Published by Elsevier
Print ISSN: 0143-4004
In questi quaderni vengono pubblicati i lavori dei docenti della Facoltà di Economia dell'Università dell'Insubria. La pubblicazione di contributi di altri studiosi, che abbiano un rapporto didattico o scientifico stabile con la Facoltà, può essere proposta da un professore della Facoltà, dopo che il contributo sia stato discusso pubblicamente. Il nome del p roponente è riportato in nota all'articolo. I punti di vista espressi nei quaderni della Facoltà di Economia riflettono unicamente le opinioni degli autori, e non rispecchiano necessariamente quelli della Abstract In this paper we discuss sensitivity of forecast with respect to the information set considered in prediction; we define a sensitivity measure called impact factor, IF. We calculate this measure in VAR processes integrated of order 0, 1 and 2. For VAR processes this measure is a simple function of the impulse response coefficients. For integrated VAR systems this measure is shown to have a direct interpretation in terms of long-run forecasts. Various applications of this concept are reviewed, including one on the interpretation and effectiveness of economic policies and one on the sensitivity of forecasts with respect to data revisions. A unified approach to inference on the IF is given, showing under what circumstances standard asymptotic inference can be conducted also in systems integrated of order 1 and 2.
Betamethasone is frequently administered to pregnant women at risk of premature labor to accelerate fetal lung maturation. Maternally administered betamethasone produces pronounced changes in the fetal peripheral vasculature, raises fetal blood pressure and produces fetal growth restriction. Endothelial nitric oxide synthase (eNOS) plays an important role in regulating vascular tone. We hypothesized that effects of betamethasone on the fetal vasculature include decreased eNOS activity. We determined a significant depression of total placental eNOS protein measured by ELISA (betamethasone treated vs control, 0.48 +/- 0.28 vs 1.57 +/- 0.45, p < 0.05) and immunohistochemistry in both syncytiotrophoblast and vascular endothelium. Following betamethasone exposure, eNOS mRNA and enzyme activity showed decreasing trends which were not statistically significant. eNOS protein was higher in the placentas of both control and betamethasone treated baboons in the presence of a female fetus compared with a male fetus. The same effect of the sex of the fetus was observed in the betamethasone group for eNOS mRNA. In conclusion, maternally administered betamethasone produces a consistent decrease in several indices of placental eNOS function that may play a role in the altered cardiovascular dynamics and fetal growth retardation produced by betamethasone administration in late pregnancy.
We sought to detect the presence of receptors for 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] in placental tissues of five late gestational pregnant sheep and to quantitate their biochemical properties and abundance. Cytosol prepared from cotyledonary tissue was found to contain two [3H]-1,25(OH)2D3 binding macromolecules that sedimented at 3.2 S and 4.1 S, respectively, on linear (4-20 per cent) hypertonic sucrose gradients. The 4.1 S component cosedimented with serum that had been prelabelled with [3H]-25-hydroxyvitamin D3 (25-OHD3) and was present in cytosols despite extensive washing of the tissue prior to homogenization. Concurrent incubation of the cytosol with [3H]-1,25(OH)2D3 and a tenfold molar excess of radioinert 25-OHD3 resulted in complete resolution of the 3.2 S macromolecule and disappearance of the 4.1 S binding component. The binding of [3H]-1,25(OH)2D3 to the 3.2 S component was completely abolished by coincubation with a 100-fold molar excess of radioinert 1,25(OH)2D3 and was replaced by a well resolved peak in the 4.1 S region. Scatchard analysis of cytosol binding to [3H]-1,25(OH)2D3 in the presence of a tenfold molar excess of radioinert 25OHD3 revealed a single class of non-interacting saturable binding site in the cotyledon and the endometrium of high affinity and low capacity. The mean +/- s.e. of the dissociation constant of the cotyledonary receptor of 0.21 +/- 0.06 nM was not different from that of 0.16 +/- 0.03 nM for the endometrial receptor. However, the abundance of the cotyledonary receptor was fourfold higher than that in the endometrium (110 +/- 20 versus 28 +/- 7 fmol/mg protein). Since it is not possible to completely separate endometrial tissue from cotyledonary tissue, the low abundance of receptor in endometrial cytosols may merely represent contamination of endometrial tissue with cotyledonary tissue. Further analysis of the [3H]-1,25(OH)2D3 occupied receptor in cotyledonary cytosols showed that it bound to DNA cellulose and was eluted with 0.16 M KCl. This in vitro binding of [3H]-1,25(OH)2D3 to DNA was confirmed in vivo by the finding of preferential nuclear targetting of [3H]-1,25(OH)2D3 (56 per cent of total cellular activity), 4 h after fetal intravenous administration of [3H]-1,25(OH)2D3 to five chronically catheterized fetal sheep. Total placental uptake of [3H]-1,25(OH)2D3 at this time amounted to 3.7 +/- 0.9 per cent of the injected dose. Preliminary analysis of ovine placental cytosols revealed a calcium binding protein of similar molecular weight to that found in the ovine intestine and in the intestine and placenta of rodents.(ABSTRACT TRUNCATED AT 400 WORDS)
In this study, we determined if vitamin D could inhibit oxidative stress-induced thromboxane production by placental trophoblasts. Trophoblast isolated from normal placentas were stimulated with CoCl2, a hypoxic mimicking agent, with or without pretreatment of 1,25(OH)2D3. Soluble phospholipase-A2, metabolites of thromboxane-A2 and prostacyclin, and 8-isoprostane were measured. Expression of cyclooxygenase-1 (COX-1), COX-2, and heme oxygenase-1 (HO-1) were determined. We found that pretreatment of trophoblasts with 1,25(OH)2D3 significantly reduced 8-isoprostane and the ratio of thromboxane-A2 to prostacyclin production, and blocked COX-2 expression induced by CoCl2. These results provide evidence of the beneficial effects of vitamin D on placental trophoblasts.
Glycosylation controls diverse protein functions and regulates various cellular phenotypes. Trophoblast invasion is essential for normal placental development. However, the role of glycosylation in human placenta throughout pregnancy is still unclear. The β-1,4-galactosyltransferase III (B4GALT3) has been found to regulate cancer cell invasion. We therefore investigated the expression of B4GALT3 in placenta and its roles in trophoblast. B4GALT3 protein expression was examined by quantitative Western blotting analysis in human placentas. For identification of B4GALT3-positive cells in normal human placenta, immunohistochemistry and immunofluorescence methods were used. To investigate effects of B4GALT3 on extravillous trophoblast (EVT)-like cell and primary EVT cells, we analyzed cell growth, adhesion, migration, and invasion in mock and B4GALT3-transfected cell. B4GALT3 expression significantly increased in third trimester human placenta. Immunostaining revealed that B4GALT3 expressed in placental villous cytotrophoblast, syncytiotrophoblast, and a subpopulation of EVT cells throughout pregnancy. Interestingly, we found increases in the expression level and percentage of B4GALT3-positive cells in third trimester EVT, but not in syncytiotrophoblasts and cytotrophoblasts of placental villi. Overexpression of B4GALT3 in HTR8/SVneo cells and primary trophoblast cells significantly suppressed cell migration. In addition, B4GALT3 suppressed cell invasion, and enhanced cell adhesion to laminin in HTR8/SVneo cells. Notably, we found that B4GALT3 modified glycans on β1-integrin, suppressed focal adhesion kinase (FAK) signaling, and enhanced β1-integrin degradation. We propose that B4GALT3-mediated glycosylation change not only enhances β1-integrin binding to laminin, but also attenuates β1-integrin stability. Our findings suggest that B4GALT3 is a critical regulator for suppressing EVT invasion in the late stages of pregnancy. Copyright © 2015 Elsevier Ltd. All rights reserved.
