-9 had the highest enzymatic activity at the contact region of fetal and maternal parts, suggesting the importance of MMP-9 in separation of the placenta from the uterine wall during labor. MMP-3 had a similar localization to MMP-9, suggesting that besides gelatinases like MMP-2 and -9, MMP-3 (stromelysin-1) may also have important roles during labor. This study describes the site-specific distribution and activities of MMPs and therefore might help in elucidating the molecular mechanisms in pathologies such as premature rupture of membranes.

Expression of the blood-group-related antigens Sialyl Lewis a, Sialyl Lewis x and Lewis y in term placentas of normal, preeclampsia, IUGR- and HELLP-complicated pregnancies

Lewis antigens belong to the blood group of antigens and mediate cellular adhesion through interaction with selectins. Invasive trophoblasts use an array of adhesion molecules to facilitate cell-cell and cell-extracellular matrix interactions. Here, we examined immunohistochemically the expression of Sialyl Lewis a (sLe(a)), Sialyl Lewis x (sLe(x)) and Lewis y (Le(y)) in term placentas obtained from cases of normal, intrauterine growth retardation (IUGR), preeclamptic (PE) and hemolysis, elevated liver enzymes and low platelets syndrome (HELLP) pregnancies. We report the expression of sLe(x) in third trimester extravillous trophoblasts (EVT). sLe(x) was significantly decreased in IUGR and moderately decreased in PE compared to normal placentas. sLe(x) was additionally found in syncytiotrophoblast, without however any significant differences in staining intensity between normal and pathological cases. sLe(a) was restricted to amnion epithelium. Finally, Le(y) was expressed in cytotrophoblasts and villous endothelial cells. Le(y) expression was significantly upregulated in IUGR and HELLP, whereas there was a trend toward increase in PE compared to normal placentas. The present study suggests that downregulation of sLe(x) in EVT might be associated with IUGR and PE. Furthermore, Le(y), which was recently described as a potent angiogenic factor, is upregulated in placental villi in conditions associated with placental malperfusion.

The architecture of first trimester chorionic villous vascularization: a confocal laser scanning microscopical study

The aim of this study was to investigate normal chorionic villous vascularization using CD31 immunofluorescence and confocal laser scanning microscopy (CLSM) to elucidate the spatial arrangement in terms of connections between vessels and cords and their branching patterns compared to deficient chorionic villous vascularization in complicated pregnancies. A descriptive morphologic study using CLSM after CD31 immunofluorescence staining of placental biopsies from normal pregnancies (n = 20), complete hydatidiform molar pregnancies (CHMs; n = 3) and empty sacs (n = 3), with a well documented gestational age (GA). In this three-dimensional study, first trimester chorionic villi were occupied by a complex network of mainly cords with redundant connections as early as 5(+5) weeks GA. With increasing GA cords transform into vessels. From about 9 weeks GA onwards, vascular development is characterized by the presence of two large vessels located centrally and surrounded by and connected to a capillary network. In first trimester CHM and empty sacs, we observed a primitive network of mainly cords. This first visualization of the spatio-temporal patterns of blood vessel formation in placental villi is characterized by the development of the vasculosyncytial membrane from a complex network of cords and can be regarded as the placental development before it becomes functional at the end of organogenesis.

Delineation of an Isodicentric Y Chromosome in a Mosaic 45,X/46,X,idic(Y)(qter-p11.3:: p11.3-qter) Fetus by SRY Sequencing, G-banding, FISH, SKY and Study of Distribution in Different Tissues

Many factors such as genetic, developmental and hormonal are involved in mammalian sex determination. The relative importance and the mutual interactions among those factors are obscure. Study of cytogenetic mosaicism involving sex chromosomes may help to further unravel the mysterious process. We report a fetus with a mosaic karyotype, 45,X/46,X,idic(Y)(qter-p11.3::p11.3-qter), with unambiguous male external genitalia and a defect in the interventricular septum of the heart. Genotype of this fetus was extensively studied by technologies including sequencing of SRY (sex-determining region on the Y chromosome) gene, G-banding, FISH (fluorescence in situ hybridization) and SKY (spectral karyotyping). A markedly higher percentage of Y-containing cells was observed in the gonads (55%) than in the amniotic fluid (17%) and placental villi (11%), which was considered to be the major reason why the fetus did not have ambiguous genitalia.

