Hormones and human trophoblast differentiation: a review

In the human, fetal cytotrophoblastic cells play a key role in the implantation process and in placental development. With the progression of placentation, two pathways of differentiation lead to the formation of two distinct phenotypes. In the villous trophoblast (fusion phenotype), the trophblast differentiates from the fusion of mononuclear cytotrophoblastic cells into a syncytium, the syncytiotrophoblast. Bathing the maternal blood, the syncytiotrophoblast is involved in maternal-fetal exchanges and in placental endocrine functions. In the extravillous trophoblast (proliferative/invasive phenotype), the cytotrophoblastic cells proliferate and migrate into the decidua, remodeling the pregnant endometrium and its vasculature. This review summarizes our current knowledge of the key step of villous differentiation-the cell-cell fusion of the cytotrophoblastic cells--and on the invasion process of extravillous trophoblast. Experimental evidence demonstrates that the genetic differentiation/invasion programs of cytotrophoblastic cells could be modulated by their environment: oxygen, extracellular matrix, and soluble factors (cytokines, growth factors, and hormones). Cytotrophoblastic cells fusion and the functional differentiation of villous trophoblast are specifically stimulated by estradiol, glucocorticoids, and human chorionic gonadotropin (hCG) whereas progesterone is ineffective. Because these hormones are temporally secreted in large amounts and present at the fetomaternal interface, they are in good position to play a physiologic role in trophoblast differentiation. hCG may be important very early in pregnancy, when production of this glycoprotein is maximal, whereas estrogen increasingly produced by the fetoplacental unit and cortisol secreted from the fetal adrenal may be implicated in the end-stage maturation and aging of the trophoblast.     

单增李斯特菌穿越生理屏障机制的研究进展

单核细胞增生性李斯特菌(Listeria monocytogenes,LM)是一种胞内寄生的革兰阳性短杆菌,属于食源性病原体,能够引起免疫力低下的人或动物患李斯特菌病,被列为四大食品病原菌之一.LM在不同生理屏障中的内化作用是介导感染的关键环节,且LM已进化出多种策略来入侵宿主细胞和适应胞内的传播.感染后的临床表现主要...     

Role of placenta in preeclampsia

Preeclampsia, which manifests itself as hypertension, proteinuria, and edema in pregnancy, requires the presence of trophoblast tissue but not a fetus. It is characterized by abnormal trophoblast invasion of the spiral arteries of the decidua and myometrium leading to a failure to establish an adequate uteroplacental blood flow and, therefore, is thought to give rise to relatively hypoxic trophoblast tissue. This, in turn, may promote an exaggerated state of oxidative stress in the placenta. This hypoxia/oxidative stress may then further attenuate trophoblast invasion but also alters placental villous angiogenesis leading to a poorly developed fetoplacental vasculature with abnormal reactivity. Oxidative stress per se may also affect vascular reactivity, blood flow, and oxygen and nutrient delivery to the fetus, which ultimately may be compromised. The synthetic and transport functions of the syncytiotrophoblast may also be altered, and there is an increased rate of trophoblast apoptosis. The linkage among abnormal trophoblast invasion, trophoblast dysfunction, and the maternal disease remains unidentified. The presumptive humoral factor that is released by the preeclamptic placenta to cause maternal disease remains elusive. Current therapies to prevent preeclampsia aim toward preventing the maternal syndrome, not preventing the primary pathophysiology.     

Immunohistochemical localization of 3 beta-hydroxy-5-ene-steroid dehydrogenase/delta 5----delta 4 isomerase in human placenta and fetal membranes throughout gestation.