A differential expression of class II major histocompatibility complex (MHC) and Thy 1.2 antigens was detected on two morphologically distinct cell populations in short-term cultures of murine decidual tissue. Stromal type decidual cells expressed Thy 1.2, albeit transiently, and consistently lacked class II antigens. By contrast decidual macrophages expressed class II antigens and lacked Thy 1.2 antigens. Stromal type decidual cells, after culture in the presence of indomethacin, displayed no evidence of prostaglandin-mediated modulation of class II expression. These findings suggest that class II positive decidual macrophages are responsible for the antigen-presenting capacity of decidua.
Placental insufficiency is a major cause of fetal growth restriction (FGR) and accumulating evidence indicates several aspects of placental morphology are altered in this condition. MRI provides quantitative indices that may be used in non-invasive assessment of the human placenta, such as relaxation time measurements, T1 and T2. We hypothesised that placental relaxation times relate to alterations in placental tissue morphology and hence may be useful in identifying the changes associated with FGR. We report on the first phase of testing this hypothesis, in a study of women in normal pregnancy. To assess relaxation time measurements in the placenta in normal pregnancy and correlate these with gestational age and stereological analyses of placental morphology following delivery. 30 women underwent MRI examination (1.5 T) between 20 and 41 weeks gestation. Placental T1 and T2 measurements were acquired from a mid-depth placental region, co-localised to a structural scan. Fixed, wax-embedded sections of these placentas collected at delivery were stained with hematoxylin/eosin and subjected to stereological analysis. Placental T1 and T2 show a significant negative correlation with gestation, (Pearson correlation p=0.01, 0.03 respectively). 17 placentas were analysed stereologically. In the group as a whole there was no significant correlation between T1 and T2 and morphological features. However, in a subset of 7 pregnancies scanned within a week of delivery, a significant positive correlation was observed between the fibrin volume density and the ratio of fibrin: villous volume densities and T2 (Spearman correlation p=0.02, 0.03 respectively). The correlations between placental T1 and T2 and gestation show that these variables are clearly influenced by changes in placental structure. Fibrin might be a key component but further work is needed to fully elucidate the major structural influences on placental T1 and T2.
Exposure to low-dose radiation is widespread and attributable to natural sources. However, occupational, medical, accidental, and terrorist-related exposures remain a significant threat. Information on radiation injury to the feto-placental unit is scant and largely observational. We hypothesized that radiation causes trophoblast injury, and alters the expression of injury-related transcripts in vitro or in vivo, thus affecting fetal growth. Primary human trophoblasts (PHTs), BeWo or NCCIT cells were irradiated in vitro, and cell number and viability were determined. Pregnant C57Bl/6HNsd mice were externally irradiated on E13.5, and placentas examined on E17.5. RNA expression was analyzed using microarrays and RT-qPCR. The experiments were repeated in the presence of the gramicidin S (GS)-derived nitroxide JP4-039, used to mitigate radiation-induced cell injury. We found that survival of in vitro-irradiated PHT cell was better than that of irradiated BeWo trophoblast cell line or the radiosensitive NCCIT mixed germ cell tumor line. Radiation altered the expression of several trophoblast genes, with a most dramatic effect on CDKN1A (p21, CIP1). Mice exposed to radiation at E13.5 exhibited a 25% reduction in mean weight by E17.5, and a 9% reduction in placental weight, which was associated with relatively small changes in placental gene expression. JP4-039 had a minimal effect on feto-placental growth or on gene expression in irradiated PHT cells or mouse placenta. While radiation affects placental trophoblasts, the established placenta is fairly resistant to radiation, and changes in this tissue may not fully account for fetal growth restriction induced by ionizing radiation.
The aim was to determine whether nutritionally mediated restriction of placental growth alters foetal body growth, pituitary gonadotrophin gene expression and gonadal development at Day 103 of gestation. Embryos recovered from adult ewes inseminated by a single sire were transferred, singly, into the uteri of adolescent recipients. After transfer, adolescent ewes were offered a high (H, n=16) or moderate (M, n=12) level of a complete diet. Ewes were slaughtered at 103+/-0.2 days of gestation and foetal blood, brain, pituitary and gonads were collected. Mean placental weight was lower (P< 0.01) in H than in M groups but foetal weight and reproductive organ weights were similar. Maternal nutrition did not influence LHbeta or FSHbeta mRNA expression in either sex but FSHbeta mRNA expression was higher (P< 0.001) in female (n=11) than in male (n=17) foetal pituitaries. Mean foetal plasma gonadotrophin concentrations were not influenced by dietary intake in either sex. Plasma progesterone concentrations were lower (P=0.001) in foetuses derived from H compared with M intake dams. Compared with M foetuses (n=5), ovaries from H foetuses (n=6) had fewer primordial follicles (P< 0.05) and fewer follicles in total (P< 0.005). In contrast, maternal nutritional status did not influence either seminiferous cord or Sertoli cell numbers in male foetuses (H, n=10; M, n=7). It is concluded that high maternal nutrient intakes restricted placental growth and altered foetal ovarian follicular development prior to the end of the second third of gestation. The latter effect was independent of gonadotrophin secretion.Crown
Thrombosis of umbilical cord vessels is associated with a high perinatal mortality. Most cases are venous thrombi, arterial thrombi are uncommon. To clarify clinical and pathological features of this entity, we reviewed 11 cases with umbilical artery thrombosis. The gestational ages were 33-40 (mean 36.8) weeks. Three cases (38%) were associated with severe intrauterine growth retardation and two cases (25%) led to intrauterine fetal deaths. All cases had completely occlusive thrombi of one umbilical artery. The arteries with thrombosis demonstrated partial necrosis of vascular wall. The pathogenesis is unclear, but nine cases (82%) had cord abnormalities (long cord, peripheral cord insertion, short cord with twist, funisitis). This paper is the first report of clinical and pathological features of umbilical artery thrombosis. The results of this study confirm the clinical importance and unique histological findings of umbilical artery thrombosis. The recognition of umbilical artery thrombosis is necessary to establish the diagnosis and treatment of this condition.
