Inhibin subunits mRNA expression level in human placenta from normal and Down's syndrome pregnancies

In order to study the mechanisms leading to increased inhibin A and activin A in maternal serum with advancing gestation and increased inhibin A in Down's syndrome pregnancy, the mRNA expression level of inhibin and activin subunits was quantitatively studied in human placenta using Northern blot and semiquantitative RT-PCR analysis. The corresponding protein level was also determined by specific ELISAs for inhibin A, inhibin B, activin A and inhibin pro alphaC in placental extracts. Normal placenta (n=27) showed a slight significant increase in alpha and betaA subunit mRNA levels in term pregnancy, with no change of the corresponding protein level. Placenta from Down's syndrome pregnancies (n=6) did not differ from controls in either mRNA expression or corresponding protein levels. In conclusion, there is a dissociation between inhibin and activin subunit mRNA levels and the corresponding protein levels in maternal serum, and Down's syndrome inhibin A increase is not explained by mRNA expression upregulation. In an additional study, ovarian cortex tissue from term pregnancies (n=3) were examined. Only the alpha subunit mRNA was expressed, at a higher level than in the placenta, suggesting that ovary could be a source of inhibin pro alphaC during pregnancy.     

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.     

Retinoid binding proteins-expression patterns in the human placenta.

The present study examined the expression and occurrence of different retinoid binding proteins in human first trimester and term placenta. At both stages, messenger RNA for the serum transport vehicle for retinol, retinol-binding protein (RBP), was detected only in decidual cells of the basal plate. In contrast, immunoreactive RBP (irRBP) was present in syncytiotrophoblast, core mesenchyme and lumen of vessels in placental villi and in mesenchyme and decidual cells of the basal plate. In villi of term placentae, however, staining for irRBP was lost in syncytiotrophoblasts and villous core mesenchyme. A putative placental RBP-receptor, approx 60-65kDa, was detected in the villous syncytiotrophoblast of both stages investigated. Immunoreactivity for the cellular retinol binding protein type I (CRBP I), was found in villous stromal cells and in decidual cells of the basal plate in sections of first trimester and term placenta. These results may suggest that maternal RBP-retinol is transferred across the chorionic villi to the fetal/villous circulation and that villous absorption of the complex is mediated via a placental RBP-receptor. Moreover, binding and possibly also metabolism of retinol may occur in the CRBP I positive villous stromal cells and decidual cells of the basal plate. In the latter, release of placental RBP-retinol may also be anticipated.     

Determination of the concentration of chorionic gonadotropin (HCG) and its free subunits in placental tissue during various periods of normal pregnancy

The aim of this study was to estimate HCG and its free subunits (alpha and beta) content in placental tissue and to determine the characteristic pattern of their ratio changes in the course of normal pregnancy. For this we used 47 samples of placental extracts. The tissue was extracted and gel filtrated on a calibrated Sephadex-G-100 column. Crude placental extracts and each fraction after chromatography were radioimmunoassayed for HCG, alpha HCG and beta HCG in a double antibody, homologous radioimmunoassay system. We obtained the following results: Peak of native HCG secretion occurred in 6-8 gestational week thereafter its concentration in placenta decreases. The absolute amount of free alpha HCG increased after the first trimester like to native HCG, but the relative quantity of alpha HCG exceeded that for HCG. In extracts from mature placenta quantity of free alpha HCG was about 5-10-fold more than HCG. In chorionic extracts from the first trimester we have proved the free beta subunit of HCG. It has not been found in extracts from mature placenta.     

Effect of hypoxia on placental activin A,inhibin A and follistatin synthesis

Placental activin A and inhibin A output is increased in pre-eclampsia, a condition characterized by placental hypoxaemia, whereas follistatin secretion is unaltered. We investigated whether hypoxia was the basis for elevated placental activin A and inhibin A output. First trimester and term placental explants were grown in 5-6% dissolved O(2) (n=10/trimester) and 200 microM cobalt chloride (CoCl(2),n =6/trimester) to simulate environmental and cellular hypoxia respectively, for up to 72 h. Activin A, inhibin A and follistatin production were compared with control cultures grown in standard media at 20% O(2). In first trimester and term placenta, activin A output declined significantly under 5-6% O(2) (P=0.006 and 0.001 after 48 h respectively). Inhibin A declined significantly under 5-6% O(2), mainly in first trimester placenta (P=0.03, 24h). CoCl(2) significantly elevated activin A production in term placenta (P=0.003, 48 h), whereas inhibin A output was unaffected. Neither low O(2) or CoCl(2) altered follistatin output from first trimester or term placenta. These findings suggest that there may be novel O(2) sensing mechanism/s that down regulate activin A and inhibin A in the placenta and that low O(2) is not the mechanism behind increased placental inhibin A or activin A output in pre-eclampsia.     