The regulation of steroid production by the placenta and fetal membranes is important for both the maintenance of pregnancy and the timing of parturition. 3 beta-Hydroxy-5-ene-steroid dehydrogenase/delta 5----delta 4-isomerase (3 beta HSD) catalyzes an obligatory step in the biosynthesis of steroid hormones. We have determined the localization of 3 beta HSD in the human placenta, fetal membranes, and umbilical cord throughout gestation by immunohistochemical analysis, using a polyclonal antibody raised in rabbits against a purified preparation of human placental 3 beta HSD. In placenta, immunoreactive (IR-) 3 beta HSD was localized in the syncytiotrophoblast and intermediate trophoblast cells at both villous and extravillous sites, but not in cytotrophoblast cells from 6 weeks gestation to term. At 6-7 weeks gestation, IR-3 beta HSD was distributed in the cytoplasm of syncytiotrophoblast in about half of placental villi. By 12-14 weeks, the syncytiotrophoblast of all placental villi stained positively for 3 beta HSD. In the fetal membranes, strong IR-3 beta HSD staining was found in the trophoblast and reticular layers of chorion and in invasive trophoblast cells in decidua, and weakly in decidual stromal cells and amniotic epithelium. No IR-3 beta HSD was found in amnion on the placental plate, but in the umbilical cord, IR-3 beta HSD was present in the amniotic epithelium and also in fibroblast cells in Warton's jelly. These observations demonstrate that the localization of 3 beta HSD immunoreactivity and, therefore, the presumed sites of delta 5- to delta 4-steroid interconversion throughout gestation are principally the syncytiotrophoblast and intermediate trophoblast cells in placenta and the trophoblast cells in chorion and decidua in fetal membranes.     

Functional differentiation of the placental syncytiotrophoblast: Effect of estrogen on chorionic somatomammotropin expression during early primate pregnancy

Estrogen stimulates morphological and functional (i.e. steroidogenesis) differentiation of the primate placental trophoblast, and with advancing gestation there is an increase in estrogen and placental chorionic somatomammotropin (CS) mRNA and protein levels. To examine whether CS formation is regulated by estrogen, placental villous trophoblast CS was determined in baboons in which estradiol levels in uterine vein were increased 2- to 3-fold (P < 0.01) on d 60 of pregnancy (term = 184 d) by administration of aromatizable androstenedione on d 30-59 or estradiol benzoate on d 45-59 of gestation. Androstenedione and estradiol treatment resulted in a 75% decrease (P < 0.01) in placental whole villous CS-3 mRNA and CS protein levels, determined by Northern and Western blot analysis, on d 60, and a corresponding decrease in syncytiotrophoblast CS protein and maternal serum CS levels. In contrast, placental villous Delta(5)-3beta-hydroxysteroid dehydrogenase, 11 beta-hydroxysteroid dehydrogenase-2, and P-450 aromatase protein levels were unaltered by androstenedione or estradiol treatment. Collectively, these results suggest that, in elevated levels, estrogen suppressed CS formation by villous syncytiotrophoblast during the first one third of primate pregnancy. Therefore, estrogen has very different and specific actions on steroid and peptide hormone biosynthesis within the placental trophoblast, which we propose are important in regulating placental function and promoting fetal-placental development in the primate.     

Expression of betaglycan in pregnant tIssues throughout gestation

BACKGROUND: Betaglycan is a membrane-anchored proteoglycan involved in mediating the passage of transforming growth factor-beta (TGF-beta), inhibin and activin activities into cells. TGF-beta and inhibin-related proteins are growth factors that are expressed by several tIssues and in pregnancy. They have a function in modulating the growth, differentiation and invasion of the placental trophoblast. OBJECTIVE: To evaluate whether betaglycan is expressed by intrauterine tissues throughout gestation. DESIGN AND METHODS: Expression of betaglycan mRNA and protein was evaluated (by RT-PCR and immunohistochemistry, respectively) in trophoblast, decidua and fetal membranes collected during the first (n=6 elective terminations of pregnancy, between 8 and 12 gestational weeks) and third (n=6 elective caesarean sections, between 39 and 40 weeks) trimesters of pregnancy. RESULTS: Betaglycan mRNA was expressed by all gestational tIssues, independently of gestational age. Immunoreactive protein was found in decidual cells and in some chorionic, but not epithelial, amniotic cells. With respect to the placental localization, syncytiotrophoblast, but not cytotrophoblast, cells were intensively stained both in the placental bed and in the villous trophoblast, and in some cells within the stroma of terminal villi, of the first and third trimesters of pregnancy. Immunoreactive betaglycan was demonstrated in the endothelial cells of decidual vessels in both the first and third trimesters of pregnancy, whereas endothelial cells of fetal blood vessels in the villous were clearly represented only in first trimester samples, not in those of term placenta. CONCLUSIONS: Betaglycan mRNA and peptide are expressed by the trophoblast, the decidua and the fetal membranes, but the localization of the peptide in vessel walls is dependent on gestational age.     