11beta-hydroxysteroid dehydrogenase type 1 and type 2 may be important in the process of human parturition and the regulation of fetal growth, by the modulation of cortisol concentrations in the fetal compartment. Changes in the expression and activity of these enzymes in late gestation have not been well described. This study has examined the gene expression of placental 11beta-HSD1 and 2, activity of 11beta-HSD2 and fetal cortisol concentrations during the final few weeks of human pregnancy and with the onset of labour. Placental 11beta-HSD2 activity decreased significantly between 38 and 40 weeks. There were no significant changes in mRNA abundance or protein expression with gestational age or labour. Placental 11beta-HSD1 mRNA abundance significantly increased with spontaneous labour. Fetal cortisol concentrations increased significantly with spontaneous labour. This study is the first to describe a decrease in 11beta-HSD2 activity in the last few weeks of human gestation. This decrease in type 2 activity, along with an increase in 11beta-HSD1 gene expression may be a mechanism by which cortisol concentrations rise at term to regulate fetal maturation and activate pathways associated with labour.
This study was designed to examine the cellular localization and developmental regulation of 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD) type 1 gene expression in the ovine placenta. Placental tissues were collected at discrete times between days 59 and 143 of pregnancy (term = 145 days). Levels of 11 beta-HSD1 mRNA were determined by Norther blot analysis. The level of both dehydrogenase and reductase activities of 11 beta-HSD1 was assessed by a radiometric conversion assay using cortisol and cortisone as physiological substrates. The cellular localization of 11 beta-HSD1 protein was determined by standard immunohistochemical technique using a polyclonal antibody specific for the ovine protein. High levels of 11 beta-HSD1 mRNA were detected in the placenta by day 59, and there was a trend towards a decrease between days 98-103 and 125-128 (P = 0.06). The level of placental 11 beta-HSD1 mRNA remained unchanged thereafter. Levels of both 11 beta-HSD1 dehydrogenase and reductase activities followed a similar pattern except that in both cases there was a significant decrease between 98-103 and 125-128 (P < 0.05). Moreover, under the present assay conditions, the dehydrogenase activity was always predominant, suggesting that the net effect of placental 11 beta-HSD1 activity would lead to glucocorticoid inactivation. Thus, the decreased 11 beta-HSD1 activity in the placenta at days 125-128 was consistent with, and may help to explain, the apparent increase in the placental transfer of cortisol from mother to fetus during that time. Throughout pregnancy, intense 11 beta-HSD1 immunoreactivity was detected in fetal trophoblastic cells, maternal stromal cells and blood vessels. In contrast, maternal syncytium was immunonegative before day 125, but became immunopositive thereafter. The observed predominant direction of 11 beta-HSD1 activity in vitro and its pattern of localization in the ovine placenta are consistent with the hypothesis that placental 11 beta-HSD protects the fetus from adverse effects of maternal glucocorticoids by inactivating glucocorticoids locally.
We developed an in vitro suspension co-culture system to examine the interaction of 1st, 2nd and 3rd trimester purified cytotrophoblasts with human endometrium. Endometrium explants were added to cytotrophoblast cell suspensions and placed on an angled gyrating platform in a 37 degrees C incubator. When endometrium was cultured alone it was able to remain viable for up to 3 days. When trophoblasts were cultured alone, they formed small and large aggregates, and occasionally spherical shells with hollow centers. When trophoblasts and endometrium were cultured together, the trophoblasts adhered to the exposed stromal surfaces of the tissue fragments. The surface epithelium was not receptive to trophoblast attachment except in one experiment when day 19 endometrium was used for the co-incubation, suggesting that surface attachment is usually restricted. A common finding was the presence of an acellular zone in the endometrium only adjacent to the attached trophoblasts. We speculate that this zone may be caused by proteolysis and resynthesis of ECM proteins by the trophoblasts. Based on our results, this in vitro suspension should prove useful for examining those factors which: (1) induce endometrial permissiveness, (2) promote paracrine effects on the endometrium, and (3) facilitate human trophoblast invasion.
To understand the role of vascular endothelial growth factor (VEGF) in placental development, we examined immunohistochemically 56 placentae ranging from 6--41-weeks gestation using rabbit antibody to a synthetic multiple antigen peptide (MAP), composed of N-terminal amino acid residues 1--20 of human VEGF. In the present study, syncytiotrophoblast and invading extravillous trophoblasts ubiquitously expressed VEGF throughout gestation. However, the expression of VEGF in syncytiotrophoblasts was uneven in the first trimester and most intense at the sprouting sites. In addition, some stromal cells in the villi and decidual cells were also positive in the first trimester. A morphometrical analysis of the ratio of the VEGF-positive cell area to the capillary area in the terminal villi statistically revealed a critical point of change at 16 weeks' gestation. These results provide further evidence to support the hypothesis that VEGF, locally expressed by trophoblasts, stromal cells in villi and decidual cells, may play an important role in the physiological growth and function of the vascular system in the villous stroma and basal plate during placental development and maturation.
This paper presents an immunohistochemical study with a monoclonal mouse antibody GZ 112, an IgG1 kappa, which is directed against an antigen expressed in first trimester placenta by all proliferative and invasive extravillous trophoblast populations including a population of Langhans cells that represent extravillous stem cells. Additionally, the GZ 112 antigen is associated with morphological changes of spiral arteries preceding local trophoblast invasion. In term placentae, GZ 112 also strongly reacts with all extravillous trophoblast populations, but additionally recognizes partly villous cytotrophoblast and syncytiotrophoblast too, displaying a heterogeneous staining pattern. GZ 112 is directed against a 42-KDa antigen. Intracytoplasmic network-like staining and cross-reactivity with various human surface and glandular epithelia indicate a cytokeratin intermediate filament or a cytokeratin intermediate filament associated molecule as antigen.
Although rat is the most widely used model of glucocorticoid programming of the fetus, the role of rat placental 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2) in the transplacental pharmacokinetics of the naturally occurring glucocorticoid, corticosterone, has not yet been fully elucidated. In this study, expression of 11beta-HSD2 in the rat placenta on two different gestation days (16 and 22) was examined using quantitative RT-PCR and Western blotting, and dually perfused rat term placenta was employed to evaluate its functional capacity to transfer and metabolize corticosterone. Marked decrease in placental expression of 11beta-HSD2 toward term was observed on both mRNA and protein levels. In perfusion studies, increasing maternal corticosterone concentration from 3 to 200 nM resulted in the fall of 11beta-HSD2 conversion capacity from 64.3 to 16.3%, respectively. Enzyme saturation occurred at about 50 nM substrate concentration. When delivering corticosterone (3 or 100 nM) from the fetal side, a similar decline of 11beta-HSD2 conversion capacity was observed (66.5% and 48.5%, respectively). Addition of carbenoxolone (10 or 100 microM), a non-specific 11beta-HSD inhibitor, to maternal perfusate decreased conversion capacity from 66.7 to 12.6 or 8.1%, respectively. Similarly potent inhibitory effect was observed in feto-maternal studies. Neither saturation nor inhibition of 11beta-HSD2 was associated with transformation of corticosterone in metabolites other than 11-dehydrocorticosterone. These data suggest that 11beta-HSD2 is the principal enzyme controlling transplacental passage of corticosterone in rats and is able to eliminate corticosterone in both maternal and fetal circulations.