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.

Expression and Localization of Cellular Prion and COMMD1 Proteins in Human Placenta throughout Pregnancy

Copper is an essential trace element for successful pregnancy. However, the mechanisms by which copper is transported from maternal circulation to the fetus have not been clearly elucidated. Two proteins, cellular prion (PrP C) and COMMD1, are known to be responsible for prion diseases and canine copper toxicosis, respectively, and are thought to play a role in copper homeostasis. However, their placental expression and localization throughout human gestation are still unknown. In this study, we used quantitative RT-PCR, western blotting and immunohistochemistry to investigate in detail the expression and localization of PrP C and COMMD1 proteins in human placenta throughout pregnancy. Our results show that both proteins are expressed in human placenta. PrP C showed the highest mRNA and protein expression levels during the first trimester of pregnancy. PrP C and COMMD1 proteins are similarly localized within the placental villi. Both proteins are present in the syncytiotrophoblast, the cytotrophoblast, vascular endothelial cells and Hofbauer cells. These data offer some insights into possible roles for PrP C and COMMD1 within the placenta.

Significance of PAM Histochemical Reaction in Delineating Macrophages

Periodic acid methenamine silver staining has been used for proof of the existence of carbohydrates in both paraffin and resin embedded tissues. The staining was applied to delineate macrophages that have been well known as cells of mononuclear phagocyte system by stainable nature for lysosomes, and it is found to be useful method for detection of macrophage. Fetal macrophage, which is often observed in placental villi, is called the highly vacuolated cell. Vacuoles in the cytoplasm seem to be empty with light microscopic staining, but it is clarified that they contain a full amount of carbohydrate. The present result leads us to the necessity of reconsidering the functional aspect of fetal macrophage.

45 Effect of fibrin matrices on increased proliferation and spreading of pre-term trophoblasts and placental villi modulation of coagulation and angiogenesis functions

45 Effect of fibrin matrices on increased proliferation and spreading of pre-term trophoblasts and placental villi modulation of coagulation and angiogenesis functions

Circulating calcitonin gene-related peptide and its placental origins in normotensive and preeclamptic pregnancies

The present study was designed to determine plasma calcitonin gene-related peptide concentration in both maternal and fetal circulations in normotensive and pre-eclamptic pregnancies and investigate whether placenta is 1 of its origins. Maternal blood, cord blood, and villous tissue were collected from women in normotensive pregnancies and complicated with pre-eclampsia. Calcitonin gene-related peptide concentrations were determined by radioimmunoassay. Cellular localizations of calcitonin gene-related peptide messenger ribonucleic acid and protein expressions in placental villi were determined by in situ hybridization and immunohistochemistry. The following results were reached: (1) maternal plasma calcitonin gene-related peptide concentrations increased with advancing gestation but fell after delivery; (2) both maternal and cord plasma calcitonin gene-related peptide concentrations were positively correlated with the infant birth weights; (3) compared with normotensive pregnancies, calcitonin gene-related peptide levels in both maternal and cord plasma decreased in pregnancies with pre-eclampsia; (4) in normotensive pregnancies, the plasma calcitonin gene-related peptide of the umbilical vein was higher than the umbilical artery, but no significant differences between vein and artery in pre-eclampsia; (5) calcitonin gene-related peptide messenger ribonucleic acid and protein were expressed by syncytiotrophoblast cells and villous vascular endothelial cells in normotensive pregnancies, but only weak or absent staining was observed in pre-eclamptic placentas; and (6) calcitonin gene-related peptide is secreted by villous tissue in explant culture in a time-dependent manner, but less calcitonin gene-related peptide was produced by villous tissues from patients with pre-eclampsia. Calcitonin gene-related peptide may play potential roles in maternal hemodynamic adaptation and fetal growth. Decreased circulating calcitonin gene-related peptide levels may be involved in maternal-fetal pathophysiology of pre-eclampsia. It is novel that placenta villous tissues might be one of the potential sources of calcitonin gene-related peptide during pregnancy.

Coeliac disease derived autoantibodies bind to the targets in intact placental villi and affect tissue transglutaminase activity

Coeliac disease derived autoantibodies bind to the targets in intact placental villi and affect tissue transglutaminase activity