From the Cover: Syncytin-A knockout mice demonstrate the critical role in placentation of a fusogenic,endogenous retrovirus-derived,envelope gene

In most mammalian species, a key process of placenta development is the fusion of trophoblast cells into a highly specialized, multinucleated syncytiotrophoblast layer, through which most of the maternofetal exchanges take place. Little is known about this process, despite the recent identification of 2 pairs of envelope genes of retroviral origin, independently acquired by the human (syncytin-1 and syncytin-2) and mouse (syncytin-A and syncytin-B) genomes, specifically expressed in the placenta, and with in vitro cell–cell fusion activity. By generating knockout mice, we show here that homozygous syncytin-A null mouse embryos die in utero between 11.5 and 13.5 days of gestation. Refined cellular and subcellular analyses of the syncytin-A-deficient placentae disclose specific disruption of the architecture of the syncytiotrophoblast-containing labyrinth, with the trophoblast cells failing to fuse into an interhemal syncytial layer. Lack of syncytin-A-mediated trophoblast cell fusion is associated with cell overexpansion at the expense of fetal blood vessel spaces and with apoptosis, adding to the observed maternofetal interface structural defects to provoke decreased vascularization, inhibition of placental transport, and fetal growth retardation, ultimately resulting in death of the embryo. These results demonstrate that syncytin-A is essential for trophoblast cell differentiation and syncytiotrophoblast morphogenesis during placenta development, and they provide evidence that genes captured from ancestral retroviruses have been pivotal in the acquisition of new, important functions in mammalian evolution.     

Placental glucose transfer and fetal growth

One of the primary regulators of maternofetal glucose transfer is the density of glucose transporter proteins in the placenta. These transporters, members of the GLUT gene family of facilitated-diffusion transporters, are embedded in the microvillous (maternal-facing) and basal (fetal-facing) membranes of the syncytiotrophoblast, the main placental barrier layer. Eight members of this family have been described in human placental tissue, but only GLUT1 protein has been identified in the syncytium, where its distribution is asymmetric. The microvillous membrane contains markedly more transporter than the basal, and, as a result, the basal membrane acts as the rate-limiting step in transplacental glucose transport; thus, changes in the density of basal membrane GLUT1 will have a significant impact on transplacental glucoseflux. What little is known about syncytial GLUT1 expression is restricted to factors associated with fetoplacental growth and metabolism; GLUT is inversely regulated by glucose concentration and basal membrane GLUT1 is positively regulated by insulin-like growth factor I, placental growth hormone, and hypoxia. In vivo, basal membrane GLUT1 is upregulated over gestation, increased in diabetic pregnancy, and decreased in chronic hypoxia, while microvillous membrane GLUT1 is unaffected. The contrast between in vitro and in vivo regulation and the specific changes in GLUT1 distribution suggest more complex regulatory interactions than those yet described.     

The pleiotropic function of PPAR gamma in the placenta

At different stages of placental development the cytotrophoblasts differentiate into specialized cells that are vital for specific placental tasks. These types include the invasive trophoblasts, which are responsible for invasion of the placenta into the uterine wall, and syncytiotrophoblasts, which form a barrier between the maternal and fetal circulations, govern trans-placental transport of gas, nutrient and waste, and produce placental hormones. Recent research illuminated the role of the nuclear receptor peroxisome proliferator-activated receptor-gamma (PPAR gamma) in the areas of adipocyte and macrophage biology, insulin action, bioenergetics and inflammation. It was somewhat surprising that PPAR gamma was also found to play a pivotal role in placental biology. In this review we summarize recent data, which show that PPAR gamma is expressed in the placenta, particularly in trophoblasts, and is essential for placental development, trophoblast invasion, differentiation of cytotrophoblasts into syncytium, and regulation of fat accumulation in trophoblasts. PPAR gamma may also play a role in modulating fetal membrane signals toward parturition. The data presented here underscore the need for a focused investigation of the unique aspects of PPAR gamma function in trophoblasts, which may have direct implications for the use of PPAR gamma ligands during pregnancy.     