We have shown that the placenta, via metabolism of maternal cortisol and cortisone by the 11beta-hydroxysteroid dehydrogenase (11beta-HSD) enzymes types 1 and 2 in the syncytiotrophoblast, regulates the maturation of the fetal pituitary adrenocortical axis in the baboon. Because the timing and regulation of fetal adrenal development by fetal ACTH in the human seem to parallel that in the baboon, we propose that the placental 11beta-HSD-1 and -2 system also has a role in regulating the development of the fetal pituitary adrenocortical axis during human pregnancy. However, although the human placenta has been shown to express the 11beta-HSD-2, it remains to be determined unequivocally whether 11beta-HSD-1 protein is present in the human placental syncytiotrophoblast. To answer this question, enriched fractions of syncytiotrophoblast were prepared from human and baboon term placentae and proteins probed with polyclonal antibodies directed to amino acids 22-36 or 66-77 of human 11beta-HSD-1. The 11beta-HSD-1 was detected by Western blot analysis as a 32-kDa protein in human and baboon syncytiotrophoblast and as a 34-kDa protein in adult baboon liver. Localization of the 11beta-HSD-1 to the syncytiotrophoblast was confirmed by immunocytochemistry following antigen retrieval. These results show that both human and baboon placental syncytiotrophoblast expressed the 11beta-HSD-1, as well as the 11beta-HSD-2, proteins. Because 11beta-HSD-1 can function as a reductase, the expression of 11beta-HSD-1 in human syncytiotrophoblast would be consistent with the ability of this tissue to convert cortisone to cortisol and provide a means by which transplacental transport of cortisol could regulate the fetal pituitary adrenocortical axis in the human, as recently shown experimentally in the non-human primate baboon model.
The human placenta contains two types of 11beta-hydroxysteroid dehydrogenase (11beta-HSD). The exclusive oxidase 11beta-HSD2 has been suggested to protect the fetus from high levels of maternal glucocorticoids by converting cortisol to inactive cortisone. Perfused term human placenta was used to examine the activity of the oxoreductase 11beta-HSD1 and to determine the regulation of cortisol effects on placental vascular tone and corticotropin-releasing hormone (CRH) output by 11beta-HSD. Radioimmunoassay showed that there was substantial cortisol (295+/-57 nM) detected in the fetal vein upon perfusion of cortisol (2 microM; perfusion rate, 12 ml/min) into the maternal intervillous space. Output of cortisol increased to 559+/-22 nM on the fetal side (P<0.05) with concurrent perfusion of carbenoxolone (CBX; 1 microM), a non-specific 11beta-HSD inhibitor. Cortisol formation increased in a dose-dependent manner with infusion of cortisone (0.1-2 microM) into the maternal intervillous space reaching 15 and 23 nM in fetal and maternal venous outflows respectively at 2 microM cortisone perfusion. There was no significant effect of cortisol either alone or in combination with CBX on the fetal arterial perfusion pressure, but cortisol perfusion increased CRH output into the fetal vein. It is concluded that activities of both 11beta-HSD1 and -2 are demonstrable in perfused human placenta in vitro, and these enzymes affect transplacental glucocorticoid transfer. These activities may provide a precise mechanism to control the passage of maternal glucocorticoids to the fetal circulation, and to regulate glucocorticoid effects within the placenta.
Common pregnancy complications are associated with impaired placental development. This study aimed to characterise the ontogeny of structural correlates of rabbit placental function, its expression of genes encoding components of the renin-angiotensin system (RAS), as well as 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2) mRNA since these are known to be expressed by the placenta and are associated with pregnancy complications, including preeclampsia and intrauterine programming. Placentae were collected at gestational age (GA) 14, 21 and 28 (term=32 days). Gene expression was analysed using real time PCR and placental structures were quantified via image analyses. The volume densities and volumes of trophoblast, fetal capillaries, maternal blood space, surface density and surface area of trophoblast all progressively increased, while the arithmetic mean barrier thickness of trophoblast decreased across gestation. Maternal plasma renin activity (PRA) was positively correlated with volumes of trophoblast and maternal blood space, surface density and surface area of trophoblast. Placental renin mRNA declined ( downward arrow62%; P<0.01) across gestation and was negatively correlated with maternal PRA (GA0), fetal and placental weights, placental angiotensin type 1 and 2 receptors (AT(1)R and AT(2)R) mRNA and volume of trophoblast. AT(1)R mRNA expression was increased by 92% (P<0.001) across gestation. AT2R mRNA expression was approximately 81% (P<0.01) greater at GA14 compared to GA21. Placental 11beta-HSD2 mRNA expression was approximately 74% greater (P<0.01) at GA21 than GA14, but by GA28 was similar to that at GA14. These data show that changes in placental gene expression are associated with key events in placental and fetal development, indicating that the rabbit provides a good model for investigations of pregnancy perturbations that alter the RAS or programme the fetus.
The present study was undertaken to determine (1) if hypoxia-induced down-regulation of placental 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2; encoded by HSD11B2 gene) activity and protein in human trophoblast cells during in vitro differentiation was mediated at the level of HSD11B2 gene transcription; and (2) whether the reduced placental 11beta-HSD2 in pregnancies complicated with fetal growth restriction (FGR) was a consequence of intrinsic abnormalities in trophoblast cells. Trophoblast cells were isolated from uncomplicated pregnancies and those complicated with FGR at term, and cultured for up to 72 h under normoxic (20% oxygen) or hypoxic (1% oxygen) conditions. Under normoxia, 11beta-HSD2 activity and protein increased progressively over the 72 h culture period, which was accompanied by a corresponding rise in 11beta-HSD2 mRNA. As demonstrated previously, hypoxia blocked the increase in levels of both 11beta-HSD2 activity and protein within the first 24h. In contrast, although hypoxia also prevented the rise in 11beta-HSD2 mRNA, it did not do so until 48 h. This time-dependent effect of hypoxia on placental 11beta-HSD2 activity/protein and mRNA suggests a dual mechanism of action whereby hypoxia may induce a rapid down-regulation of 11beta-HSD2 protein synthesis, which occurs initially at the level of translation, and later extends to the level of transcription. Indeed, transient transfection studies demonstrated that hypoxia diminished HSD11B2 promoter activity. When trophoblast cells isolated from FGR placentas were cultured and allowed to differentiate under the same conditions, they not only exhibited a similar pattern of 11beta-HSD2 activity and mRNA expression but also responded to hypoxia similarly to those from normal placentas. This suggests that the reduced placental 11beta-HSD2 in FGR is not due to intrinsic abnormalities in trophoblast cells, but likely a result of extrinsic factors associated with FGR.
