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.     

Placental ABCA1 expression is reduced in primary antiphospholipid syndrome compared to pre-eclampsia and controls

The ATP binding cassette transporter A1 (ABCA1) mediates cellular cholesterol and phospholipid efflux, and is implicated in phosphatidylserine translocation and apoptosis. Loss of functional ABCA1 in null mice results in severe placental malformation. This study aimed to establish the placental localisation of ABCA1 and to investigate whether ABCA1 expression is altered in placentas from pregnancies complicated by pre-eclampsia and antiphospholipid syndrome. ABCA1 mRNA and protein localisation studies were carried out using in situ hybridization and immunohistochemistry. Comparisons of gene expression were performed using real-time PCR and immunoblotting. ABCA1 mRNA and protein was localised to the apical syncytium of placental villi and endothelia of fetal blood vessels within the villi. ABCA1 mRNA expression was reduced in placentas from women with APS when compared to controls (p<0.001), and this was paralleled by reductions in ABCA1 protein expression. There were no differences in ABCA1 expression between placentas from pre-eclamptic pregnancies and controls. The localisation of ABCA1 in human placenta is consistent with a role in cholesterol and phospholipid transport. The decrease in ABCA1 protein in APS may reflect reduced cholesterol transport to the fetus affecting the formation of cell membranes and decreasing the level of substrate available for steroidogenesis.     

Villous histomorphometry and placental bed biopsy investigation in Type I diabetic pregnancies

Insulin-dependent diabetes mellitus (Type I) is associated with disregulation of the glucose and oxygen metabolic pathways during pregnancy, both of which affect placental villous development. Term complete placentas and placental bed biopsies, between 37 and 40 weeks, from 12 singleton pregnancies complicated by Type I diabetes were collected following delivery by elective Caesarean section. The controls consisted of 10 term placentas from uncomplicated pregnancies delivered by elective Caesarean section. Villous morphology was investigated using unbiased histomorphometric techniques, in relation to the degree of transformation of the spiral arteries and the presence of fetal macrosomia. A significant increase in fetal and placental weights, placental volume, volumes of the intervillous space and the trophoblast was found in the diabetic group compared to the controls. A significant reduction in the villous membrane specific diffusing capacity was observed between the diabetic and control groups (1.32 vs 1.72 cm3 min(-1)mmHg(-1)kg(-1), P=0.032). A significant increase in the volume of the intermediate and terminal villi, the surface area of the villi and of the fetal capillaries, and the harmonic thickness of the villous membrane was found in the macrosomic subgroup compared to the controls. There were no differences between the hypertensive subgroup with histological evidence of partial transformation of the spiral arteries and the controls. These data indicate that placental development in insulin-dependent diabetic pregnancies is affected differentially when pregnancies complicated by fetal macrosomia are separated from those complicated by maternal hypertensive disorders with partial transformation of the spiral arteries. The reduction in the specific diffusing capacity of the villous membrane may contribute to the fetal hypoxia and increased fetal and neonatal morbidity associated with diabetes.     

Elastic fibres in the human placenta

Recent studies described the presence of elastic fibres within stem villi of human term placenta. This particular study focuses on changes of the elastic fibre system in placental villous tree from placentas of different gestational ages. The presence of elastic fibres was researched by light and electron microscopy in first-, second-trimester and term human placentas. Light microscopic analysis revealed elastic fibres in the stroma of main stem villi and in large vessels present in the chorionic plate of term placentas. Transmission electron microscopy revealed a wide variation in microfibrillar component deposition in placentas from all the gestational ages studied. Although minimal amount of the microfibrillar component was observed only in some main stem villi of second-trimester placentas, abundant microfibrillar material was present in all types of placental villi in placental sections from term pregnancies. The amorphous component was only occasionally identified in sections from the chorionic plate of second trimester placentas. In ultra-thin sections from placental tissues from the 37th to the 42nd weeks of gestation, we can observe the amorphous component of the elastic fibres. Bundles of microfibrils with scanty participation of the amorphous component of the elastic fibres can be observed in stem villi. In mature intermediate villi, cells and their processes with distinct plasma membranes were seen close to some bundles of microfibrillar component, at times with small spots of the amorphous component. This study shows that elastic fibres of the villous stroma, are mainly composed of the microfibrillar component, while the amorphous component appears more frequently in advanced stages of villous differentiation of term placentas.     