Effect of IPs, cAMP, and cGMP on the hPL and hCG secretion from human term placenta

The acute control of human placental lactogen (hPL) and chorionic gonadotrophin (hCG) secretion by the placenta remains elusive. The in vitro release of both hormones can be stimulated by calcium inflow and by albumin. To investigate the placental secretory response to putative ligand(s) present in the maternal circulation, we evaluated the coupling of the hPL and hCG releases from term placenta with intracellular signaling pathways. Addition of NaF, forskolin or sodium nitroprusside, activators of the inositol phosphates (IPs), cAMP and cGMP pathways, significantly increased their respective messengers in villous explants but failed to affect the hPL and hCG releases from syncytiotrophoblast. By contrast, albumin did not modify the IPs, cAMP and cGMP villous content but significantly stimulated the placental hormonal release. These data suggest that the hPL and hCG secretion is not regulated through the IPs, cAMP and cGMP signaling pathways.     

Human chorionic gonadotropin stimulates trophoblast invasion through extracellularly regulated kinase and AKT signaling

Chorionic gonadotropin (CG) is indispensable for human pregnancy because it controls implantation, decidualization, and placental development. However, its particular role in the differentiation process of invasive trophoblasts has not been fully unraveled. Here we demonstrate that the hormone promotes trophoblast invasion and migration in different trophoblast model systems. RT-PCR and Western blot analyses revealed expression of the LH/CG receptor in trophoblast cell lines and different trophoblast primary cultures. In vitro, CG increased migration and invasion of trophoblastic SGHPL-5 cells through uncoated and Matrigel-coated transwells, respectively. The hormone also increased migration of first-trimester villous explant cultures on collagen I. Proliferation of the trophoblast cell line and villous explant cultures measured by cumulative cell numbers and in situ 5-bromo-2'-deoxyuridine labeling, respectively, was unaffected by CG. Addition of the hormone activated ERK-1/2 and AKT in SGHPL-5 cells and pure, extravillous trophoblasts. Inhibition of MAPK kinase/ERK and phosphatidylinositide 3-kinase/AKT blocked phosphorylation of the kinases and attenuated CG-dependent invasion of SGHPL-5 cells. Similarly, the inhibitors decreased hormone-stimulated migration in villous explant cultures. Western blot analyses and gelatin zymography suggested that CG increased matrix metalloproteinase (MMP)-2 protein levels and activity in both culture systems. Inhibition of ERK or AKT diminished CG-induced MMP-2 expression. In summary, the data demonstrate that CG promotes trophoblast invasion and migration through activation of ERK and AKT signaling involving their downstream effector MMP-2. Because the increase of CG during the first trimester of pregnancy correlates with rising trophoblast motility, the hormone could be a critical regulator of the early invasion process.     

Oxygen regulation of placental 11 beta-hydroxysteroid dehydrogenase 2: physiological and pathological implications