Proper glucocorticoid exposure in utero is vital for normal fetal organ maturation, but excess glucocorticoids are detrimental to fetal growth and can even predispose the individuals to the high risk of having certain diseases in adulthood. The fetus is protected from 10 times higher maternal glucocorticoid levels by the placental enzyme 11beta-hydroxysteroid dehydrogenase 2 (11beta-HSD2), which converts biologically active cortisol to inactive cortisone. Thus it is of primary importance to understand how this enzyme is regulated. Activation of cAMP/PKA pathway is known to upregulate 11beta-HSD2 expression in placental syncytiotrophoblasts, however the endogenous hormones utilizing this pathway remain largely unknown. By using cultured human placental syncytiotrophoblasts, we demonstrated that inhibition of protein kinase A with H89 attenuated 11beta-HSD2 expression in the syncytiotrophoblasts, suggesting endogenous factors from the syncytiotrophoblasts using this pathway to maintain 11beta-HSD2 expression in the syncytiotrophoblasts. Neutralization of human chorionic gonadotropin (hCG) secreted by the syncytiotrophoblasts with hCG antibody decreased 11beta-HSD2 promoter activity, mRNA and protein expression as well as intracellular cAMP level, while treatment of the syncytiotrophoblasts with exogenous hCG increased 11beta-HSD2 expression, which was attenuated by H89. Furthermore, we found that cortisol increased both hCG expression and secretion. The up-regulation of 11beta-HSD2 expression by cortisol was significantly attenuated by co-treatment with hCG antibody or H89 in the syncytiotrophoblasts. In conclusion, hCG is an important paracrine or autocrine hormone maintaining 11beta-HSD2 expression and the up-regulation of 11beta-HSD2 expression by cortisol may be mediated in part by hCG in the syncytiotrophoblasts.
The placental 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2) serves as a functional barrier to protect the fetus from excessive exposure to high levels of maternal cortisol. There is evidence that placental 11beta-HSD2 is reduced in pregnancies complicated with intrauterine growth restriction (IUGR), but the relationship between the two is uncertain owing to other maternal complications often associated with this pathological condition of pregnancy. To gain insight into the role of placental 11beta-HSD2 in the pathogenesis of IUGR, we studied variations in the activity and expression of this important enzyme as well as its functional indicator, the ratio of cortisone to cortisol in umbilical cord blood, in a cohort of 12 term deliveries complicated with idiopathic IUGR and 12 term controls. We showed that both placental 11beta-HSD2 activity and mRNA were reduced in IUGR. This was accompanied by a decrease in the ratio of cortisone to cortisol in the umbilical artery, suggesting that not only placental but also fetal 11beta-HSD2 activity may be compromised in idiopathic IUGR. Given that we previously identified the nuclear receptor PPARdelta as a potent suppressor of placental 11beta-HSD2, we also tested but found no evidence to support the hypothesis that placental PPARdelta expression is increased in IUGR thereby contributing to the molecular mechanisms that underlie the attenuated placental 11beta-HSD2. Taken together, our present findings provide evidence suggesting a role for an attenuated placental as well as fetal 11beta-HSD2 in the pathogenesis of IUGR.
Intrauterine fetal death is an agonizing, often unpredictable event. Autopsies of stillborn fetuses, including placentas, are performed to clarify the cause of death. Autopsy results are not always easily understood by the patients or their healthcare providers. To evaluate placental causes of death in stillbirths based on autopsy and placental findings that are related to maternal underperfusion, fetal underperfusion, or inflammatory etiologies in hierarchical order. Retrospective review of 120 autopsy reports of singleton stillborn fetuses and placentas from 23 to 40 weeks of gestation. Among the placental causes of death there were 54(51%) cases with direct cause or major contributor to death in the etiology of maternal vascular supply abnormalities, 28(26%) cases in the etiology of fetal vascular supply abnormalities and 13(12%) in the etiology of inflammatory lesions. Maternal vascular supply abnormalities were more common in preterm stillbirths and fetal vascular supply abnormalities were more common among term stillbirths. In 88% of stillbirths, the direct cause or a major contributor to death was found in the placentas. The incidence of unexplained death was 8%. Pathological analysis of the placenta is essential for clarifying causes of stillbirths. Using specific simplified categories for abnormal placental findings may increase the benefits of the autopsy report.
Invasive equine trophoblast cells of the chorionic girdle express high levels of paternally inherited Major Histocompatibility Complex (MHC) class I antigens prior to migration into the endometrium to form the so-called endometrial cups. Three groups of experiments were performed to determine if maternally inherited MHC class I antigens are expressed on chorionic girdle cells. Results indicated that maternally and paternally inherited MHC class I antigens are co-dominantly expressed by cells of the invasive equine trophoblast, and therefore, that the expression of polymorphic equine MHC class I genes does not appear to be affected by genomic imprinting in this tissue. The demonstration that cells of the chorionic girdle were immunogenic supports the hypothesis that invasion of the maternal endometrium by chorionic girdle cells stimulates the production of anti-paternal alloantibodies normally observed in early horse pregnancy. The co-dominant expression of MHC class I antigens by invasive chorionic girdle cells has important implications for the mechanism of recognition of allogeneic fetal MHC class I antigens by the maternal immune system.
Trophoblast cells isolated from term human placenta and maintained as an adherent culture express surface receptors for transferrin as indicated by quantitative binding studies using 125I-labelled transferrin. The Kd was 5.3 x 10(-9) M. About 36 per cent of the total cell receptor population was found at the cell surface, the remainder being intracellular. Both 125I-labelled and 59Fe-labelled transferrin were internalized by receptor-mediated endocytosis with similar rates. Pulse-chase experiments showed that 125I-labelled transferrin was recycled and released back to the medium, whereas 59Fe accumulated intracellularly and was released slowly. Polyacrylamide gel electrophoresis followed by autoradiography revealed that 59Fe was accumulated by cells largely in the form of ferritin. A small intracellular pool of low molecular weight 59Fe was also detected. In the presence of monensin, the transfer of 59Fe to ferritin was greatly reduced. The nature and amount of 59Fe released from cells could be modulated by the incubation conditions. In the absence of chelating agents and iron salts, released 59Fe was found to be associated with a low molecular weight fraction as well as with transferrin and ferritin. The low molecular weight 59Fe readily formed a complex with added chelators such as apotransferrin, DTPA or desferrioxamine. The release of 59Fe could be increased by repeatedly changing the medium during the course of the incubation. 59Fe release from trophoblast cells exceeded the release of lactate dehydrogenase and also exceeded the release of 59Fe from 3T3 fibroblasts, suggesting a cell-specific process.