A comparison in concentration of heat shock proteins (HSP) 70 and 90 on chorionic villi of human placenta in normal pregnancies and in missed miscarriages

PURPOSE: To investigate the role of heat shock protein (HSP) on the chorionic villi of human placental cells and to compare the concentration of placental HSP70 & 90 in term deliveries and in missed miscarriages. MATERIALS AND METHODS: Fifty products of conception from women who experienced first trimester missed miscarriage and 50 placentas from women who gave birth at term were studied. An immunohistochemical investigation was carried out with which we marked the localization of heat shock proteins 70 and 90 on the syncytiotrophoblastic, cytotrophoblastic, stromal and blood vessel cells, using specific antibodies which can detect the presence of those proteins on light microscopy. We compared their expression with the normal placental tissue of term pregnancies and with material acquired from first trimester missed miscarriages. An indirect immunoperoxidase method was applied using polyclonal antibodies against HSP70 and HSP90 on formalin-fixed paraffin-embedded tissues. RESULTS: Expression of HSP90B was increased in chorionic villi of first trimester missed miscarriages concerning syncytiotrophoblasts, cytotrophoblasts, vessel and stroma cells compared to full-term placentas. There was a statistically significant increase of HSP90A expression in chorionic villi of first trimester missed miscarriages, concerning only the cytotrophoblast cells, compared to full-term placentas. Expression of HSP70 cognate protein was significantly increased in chorionic villi of first trimester missed miscarriages, concerning syncytiotrophoblastic cells only, compared to full-term placentas. Finally, HSP70 inducible protein was significantly increased in chorionic villi of first trimester missed miscarriages concerning syncytiotrophoblasts, cytotrophoblasts, vessel and stroma cells compared to full-term placentas. CONCLUSIONS: The results of the present study have sufficiently shown that there is an increase of HSP70 & 90 expression in chorionic villi of first trimester missed miscarriages compared to full-term placentas and this increase may have an important implication on the miscarriage process.     

Sites of mRNA expression of the cystic fibrosis (CF) and multidrug resistance (MDR1) genes in the human placenta of early pregnancy: No evidence for complementary expression.

The aims of this study were to establish the sites of mRNA expression of both the cystic fibrosis (CF) and multidrug resistance (MDR1) genes in human placental sections from early pregnancy (first, early and mid-second trimesters). Riboprobes specific for each of these two genes were generated and used for in situ hybridization experiments. The results show parallel mRNA expression for the CF and MDR1 genes, with the signal detected in the syncytiotrophoblast and cytotrophoblast cells of the placental villi. Other cell types within the villous core were negative. Similar results were obtained at all stages of pregnancy studied.     

Morphometric placental villous and vascular abnormalities in early- and late-onset pre-eclampsia with and without fetal growth restriction

OBJECTIVE: To evaluate placental morphology in pregnancies complicated by early- and late-onset pre-eclampsia (PET) with and without fetal growth restriction (FGR) using stereological techniques. DESIGN: A total of 69 pregnant women were studied. Twenty women had pregnancies complicated by PET, 17 by FGR and 16 by both PET and FUR; the remaining 16 were from gestational-age-matched controls. Each group was further classified into early onset (<34 weeks) and late onsets (>34 weeks) based on gestational ages. SETTING: NPIMR at Northwick Park and St Marks Hospital. POPULATION: placentae from pregnant women. METHODS: Formalin-fixed, wax-embedded sections stained with anti-CD34 antibodies and counterstained with haematoxylin. MAIN OUTCOME MEASURES: Volumes, surface areas, lengths, diameters and shape factors of the villous tissues and fetal vasculature in the intermediate and terminal villi of all the groups studied. RESULTS: Terminal villi volume and surface area were compromised in early-onset PET cases, late-onset PET had no impact on peripheral villi or vasculature features. The morphology of the vascular and villous subcomponents in the intermediate and terminal villi was significantly influenced by late-onset FGR, whereas early-onset FGR caused a reduction in placental weight. Length estimates were not influenced by PET, FGR or age of onset. Intermediate arteriole shape factor was significantly reduced in late-onset FGR. CONCLUSIONS: Isolated early-onset PET was associated with abnormal placental morphology, but placentas from late-onset PET were morphologically similar to placentas from gestational-age-matched controls, confirming the existence of two subsets of this condition and supporting the hypothesis that late-onset PET is a maternal disorder and not a placental disease.     