Preterm fetal growth restriction is associated with increased parathyroid hormone-related protein expression in the fetal membranes

OBJECTIVE: Parathyroid hormone-related protein has roles in normal fetal growth, placental calcium transport, and vascular tone regulation; these factors are compromised in growth-restricted fetuses. Our objective was to determine whether intrauterine parathyroid hormone-related protein expression was increased in association with fetal growth restriction. STUDY DESIGN: The expression of parathyroid hormone-related protein was examined in intrauterine tissues from women with idiopathic fetal growth restriction with preterm (n = 8-10) and term (n = 8-10) gestations and from gestation-matched control subjects. The abundance and immunoreactive content of parathyroid hormone-related protein messenger ribonucleic acid were determined by Northern blot and radioimmunoassay, respectively, in the placenta, amnion, and chorion-decidua. RESULTS: The expression of parathyroid hormone-related protein messenger ribonucleic acid was increased in the amnion (placental and reflected) in association with preterm fetal growth restriction (P <.05). Both parathyroid hormone-related protein messenger ribonucleic acid and protein expression were increased in chorion-decidua in association with preterm fetal growth restriction (P <.05). In term gestations both parathyroid hormone-related protein messenger ribonucleic acid and protein expression were greater in amnion over placenta than in reflected amnion (P <.05); these in turn were greater than those in chorion-decidua (P <.05). No significant changes were detected in parathyroid hormone-related protein messenger ribonucleic acid or in protein expression in association with term fetal growth restriction. CONCLUSION: Either parathyroid hormone-related protein messenger ribonucleic acid or protein expression, or both, was increased in the fetal membranes in association with fetal growth restriction in preterm but not term gestations, suggesting that parathyroid hormone-related protein may be involved in the pathogenesis of preterm fetal growth restriction.     

Placental,maternal serum and amniotic fluid molecular weight forms of inhibin A and pro-alphaC

Previous studies have identified the presence of unidentified small molecular weight (mol wt) forms of inhibin and the pro-alphaC region of the inhibin alpha subunit in serum from women during late pregnancy. The aim of this study was to investigate if these gestational-related changes in mol wt forms arose from changing placental production. Pooled placental extracts, derived from normal healthy singleton pregnancies in the 1st, 2nd and 3rd trimesters of pregnancy, were fractionated by a combined immunoaffinity chromatography, preparative PAGE and electroelution procedure. Inhibin A, inhibin B and the pro-alphaC region of the inhibin alpha subunit were determined in the eluted fractions by specific ELISAs, with the profiles of immunoactivity characterized in terms of molecular size and percentage recovery. Inhibin B was undetectable in all samples. Mol wt peaks of 36k, 75K and 97K for inhibin A and 29k, 55K and 97K for pro-alphaC were detected in placental extracts across all three trimesters. The relative abundancy of small mol wt inhibin A forms (<30K) present in the placenta increased significantly in the third trimester placenta, increasing from 0.3 per cent in the first trimesters to 6 per cent in the third trimester (P=0.01, chi-squared test). The relative abundances of various mol wt forms of pro-alphaC was similar at all three gestations (P=0.67). In serum, small mol wt inhibin A and pro-alphaC forms accounted for 23.4 per cent and 37.4 per cent of inhibins, respectively, in the third trimester.These data suggest that the presence of small mol wt forms of both inhibin A and pro-alphaC in maternal serum is only partially attributed to placental production and/or secretion. We conclude that inhibin A and pro-alphaC inhibins in maternal serum are processed in late pregnancy by more than one mechanism to form low mol wt circulating forms of, as yet, undetermined structure.     

Notch1蛋白在正常妊娠早中晚期胎盘绒毛中的表达变化及其意义

目的:检测Notch1蛋白在正常妊娠早、中、晚期胎盘绒毛中的表达水平变化,探讨Notch1在胎盘形成中的作用及意义。方法:采用免疫组织化学SP法、Real-timePCR、Western blot分别检测20例正常早期绒毛组织、15例中期妊娠和20例晚期妊娠胎盘组织中Notch1 mRNA及蛋白的表达水平。结果:Notch1蛋白在正常早、中、晚期妊娠过程中均有不同程度的表达,随着妊娠的进展其表达水平呈下调趋势,但无统计学差异(P>0.05)。正常早期绒毛组织中Notch1 mRNA的表达水平显著高于妊娠中、晚期胎盘组织(P<0.05)。结论:Notch1蛋白可能参与正常妊娠早期绒毛的分化及胎盘的形成过程,有利于维持妊娠的正常发展。     