Preeclampsia (PE) is a major cause of maternal and perinatal morbidity and mortality. The genesis of PE is related to deficient trophoblast invasion of maternal spiral arteries, which might result in a reduction of placental (PL) oxygen (O(2)). An absence of increased O(2) that normally occurs around the 10-12th wk of gestation results in aberrant expression of genes that might contribute to the pathophysiology of PE. We examined the expression and regulation of PL 11 beta-hydroxysteroid dehydrogenase 2 (11 beta-HSD) in normal pregnancies and in PE. Two types of 11 beta-HSD exist in the placenta, 11 beta-HSD1 and 11 beta-HSD2. 11 beta-HSD2 is thought to protect the fetus from cortisol excess. In PE, both the expression and activity of PL 11 beta-HSD2 were reduced significantly compared with those in age-matched controls. As PE is associated with a reduction of PL O(2), we next investigated whether in normal pregnancy 11 beta-HSD2 expression changes at the time of the increase in O(2). 11 beta-HSD2 was detected as early as 5 wk, with expression limited to the syncytiotrophoblast (ST). At 10-12 wk, this expression increased and was also found in the cytotrophoblast and extravillous trophoblast. These results were substantiated by Western blot. The ability of O(2) to regulate 11 beta-HSD2 was determined both in cultures of villous explant from early gestation and in term trophoblast cells after incubation under 3% or 20% O(2). Villous explants cultured under 20% O(2) showed higher enzyme activity and expression compared with 3% O(2). Term trophoblast cells also exhibited higher enzyme activity at 20% vs. 3% O(2). No change in 11 beta-HSD1 expression was observed in early pregnancy or in PE. This is the first report to suggest that 11 beta-HSD2 is O(2) dependent in first and third trimester placenta during human gestation.     

Characterization of the endocannabinoid system in early human pregnancy

In recent years, it has been demonstrated that high circulating levels of the endogenous cannabinoid anandamide, resulting from low expression of its metabolizing enzyme fatty acid amide hydrolase (FAAH), may contribute to spontaneous miscarriage and poor outcome in women undergoing in vitro fertilization. The site of action of this compound, however, has not been determined. In this study, we examined the distribution of the cannabinoid receptors, CB1 and CB2, and the endocannabinoid-metabolizing enzyme FAAH in first trimester human placenta. Here, we show that FAAH is expressed throughout the human first trimester placenta, in extravillous trophoblast columns, villous cytotrophoblasts, syncytiotrophoblasts, and macrophages. Furthermore, FAAH mRNA levels appear to be regulated during gestation, with levels peaking at 11 wk before declining again. The immune system-associated cannabinoid CB2 receptors were localized only to placental macrophages. Interestingly, the cannabinoid receptor CB1 was not identified in first trimester placenta despite having previously been shown to be present in placental tissues at term. These findings suggest that the placenta may form a barrier preventing maternal-fetal transfer of anandamide and/or modulate local levels of anandamide by regulation of FAAH expression with gestation.     

Cytoadherence of Plasmodium falciparum-infected erythrocytes in the human placenta

In Plasmodium falciparum-parasitized pregnant women, erythrocytes infected by mature stages of the parasite sequester into placental intervillous spaces. The presence of parasites in the placenta causes maternal anaemia and low birth weight of the infant. In-vitro studies suggest placental sequestration may involve the cytoadherence of infected erythrocytes to chondroitin sulphate A (CSA) and/or intercellular adhesion molecule 1 (ICAM-1) expressed by human placental syncytiotrophoblast. We identified P. falciparum receptors expressed on the surface of human syncytiotrophoblast using immunofluorescence of placental biopsies from Cameroon, a malaria-endemic area. In all placentas, a strongly positive staining was observed on the syncytiotrophoblast for CSA, but not for ICAM-1, vascular endothelium cell adhesion molecule-1, E-selectin, nor CD36. The cytoadherence ability of parasites from pregnant women and nonpregnant subjects was assessed on in-vitro cultured syncytiotrophoblast. Parasites from pregnant women bound to the trophoblast via CSA but not ICAM-1. Parasites from nonpregnant hosts either did not bind to the trophoblast culture or bound using ICAM-1. Our data support the idea that placental sequestration may result from cytoadherence to placental trophoblast and that pregnant women are parasitized by parasites that differ from parasites derived from nonpregnant host by their cytoadherence ability.     