Altered villus vessel fibronectin in preeclampsia

Fibronectin is a high molecular-weight glycoprotein found in most tissues and body fluids. Maternal plasma fibronectin levels have been shown to be elevated in preeclampsia, but little is known about placental fibronectin in preeclampsia. Fibronectin tissue distribution in placental villi was "blindly" graded by two examiners using placentas from eight nonpreeclamptic and six preeclamptic pregnancies. Selected frozen sections of normal-appearing areas from each placenta were incubated with rabbit antihuman fibronectin antiserum and then stained with fluorescein isothiocyanate-conjugated goat antirabbit immunoglobulin G and examined by fluorescent microscopy. We found less intensity of fetal vessel fibronectin staining in villi from placentas from preeclamptic pregnancies than in those of normal pregnancies (p less than 0.02, Mann-Whitney U test). It is unclear why fetal villous vessels in preeclampsia have decreased tissue fibronectin, but this may reflect an additional vascular abnormality associated with the preeclampsia syndrome.     

Localization of the extracellular Ca(2+)-sensing receptor in the human placenta

We have demonstrated using immunohistochemistry and in situ hybridization that the calcium-sensing receptor (CaR) is expressed in both villous and extravillous regions of the human placenta. CaR expression was detected in both first trimester and term placentas. In the villous region of the placenta, the CaR was detected in syncytiotrophoblasts and at lower levels in cytotrophoblasts. Local expression of the CaR in the brush border of syncytiotrophoblasts suggests a role for maternal Ca(2+) concentration in the control of transepithelial transport between the mother and fetus. In the extravillous region of the placenta, the CaR was detected in cells forming trophoblast columns in anchoring villi, in close proximity to maternal blood vessels and in transitional cytotrophoblasts. Given the importance of extravillous cytotrophoblasts in the process of uterine invasion and maintenance of placental immune privilege, the CaR represents a possible target by which the maternal extracellular Ca(2+) concentration could promote or maintain placentation. Thus, the results support hypotheses that the CaR contributes to the local control of transplacental calcium transport and to the regulation of placental development.     

No Correlation Between Ultrasound Placental Grading at 31–34 Weeks of Gestation and a Surrogate Estimate of Organ Function at Term Obtained by Stereological Analysis

We test the experimental hypothesis that early changes in the ultrasound appearance of the placenta reflect poor or reduced placental function. The sonographic (Grannum) grade of placental maturity was compared to placental function as expressed by the morphometric oxygen diffusive conductance of the villous membrane. Ultrasonography was used to assess the Grannum grade of 32 placentas at 31–34 weeks of gestation. Indications for the scans included a history of previous fetal abnormalities, previous fetal growth problems or suspicion of IUGR. Placentas were classified from grade 0 (most immature) to grade III (most mature). We did not exclude smokers or complicated pregnancies as we aimed to correlate the early appearance of mature placentas with placental function. After delivery, microscopical fields on formalin-fixed, trichrome-stained histological sections of each placenta were obtained by multistage systematic uniform random sampling. Using design-based stereological methods, the exchange surface areas of peripheral (terminal and intermediate) villi and their fetal capillaries and the arithmetic and harmonic mean thicknesses of the villous membrane (maternal surface of villous trophoblast to adluminal surface of vascular endothelium) were estimated. An index of the variability in thickness of this membrane, and an estimate of its oxygen diffusive conductance, were derived secondarily as were estimates of the mean diameters and total lengths of villi and fetal capillaries. Group comparisons were drawn using analysis of variance. We found no significant differences in placental volume or composition or in the dimensions or diffusive conductances of the villous membrane. Subsequent exclusion of smokers did not alter these main findings. Grannum grades at 31–34 weeks of gestation appear not to provide reliable predictors of the functional capacity of the term placenta as expressed by the surrogate measure, morphometric diffusive conductance.     