Production of inhibin A and inhibin B in human ovarian sex cord stromal tumors

OBJECTIVE: Our purpose was to examine the cellular localization of inhibin subunits and messenger ribonucleic acid expressions for the inhibin subunits and the serum levels of inhibin A and inhibin B in human ovarian sex cord stromal tumors. STUDY DESIGN: We examined the immunohistochemical localization of the inhibin subunits and the expression of the corresponding messenger ribonucleic acids by Northern blot analysis in a granulosa cell tumor and a Sertoli-Leydig cell tumor. We also measured serum concentrations of dimeric inhibin A and inhibin B by two-site enzyme-linked immunosorbent assay. RESULTS: Immunostaining specific for the inhibin α, βA, and βB subunits was observed in the granulosa cell tumor. In the Sertoli-Leydig cell tumor we observed immunostaining specific for the α subunit in Leydig tumor cells and that specific for the βA subunit in Sertoli tumor cells and that specific for the βB subunit in both tumor cells. Northern blot analysis revealed the presence of messenger ribonucleic acids for the α, βA, and βB subunits in the granulosa cell tumor and the Sertoli-Leydig cell tumor. The serum levels of dimeric inhibin A and inhibin B in patients were elevated preoperatively and decreased progressively after surgery. CONCLUSION: Our results suggest that inhibin A and inhibin B are produced by the human sex cord stromal tumors and that inhibins might be the useful markers of the tumors.(Am J Obstet Gynecol 1997;177:7)     

Inhibin expression in normal and pre-eclamptic placental tissue.

Serum inhibin levels increase during normal pregnancy, but are significantly higher in patients with pre-eclampsia. The aim of this study was to demonstrate possible increased expression of inhibin within the placentas of women with pre-eclampsia compared with non-pre-eclamptic controls. Cellular expression of inhibin alpha and beta A subunits was studied using immunohistochemistry on formalin-fixed, paraffin-embedded placental sections from cases of pre-eclampsia (n = 23) and gestational age-matched non-pre-eclamptic controls (n = 16). Immunohistochemistry was performed using monoclonal antibodies against inhibin alpha and beta A subunits by the indirect immunoperoxidase technique. Intensity of staining was graded by a semiquantitative scoring method. Differences in distribution and intensity of staining between control and pre-eclamptic placentas were analyzed using a nonparametric Mann-Whitney U test. Staining for both inhibin alpha and beta A was predominantly confined to the cytoplasm of syncytiotrophoblast, with weak expression within intermediate trophoblast. The intensity of staining for inhibin alpha was significantly greater in the syncytiotrophoblast of pre-eclamptic patients (mean staining intensity controls = 0.97, disease = 1.87; p < 0.001). Inhibin beta A staining was generally stronger than for the alpha subunit, and was also significantly increased in pre-eclamptic patients compared with controls (mean controls = 1.72, disease 2.19; p < 0.05). This is the first evidence for increased placental inhibin presence in pre-eclampsia, suggesting increased inhibin production within the placenta, a finding that could account for increased serum inhibin levels in pre-eclampsia.     

In-vivo concentrations of inhibins, activin A and follistatin in human early pregnancy

The aims of this study were to investigate the relationship between inhibins, activin A and follistatin in first trimester fetal fluids, maternal serum, placenta and decidua, and to investigate if these hormones are present in the circulation of the early second trimester human fetus. Amniotic and coelomic fluid, maternal serum, placental villi and decidual tissue were obtained from normal pregnancies at 8-12 weeks. Fetal blood by cardiocentesis and maternal blood were collected at 14-16 weeks gestation. Placental extracts had higher concentrations of inhibins, activin A and follistatin compared with decidual extracts. In the second trimester, inhibins and follistatin were detectable in fetal blood at 14-16 weeks gestation. Maternal serum concentrations of inhibin A (P < 0.001) and follistatin (P < 0.05) were significantly higher than fetal serum whereas inhibin B (P < 0.01) and pro-alpha C concentrations (P < 0.001) were higher in fetal serum. Inhibin B concentrations were also higher in male fetal serum samples that had higher concentrations of testosterone. The presence of all molecular forms of inhibins, activin A and follistatin in the first trimester fetal fluids, placental and decidual extracts in the first trimester confirms other reports. In the second trimester, high concentrations of inhibin B with testosterone in the fetal circulation indicate that these hormones may interact in the development of the male fetal gonads.     