High-affinity binding sites for [125I]endothelin(ET)-1 have been detected in purified membrane preparations of the fetal arteries and veins of the chorionic plate and the stem villi vessels of the human term placenta. Regardless of the vessel type, the apparent dissociation constant was found to be in the picomolar range (26----45 pM), and the Bmax value close to 600 fmol/mg protein. In stem villi vessels, ET-1, ET-2, sarafotoxin S6b and vasocontractor intestinal peptide (VIC) were approximately equipotent in their competitive displacement of [125I]ET-1 binding. The endothelin precursors, human and porcine big-endothelin, recognized ET-1 sites with low affinity (nM range), a finding which reflects their low potency as recognized vasocontractant agents. Interestingly, [125I]ET-1 binding parameters and pharmacological profiles were identical in fetal veins and arteries of the chorionic plate. Similarly, a study carried out in rat aortic membranes, revealed the presence of high affinity [125I]ET-1 binding sites with pharmacological characteristics close to those of the human stem villi vessels. In all vessels investigated, the binding pattern of ET-3 against [125I]ET-1 was of a non-competitive nature. Thus, these results demonstrate the presence of specific [125I]ET-1 binding sites along the vascular tree of the fetal side of the placenta and would support evidence currently available, favouring the existence of distinct ET-1 and ET-3 receptors. Finally, ET-1 in the human placenta may play an important physiological role as regulator of vascular resistance and/or be implicated as a pathological factor in certain pregnancy-related diseases.
The delivery of iron to the early organogenesis rat embryo has been studied, using 59Fe- and 125I-labelled rat transferrin. Rat conceptuses at 9.5 days postconception were cultured for 27 or 51 h in whole rat serum. Rat transferrin labelled with 59Fe was added for the final 0.1, 0.5, 6, 24 or 48 h of culture. Radioactivity accumulated progressively in both the embryo and the visceral yolk sac. Similar results were obtained when unconjugated 59Fe3+ was added to the rat serum used as culture medium. Both acid-soluble and acid-insoluble 59Fe were substantially present in the embryo and yolk sac after all exposure periods. When conceptuses were cultured in the presence of 125I-labelled rat transferrin, acid-soluble radioactivity was progressively released into the culture medium, but accumulation into the embryo and visceral yolk sac was slight and did not change with duration of exposure to the labelled protein. Similar findings were obtained using 125I-labelled bovine serum albumin. In these experiments, there was a close correspondence between the amount of iron accumulated by the embryo and visceral yolk sac in the final 24 h of a 51-h culture and the amount of transferrin converted into acid-soluble products in the same period. Visceral yolk sacs from 17.5-day pregnant rats were explanted and cultured in the presence of 59Fe-labelled rat transferrin, 125I-labelled rat transferrin or 125I-labelled bovine serum albumin, for periods up to 3 h. Again uptake of 59Fe increased with time of incubation, and the 125I-labelled proteins were digested to acid-soluble products which were released into the culture medium. The results indicate that transferrin delivers iron for incorporation into both the embryo and the visceral yolk sac, and are consistent with a mechanism involving receptor-mediated endocytosis of iron-laden transferrin by the cells of the visceral yolk sac. The transferrin itself appears to be quantitatively degraded, following delivery of iron to the yolk sac cells, a result that differs from findings in other cell types, in which the protein is not degraded but returns to the plasma membrane to participate in further cycles of iron acquisition and delivery.
The effects of the carboxylic ionophore, monensin, on the receptor-mediated binding and uptake of 125I-labelled IgG by the guinea-pig yolk sac have been studied in vitro. Exposure of tissue to 10 microM monensin resulted in a rapid inhibition of uptake which correlated with a time- and temperature-dependent loss of cell-surface receptor activity. Monensin appeared to bring about a change in receptor distribution since the lost activity could be detected after permeabilizing the tissue with saponin. Electron microscopic examination of monensin-treated tissue revealed that the apical plasma membrane of endoderm cells was depleted of coated and uncoated pits and that the apical cytoplasm contained numerous large vacuoles. Dilation of the Golgi apparatus was also observed. Normal surface receptor activity and ultrastructural features could be largely recovered by removal of monensin. Recovery of receptor activity was unaffected by the presence of cycloheximide. These results are consistent with a model in which IgG receptors are recycled and in which monensin blocks this process by causing receptors to be trapped intracellularly. Ammonium chloride or a combination of valinomycin and carbonyl cyanide p-(trifluoromethoxy)-phenylhydrazone also brought about a loss of surface IgG receptors, lending support to the idea that inhibition of recycling was the result of perturbation of an intracellular acidification event and implying that passage through an acidic compartment may be important for correct receptor processing.
Human placental homogenates and membrane fractions were centrifuged on continuous sucrose density gradients, with or without buoyant density perturbation of plasma-membranes by digitonin, and aliquots of each gradient fraction were assayed for a range of plasma-membrane and intracellular organelle markers, and for specific binding and inactivation of radiolabelled GnRH agonist (GnRHA), Buserelin ([D-Ser(tBu)6] GnRH 1-9 ethylamide). GnRH agonist (GnRHA) binding equilibrated in the same regions of control gradients as the plasma-membrane markers, EGF-receptor and alkaline phosphatase. Moreover, binding of 125I-labelled GnRHA and 125I-labelled chicken GnRH II (ch GnRH II) was enriched in the same regions of the gradients, indicating that both bound to the same membrane fractions. Digitonin pretreatment increased the buoyant density of all three placental plasma-membrane markers to a similar degree. Intracellular organelle markers (and hCG content) equilibrated in different regions of the gradient to placental surface-membranes, and were not perturbed appreciably by digitonin. In contrast, inactivation of 125I-labelled GnRHA was associated largely with the soluble (cytosol) fraction which failed to enter the gradient, and little tracer inactivation was observed in fractions enriched in GnRHA binding activity. Similar results were obtained with fractionated rat pituitary membranes. We conclude that: (a) placental GnRH binding sites do not represent binding of radiolabelled ligand to GnRH-degrading enzymes, (b) degradation of radiolabelled ligand is associated largely with placental cytosol fractions, and (c) GnRH binding activity appears to be associated largely with placental plasma-membranes.
A system for culturing the rat visceral yolk sac in vitro as a closed vesicle--the 'giant' yolk sac--has been employed to investigate the vectorial nature of the uptake and digestion of exogenous protein substrates. Uptake of 125I-labelled formaldehyde-denatured bovine serum albumin by such yolk sacs was found to be similar to that observed in yolk sacs removed directly from the mother at 17.5 days' gestation, provided that homologous serum was used as a culture medium. However, unlike the control yolk sacs, giant yolk sacs tended to accumulate substrate within the tissue with increasing culture time. The concentration of digestion products released to the inside of the closed vesicle was found to be greater than that released to the surrounding culture medium at time intervals up to five hours. Giant yolk sacs preloaded with 125I-labelled bovine serum albumin were found to release material to the culture medium or the inside of the vesicle almost entirely in the acid-soluble (digested) form. This system is a useful model for studying the polar nature of epithelia, particularly those involved in the uptake and transport of nutritional and/or informational macromolecules.