Expression of extracellular matrix metalloproteinase inducer in human placenta and fetal membranes at term labor

Matrix metalloproteinases (MMPs) are the main mediators of extracellular matrix (ECM) degradation during human parturition. However, the mechanisms involved in regulation of MMP production during parturition remain poorly understood. Recently, an extracellular matrix metalloproteinase inducer (EMMPRIN) has been shown to play a key role, as a local regulator, in stimulating MMP production in cancer systems. Whether EMMPRIN is expressed and stimulates MMP production in human placenta and fetal membranes is presently unknown. In this study, we investigated the expression of EMMPRIN at the levels of mRNA and protein in human term placenta and fetal membranes with or without labor. Western blot analysis showed that EMMPRIN protein was detected in term placenta and fetal membranes at two molecular masses of 40 and 65 kDa (glycosylated protein) and one of approximately 30 kDa (nonglycosylated protein). The ratio of 65 kDa EMMPRIN to total EMMPRIN significantly increased (P < 0.05) in term labor chorio-decidua and amnion compared with nonlabor chorio-decidua and amnion. Immunohistochemical analysis revealed that EMMPRIN was expressed in placental syncytiotrophoblast, amniotic epithelial cells, trophoblast cells of chorion laeve, and decidua parietalis. EMMPRIN was also detected at the mRNA level using RT-PCR in cultured placental syncytiotrophoblast, amniotic epithelial cells, and chorionic trophoblast cells. We conclude that human placenta and fetal membranes express EMMPRIN, with the potential to stimulate MMP production, thereby facilitating fetal membrane rupture and leading to detachment of the placenta and fetal membranes from the maternal uterus at the time of parturition.     

Thermodynamically reengineering the listerial invasion complex InlA/E-cadherin

Biological processes essentially all depend on the specific recognition between macromolecules and their interaction partners. Although many such interactions have been characterized both structurally and biophysically, the thermodynamic effects of small atomic changes remain poorly understood. Based on the crystal structure of the bacterial invasion protein internalin (InlA) of Listeria monocytogenes in complex with its human receptor E-cadherin (hEC1), we analyzed the interface to identify single amino acid substitutions in InlA that would potentially improve the overall quality of interaction and hence increase the weak binding affinity of the complex. Dissociation constants of InlA-variant/hEC1 complexes, as well as enthalpy and entropy of binding, were quantified by isothermal titration calorimetry. All single substitutions indeed significantly increase binding affinity. Structural changes were verified crystallographically at < or =2.0-A resolution, allowing thermodynamic characteristics of single substitutions to be rationalized structurally and providing unique insights into atomic contributions to binding enthalpy and entropy. Structural and thermodynamic data of all combinations of individual substitutions result in a thermodynamic network, allowing the source of cooperativity between distant recognition sites to be identified. One such pair of single substitutions improves affinity 5,000-fold. We thus demonstrate that rational reengineering of protein complexes is possible by making use of physically distant hot spots of recognition.     

Gestational age-dependent dual action of epidermal growth factor on human placenta early in gestation.

In order to better understand the role of epidermal growth factor (EGF) in the regulation of placental growth and function, effects of EGF on proliferative activity and differentiated function of trophoblast were examined. Explants from very early (4-5 week) placentas and early (6-7 week, 8-9 week, 10-12 week) placentas were respectively cultured under a serum-free condition in the absence or presence of EGF (100 micrograms/L) for the first 48 h, and the cultures were continued for subsequent 72 h without EGF. The proliferative potential and differentiated function of trophoblast were assessed by immunohistochemical Ki-67 staining and by determining the ability to secrete human CG (hCG) and human placental lactogen (hPL), respectively. Quantitative estimates of proliferative activity based on mean percentage of Ki-67 positive nuclei showed that EGF stimulated proliferative potential of cytotrophoblast in very early (4-5 week) placental explants. The EGF stimulation of trophoblast proliferation was apparent at a 12-h EGF-treated period. By contrast, early (6-12 week) placental explants did not respond to EGF with increase in trophoblast proliferation. Instead, in early placental explant culture EGF stimulated hCG and hPL secretion with a lag period of 72 h, whereas very early placental explants did not respond to EGF with increase in hCG and hPL secretion. These results suggest that EGF exerts gestational age-dependent dual action on the first-trimester placenta: one is to stimulate trophoblast proliferation in 4-5 week placenta and the other is to stimulate differentiated trophoblast function in 6-12 week placenta.