Epidermal growth factor binding and receptor distribution in term human placenta

Human placenta has a large number of epidermal growth factor (EGF) receptors when measured either by [125I]iodoEGF binding or by protein yield after purification. To localize EGF receptors in situ in normal human term placenta, two different light microscopic methods were used. To detect unoccupied, accessible EGF binding sites on the extracellular surface of placental cells in intact blocks of tissue, samples were incubated with [125I]iodoEGF, sectioned and autoradiography performed. To detect the total pool of intracellular and extracellular EGF receptors, placental tissue was sectioned, treated with detergent, and then anti-EGF receptor antibody was localized by immunohistoperoxidase techniques. Both [125I]iodoEGF and anti-EGF receptor antibody methods showed that EGF receptors were primarily present on syncytiotrophoblast cells of placental villi. Smooth muscle cells of placental blood vessels also contained EGF receptors. Neither connective tissue cells within the core of terminal chorionic villi nor endothelium of fetal blood vessels had detectable [125I]iodoEGF binding or immunoreactive EGF receptors. Since the quantity of placental smooth muscle cells is only a small fraction compared to trophoblast cells, we conclude that syncytiotrophoblast cells are primarily responsible for the high levels of EGF receptors found in extracts prepared from human term placenta.     

Syncytin在不同时期绒毛或胎盘组织中的表达及意义

目的:研究Syncytin在胎盘发育中的表达变化及意义。方法:采用荧光实时定量PCR(real-time FQ-PCR)方法检测正常早期妊娠绒毛组织(20例)、中期妊娠胎盘组织(15例)及晚期妊娠胎盘组织(20例)中Syncytin mRNA表达。结果:正常妊娠早、中、晚期胎盘组织Syncytin mRNA表达随孕周增加而升高,差异有统计学意义(P<0.05)。结论:Syncytin在胎盘发育过程中表达不同,推测可能参与胎盘发育之调节。     

Placental and maternal serum inhibin-A and activin-A levels in Down's syndrome pregnancies

The objective of this study was to analyse the levels of inhibin-A and activin-A in maternal serum and placental tissue from Down's syndrome (DS) pregnancies. Inhibin-A and activin-A levels were determined by specific immunoassays and individual results were expressed as multiples of the control median (MoM) at the appropriate gestation. Immunohistochemistry was used to localize inhibin alpha and beta(A)-subunits in a selection of placental sections. In DS pregnancies, median inhibin-A levels were found to be significantly elevated to 1.46 MoM (P< 0.05) in placental extracts, and 2.06 MoM (P< 0.0001) in maternal serum, when compared with uncomplicated pregnancies. Median activin-A MoMs were also elevated in placental extracts and maternal serum to 1.62 MoM (P< 0.01), and 1.26 MoM (P< 0.05), respectively. Immunohistochemistry revealed that the alpha subunit of inhibin-A and the beta(A)subunit of inhibin-A and activin-A were mainly localized to the trophoblastic layer of placental villi. Semiquantitative studies of staining intensity revealed a trend towards stronger staining of placental trophoblasts and stroma of DS tissues, although this was statistically significant only for beta(A)subunit staining of trophoblasts (P< 0.05).These results support the hypothesis that maternal serum levels of inhibin-A and activin-A are elevated due to increased production in the placenta, and increased immunostaining of trophoblasts suggests that this may be due to increased production in the trophoblasts.     

Alpha-fetoprotein gene expression in early and full-term human trophoblast

Alpha-fetoprotein (AFP) is a major serum glycoprotein synthesized during fetal life mainly by the yolk sac and the fetal liver. At term, it reaches high concentrations in the maternal intervillous blood, which is in direct contact with the placental trophoblastic microvillous membrane, and this suggests the placental origin of the AFP at the fetal-maternal interface. We used several experimental approaches to investigate the expression of AFP gene and fetal protein production in early gestation and term placentas. RT-PCR and immunological studies clearly identified AFP messenger RNA and AFP protein in the placental villi from first trimester of pregnancy. The AFP gene was also expressed in highly purified cytotrophoblasts from early placentas, and enzymo-immunoassay showed that AFP protein was synthesized and secreted by early cytotrophoblasts. AFP was also detected in the cytoplasm of these cells by immuno-cytochemistry. However, none of these methods detected any expression of the AFP gene in full-term placental villi or in cultured trophoblasts.These findings demonstrate that both AFP mRNA and protein are present in trophoblastic cells early in pregnancy. The absence of AFP gene expression in term placental villi also suggests, that the AFP at the fetal-maternal interface is attributable to a notable transplacental passage of AFP from fetal blood in late pregnancy.     