In vitro binding, internalization and release of the plasma protein ceruloplasmin was investigated using primary cultures of human placental trophoblast cells. Binding of 125I-labelled ceruloplasmin at 4 degrees C reached equilibrium by 5-6 h; binding was linear throughout all concentrations tested (1 nM-3.3 microM). Addition of greater than 5-10 microM unlabelled ceruloplasmin or a variety of other proteins (albumin, transferrin, IgG) were equally effective in displacing bound ceruloplasmin in a concentration-dependent manner. When cells were incubated at 37 degrees C, the majority of surface-bound 125I-labelled ceruloplasmin was released directly to the extracellular medium. Trypsin-resistant radioactivity increased to 18 per cent of initially bound ceruloplasmin within 1 min, declining to 5 per cent by 2 h. The acquisition of trypsin-resistant radioactivity was unaffected by the addition of a variety of metabolic inhibitors and no evidence of intracellular degradation of ceruloplasmin was found. In summary, our results suggest that the majority of ceruloplasmin binding to trophoblast cells is nonspecific, of low affinity, and easily dissociable at 4 degrees C. Only a small amount of ceruloplasmin appeared to be internalized, by an as yet unknown mechanism.
The binding and subsequent internalization of 125I-labelled transferrin has been studied in the human choriocarcinoma cell line, JAR. At 4 degrees C, binding was time- and concentration-dependent and exhibited saturation kinetics. The results indicate 1.6 x 10(6) binding sites per cell surface with a Kd of 4.12 x 10(-9) M. Experiments with saponin-permeabilized cells revealed a large intracellular receptor pool constituting 72 per cent of the total cell receptor population. Evidence of ligand internalization at 37 degrees C was obtained by showing the progressive resistance of cell-associated radioactivity to acid treatment. Pulse-chase experiments showed that after internalization, ligand resided within the cell for about 6 min before being released back to the medium in an intact form. Ligand release, but not internalization, was almost completely inhibited by exposure to monensin. Preincubation of cells at 37 degrees C in the presence of monensin and under transferrin-free conditions had no effect on the subsequent binding of 125I-transferrin. Pulse-chase experiments using 125I-labelled anti-transferrin receptor antibody suggest that receptors are internalized in the absence of added transferrin with a rate coefficient of 0.036 min-1. In the presence of unlabelled transferrin, the initial rate of labelled antibody internalization was increased (rate coefficient was 0.23 min-1) but the maximal amount of antibody internalized remained virtually unchanged. Internalized antibody accumulated intracellularly with no evidence of release or degradation.
Preeclampsia is a pregnancy-specific syndrome characterized by high blood pressure and proteinuria, which has a pathophysiology of insufficient placental blood perfusion. MicroRNA-126 (miR-126), an angiogenesis-related miRNA, has been proved to play a significant role in endothelial cells response to ischemia in vitro and in vivo. However, whether miR-126 has therapeutic potential in vasculogenesis of preeclampsia placenta remains uncertain. In this study, we focused our attention on this unsolved problem. First, we established the preeclampsia animal model and over-expressed miR-126 in vivo using a specific agomir. Then we described the effects of miR-126 on placental vasculogenesis in preeclampsia rats, including the evaluation of placental blood perfusion using microbubbles-assisted contrast-enhanced ultrasonography (CEUS), placental histology, immunohistochemistry and pregnancy outcome. Finally, we investigated the possible target gene and pathway that miR-126 modulates. Together, our results showed that preeclampsia animal with over-expressed miR-126 had higher pup weight, placenta weight and proportion of live pups. Quantification of uteroplacental perfusion by CEUS and CD34 staining of placental tissue revealed that blood volume and microvessel density increased in miR-126 treated group. MiR-126 was related to PIK3R2 down-regulation and Akt activation within placenta, which had impacts on vascularization of placenta. Therefore, miR-126 may be an efficient gene therapy to promote angiogenesis and blood perfusion in preeclampsia placenta.
To test the hypothesis that a combination of PP13, PAPP-A and first-trimester uterine artery Doppler would improve the prediction of pre-eclampsia. This is a prospective cohort study of pregnant women followed from the first-trimester to delivery. PP13 and PAPP-A were determined by immunoassay of maternal serum at 11-14 weeks', when uterine artery Doppler measurements were assessed. Cases identified with any form of pre-eclampsia were compared with a control group without pre-eclampsia. The sensitivity of each marker or their combinations in predicting pre-eclampsia for different fixed false positive rates was calculated from the ROC curves. Forty two women were diagnosed with pre-eclampsia and 410 women with pregnancies not complicated by pre-eclampsia were used as controls. For a fixed false positive rate (FPR) of 20%, PP13, PAPP-A and mean uterine artery pulsatility index identified 49%, 58% and 62% respectively, of women who developed any form of pre-eclampsia. PP13 was best in predicting early onset pre-eclampsia with a sensitivity of 79% at a 20% FPR. Combinations of the three first-trimester assessments did not improve the prediction of pre-eclampsia in later pregnancy. First-trimester PP13, PAPP-A and uterine artery PI are reasonable, individual predictors of women at risk to develop pre-eclampsia. Combinations of these assessments do not further improve the prediction of pre-eclampsia.
Placental tissue protein 13 (PP-13), one of the 56 known placental proteins identified till today, was purified from placentas obtained from women at delivery, and used to evoke antibodies against it. The purified PP-13 was lysed to peptides, which were sequenced, leading to the full-length cDNA sequencing and its expression in Escherichia coli. Sequence analysis in databases showed homology to the galectin family. Of the various antibody preparations developed, a pair of monoclonal antibodies (MAbs) coupled to the recombinant PP-13 (PP-13-R) was used for the immunodetection of PP-13 in pregnant women's serum with the solid-phase ELISA format. With a dynamic range of 25-500 pg/mL with no background in non-pregnant women's serum and men's serum, the ELISA test was suitable for the detection of PP-13 in the 1st, 2nd, and 3rd trimesters. PP-13 levels slowly increase during pregnancy. In the 1st trimester, lower than normal PP-13 levels were found in fetal growth restriction (IUGR), preeclampsia (PE), and particularly in early PE (<34 weeks of gestation). In the 2nd and 3rd trimesters, higher than normal concentrations were found in PE, IUGR and in preterm delivery (PTD). Application of PP-13 to cultured trophoblasts elicited depolarization carried by calcium ions, followed by liberation of linoleic and arachidonic acids from the trophoblast membrane, and a subsequent elevation of prostacyclin and thromboxane. These effects were negligible when PP-13 derived from the placentas of patients with IUGR, PE or PTD was used. The results are discussed in view of the potential utilization of PP-13 for early serum screening to assess the risk to develop placental insufficiency, coupled to a differential analysis of the various pathologies by analyzing cultured trophoblasts.