Expression of Fas-ligand in first trimester and term human placental villi

The expression of Fas-ligand (FasL) on trophoblast cells is thought to play a role in immune regulation during human pregnancy. However, there are some discrepancies in the published data concerning the cell types expressing FasL in the placental villi. Therefore, we examined the expression of FasL on cryosections of first trimester and term placental tissue with three different anti-sera against FasL, which are in common use. By immunohistochemistry, all three anti-sera principally gave the same staining result. In the first trimester of pregnancy, villous cytotrophoblast cells underlying the syncytium, as well as all extravillous trophoblast cells of cell columns and cell islands, gave a clear, mainly membrane-located staining, whereas the syncytiotrophoblast, which forms the borderline to the maternal blood flow, only gave a spot-like reaction in distinct areas. The same result was obtained with term placental villi; however, in this tissue, the staining of the villous cytotrophoblast cells was less pronounced. From our results, we suggest that in placental villi, an important role of FasL in immune regulation is not very conclusive because this molecule is mainly expressed on trophoblast with no access to maternal blood or tissue. This is in contrast to the uterine part of the placenta, where FasL expressing trophoblast cells are in close contact with apoptotic maternal leukocytes.     

Decreased very-low-density lipoprotein and low-density lipoprotein receptor messenger ribonucleic acid expression in placentas from preeclamptic pregnancies

OBJECTIVE: Our purpose was to investigate the levels of very-low-density lipoprotein and low-density lipoprotein receptor messenger ribonucleic acid expression in placentas obtained from normal pregnant women at various gestational stages and from patients with preeclampsia. STUDY DESIGN: The relative level of expression of very-low-density lipoprotein and low-density lipoprotein receptor messenger ribonucleic acid in each sample was determined by Northern blot analysis as a ratio of the intensity to that of glyceraldehyde-3-phosphate dehydrogenase messenger ribonucleic acid. RESULTS: The expression of very-low-density lipoprotein receptor messenger ribonucleic acid increased significantly (p < 0.05) from both the first (n = 9) and second (n = 8) trimesters to the third (n = 11) trimester. Similarly, the expression of low-density lipoprotein receptor messenger ribonucleic acid increased significantly (p < 0.05) from the first to the third trimester. The expression of both very-low-density lipoprotein and low-density lipoprotein receptor messenger ribonucleic acids in third-trimester placentas from preeclamptic pregnancies (n = 13) was significantly lower than that in normal pregnancies in the third trimester. CONCLUSION: These results demonstrate the expression of very-low-density lipoprotein and low-density lipoprotein receptor messenger ribonucleic acids in the human placenta, both of which increase at late gestational stages, and the decreased expression in cases of preeclampsia. Abnormal fetomaternal lipid metabolism resulting from low expression of these receptors in the placenta may be involved in the pathogenesis of preeclampsia. (Am J Obstet Gynecol 1996;175:1551-6.)     

TGF-β和IGF表达异常与滋养层相关疾病关系的研究

目的:探讨转化生长因子(TGF-β)和胰岛素样生长因子(IGF)等与滋养层侵入调节密切相关的细胞因子在胎盘绒毛组织中的表达,及与葡萄胎、先兆子痫等滋养层细胞相关妊娠疾病病理机制的关系。方法:采用半定量逆转录多聚酶链反应(RT-PCR)检测方法,分别以正常早孕绒毛和正常足月(自然分娩或剖宫产)胎盘绒毛组织为对照,观察葡萄胎组织与先兆子痫足月胎盘绒毛组织中TGF-β和IGF的表达。结果:5种胎盘绒毛组织中均可检测到IGF-I mRNA的表达,且先兆子痫足月胎盘绒毛组织中IGF-I的表达量明显低于正常足月胎盘绒毛组织,而葡萄胎组织中IGF-I的表达略高于正常早孕绒毛。在3种足月胎盘绒毛组织中可检测到IGF-ⅡmRNA的表达,但相互之间无明显差异。葡萄胎组织中IGF-Ⅱ的表达低于早孕绒毛。5种胎盘绒毛组织中均有TGF-β3的表达,而TGF-β1和TGF-β2的表达并未检测到。在表达量上,葡萄胎组织中TGF-β3的表达明显高于正常早孕绒毛组织,先兆子痫胎盘绒毛组织中TGF-β3的表达强于足月自然分娩的正常胎盘绒毛组织,而剖宫产胎盘绒毛组织中,TGF-β3的表达亦相对较强。结论:TGF-β3和IGF-I在胎盘绒毛组织中的表达变化,可能和葡萄胎、先兆子痫等与滋养层密切相关的妊娠疾病的发生有关。