The incidence of pre-eclampsia is significantly higher in trisomy 13 pregnancies than in normal pregnancies. Soluble fms-like tyrosine kinase-1 (sFlt-1), located on chromosome 13, is an anti-angiogenic molecule derived from the placenta and contributes to the pathogenesis of pre-eclampsia. Elevated sFlt-1 and reduced placental growth factor (PlGF) are associated with trisomy 13 pregnancies and may play a pathogenic role in the subsequent development of pre-eclampsia. Here we present a case of a trisomy 13 pregnancy without any signs of pre-eclampsia that showed alterations in circulating angiogenic factors and abnormal placental appearance. The placenta developed edematous changes and contained multiple small cysts. Histology of the placenta confirmed avascular edematous cystic villi and did not show the typical appearance of a partial mole or mesenchymal dysplasia. The sFlt-1/PlGF ratio in maternal serum (134) was much higher than that in gestational age-matched women who were normotensive (2.9-7.2; mean, 5.0). Immunostaining for Flt-1 and endoglin was more intense in our case compared with gestational age-matched controls, and at a similar level to a case of pre-eclampsia. Placental findings that showed avascular edematous cystic villi in our case may be associated with angiogenic imbalance involved in the pathogenesis of pre-eclampsia in trisomy 13 pregnancies.
Ultrastructural, immunochemical, fluorescence and stereological studies were undertaken on human villous trophoblast from 13 weeks of gestation to term. The aim was to describe and quantify morphological changes during proliferation, differentiation and apoptosis in cytotrophoblast and syncytial regions of non-aggregated and aggregated nuclei. Numbers of trophoblast nuclei increased continuously from 13 weeks. In term placentae, intrasyncytial differentiation was characterized ultrastructurally by gradual decreases in nuclear size and packing density accompanied by nucleolar regression, and increasing heterochromatinization, envelope convolution and packing density of nuclear pore complexes. In densely packed areas, nuclear profiles resembled interlocking jigsaw pieces. Occasionally, these 'pre-apoptotic' nuclei were associated with annulate lamellae. Rarely, nuclear changes terminated in apoptosis with a characteristic pattern of condensed peripheral chromatin, a central island of euchromatin, no nucleoli and no discernible nuclear pores. Apoptotic nuclei were seen singly and within dense nuclear aggregations. Similar spatial patterns of nuclei and chromatin were seen in propidium iodide-stained sections at 13-41 weeks. Whilst the relative incidence of intensely fluorescent nuclei remained constant, absolute numbers increased linearly during gestation and correlated positively with the volume of syncytial knots. Nuclei labelled for DNA fragmentation occurred very infrequently and were also found in nuclear clusters as well as singly. We suggest that nuclear differentiation in syncytium has two phases: on entering syncytium, nuclei become committed to a long programmed pre-apoptotic phase which leads to a short apoptotic execution phase. We propose further that clustered nuclei (pre-apoptotic and apoptotic) in syncytial knots probably represent the extrusion component of normal continuous epithelial turnover.
Gestational diabetes mellitus (GDM) is an increasing harm in pregnancy. Inflammatory processes in the placenta seem to have an influence on pathogenesis besides known factors like maternal BMI. Galectin-13 (gal-13) is an immunoregulatory protein, which is suspected to play a role in development of GDM in the placenta. A total of 40 placentas were obtained from women treated for gestational diabetes mellitus. Placental tissue for control group was obtained from 40 women with normal pregnancy. We investigated the protein expression of gal-13 in term placentas with immunohistochemistry and immunofluorescence. Immunohistochemical staining was analyzed with the semi-quantified IRS score. Gal-13 serum levels were performed with ELISA on a total of 20 probes from women with GDM and healthy control pregnancies in the third trimester. Gal-13 was found in syncytiotrophoblast, in nuclei of syncytiotrophoblast and trophoblast cells as well in extravillous trophoblast cells of normal placentas. In GDM placentas, gal-13 expression was significantly decreased in all of these examined cell types (syncytiotrophoblast p = 0.003, nuclei of syncytiotrophoblast p = 0.007; extravillous trophoblast cells p = 0.001). The ELISA showed a significant lower gal-13 serum level in blood from pregnant women with GDM in comparison to healthy controls. As gal-13 with its anti-inflammatory functions plays a role in regulation of maternal immune system, a lack of gal-13 may contribute to an imbalance in inflammation processes in the placenta during pregnancy and therefore influences development of GDM. Copyright © 2014 Elsevier Ltd. All rights reserved.
A method is presented for obtaining assumption-free estimates of the number of nuclei in the trophoblast of the human placenta and for defining the size of the trophoblast proliferative unit (TPU). The method relies on the disector, a stereological device for counting arbitrary particles in 3-dimensional space using pairs of parallel sections separated by a known distance. It is applied to investigate factors which contribute to trophoblast growth from 13 weeks of gestation to term. Physical disectors were sampled systematically using adjacent 4-4.6 microns thick paraffin sections. Nuclei in the trophoblast (syncytial and cellular) were counted if they appeared in an unbiased counting frame on one section but were absent from the adjacent section. Nuclear packing densities were converted to absolute numbers of nuclei by using placental volume as the reference space. At 37-39 weeks, the average placenta contained 6.4 x 10(10) trophoblast nuclei of which 90 per cent were located within the syncytium and the remainder in the cytotrophoblast. From a knowledge of total trophoblast volume, it was found that each nucleus is associated with 970 microns3 of trophoblast and each cytotrophoblast cell with 11,000 microns3. The latter may be regarded as the volume of a TPU. From 13 weeks of gestation to term, there was a ninefold increase in nuclear number but the trophoblast volumes associated with nuclei, including the size of the TPU, remained constant. Growth of trophoblast is purely hyperplastic and occurs by recruitment of new TPUs.
Expression of placental tissue protein 13 (PP13) in different human tissues was investigated by chemiluminescence Western blot analysis using monospecific anti-PP13 serum. In term placentae we detected a 16 kDa single protein band immunochemically identical to the purified PP13 antigen. After investigation of 26 types of human fetal and adult tissue, PP13 was also found in certain other normal and tumorous tissue extracts. It is not secreted into circulation as we could not find PP13 in sera of pregnant women. A full length cDNA with 578 bp insert was isolated by screening a human placental cDNA library with anti-PP13 serum. The open reading frame of the cDNA encodes for a 139-residue-long protein with a predicted molecular mass of 16.118 kDa, identical to the previously isolated and characterized PP13 antigen described in 1983. By alignment search of the protein databank PP13 is highly homologous (69 per cent) to the 16.5 kDa human eosinophil Charcot-Leyden Crystal protein, a unique dual-function lysophospholipase, a member of the beta-galactoside binding S-type animal lectin superfamily. Northern blot analysis revealed a 600 bp PP13 mRNA, detected only in placental tissue from 16 types of human healthy adult tissue. Lysophospholipase activity of PP13 was confirmed by(1)H and(31)P nuclear magnetic resonance (NMR) measurements.
Top-cited authors
Graham J Burton
  • University of Cambridge
Christopher W Redman
  • University of Oxford
Ian L Sargent
  • University of Oxford
Berthold Huppertz
  • Medical University of Graz
Eric Jauniaux
  • University College London