Differential localization of placental extracellular superoxide dismutase as pregnancy progresses

OBJECTIVE: The aim of this study was to determine placental localization and activity of extracellular superoxide dismutase, a nitric oxide modulator, during early gestation and to correlate these characteristics with fetal vascular development. STUDY DESIGN: First-trimester (n = 10) and second-trimester (n = 10) villi were obtained at elective pregnancy termination. Extracellular superoxide dismutase was localized by means of an immunoperoxidase method. Activity was measured by determining the inhibition of cytochrome c reduction at pH 10 and messenger ribonucleic acid expression by in situ hybridization. RESULTS: Extracellular superoxide dismutase was intracellular within villous trophoblasts until 17 weeks' gestation, when it relocated to the villous extracellular matrix. Activities were similar between first- and second-trimester villi. In situ hybridization confirmed extracellular superoxide dismutase messenger ribonucleic acid within trophoblasts throughout gestation. CONCLUSION: Extracellular superoxide dismutase is produced by trophoblasts early in pregnancy, but it remains intracellular until 17 weeks' gestation, which may be related to fetal vascular development.     

D2-40/podoplanin expression in the human placenta

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

Abnormal Expression of the Imprinted Gene Phlda2 in Cloned Bovine Placenta

Cloning in mammals suffers from high rates of pregnancy losses associated with abnormal placentation, mainly placentomegaly, leading to fetal death. Placental growth is dependent on the regulated expression of many genes of which imprinted genes play a fundamental role. Among them, the Phlda2 gene is expressed from the maternal allele and acts to limit placental growth in mouse and human. Here we used Northern blots, quantitative RT-PCR and in situ hybridization to analyze the expression patterns of bovine PHLDA2 and to compare its expression levels in normal and somatic cell nuclear transfer (SCNT) placentas over a range of gestational stages. PHLDA2 is not expressed in extra-embryonic tissues before d32 of gestation but the level of expression increases throughout pregnancy until term in the placental villi collected from pregnancy obtained by artificial insemination (AI). At all stages of pregnancy, PHLDA2 mRNA are specifically localized in the trophoblast mononucleated cells contrasting with lack of expression in the binucleated cells and uterine tissues. In SCNT placentas, a similar pattern of expression was observed during early pregnancy. In contrast the level of expression is significantly reduced around d200 of gestation in the placental villi from pathological clones. The reduced expression of PHLDA2 was obvious particularly in the placental villi anchored within the uterine crypts with expression confined to the trophoblast of the chorionic plate. Altogether, these results highlight a similarity in expression patterns for PHLDA2 bovine and human where expression is localized to the trophoblast throughout pregnancy and parallels the continuous growth of the placenta. Moreover, the lack of expression in the fetal villi from oversized bovine cloned placenta is consistent with the function of PHLDA2 in restraining placental growth and underlines an aberrant expression of this gene after somatic cloning.     

Reduced amount of cytochrome c oxidase subunit I messenger RNA in placentas from pregnancies complicated by preeclampsia

BACKGROUND: Cytochrome c oxidase is a marker enzyme of the mitochondrial inner membrane. A change in the structure and activity of cytochrome c oxidase may alter the electron transport in the inner membrane, leading to insufficient adenosine triphosphate (ATP) production. ATP is essential for maintaining the function of cells. The aim of this study was to compare the expression of cytochrome c oxidase subunit I mRNA in placentas from normal and preeclamptic pregnancies. METHODS: By means of in situ hybridization, frozen sections of placentas from 23 women with preeclampsia and 29 women with uneventful pregnancies were examined. Digoxigenin (DIG)-labeled probes were used to detect the expression of cytochrome c oxidase subunit I mRNA in the placentas. The expression density was assessed by using an image disposal and analysis system. RESULTS: Positive expression of cytochrome c oxidase subunit I mRNA was found in the cytoplasm of villous syncytiotrophoblasts. The mean light density of cytochrome c oxidase subunit I mRNA in placental villi of normal pregnant women was 0.2638, and 0.1763 in women with preeclampsia, a statistically significant difference (P < 0.05). The number density of cytochrome c oxidase subunit I mRNA in placental villi was also significantly reduced in preeclamptic women compared with the control group (P < 0.05). CONCLUSIONS: Our study demonstrates a reduced amount of cytochrome c oxidase subunit I mRNA in preeclamptic placentas compared to control placentas. We hypothesize that a reduced expression may play a role in the pathophysiology of preeclampsia.     

Automated image analysis of placental villi and syncytial knots in histological sections

IntroductionDelayed villous maturation and accelerated villous maturation diagnosed in histologic sections are morphologic manifestations of pathophysiological conditions. The inter-observer agreement among pathologists in assessing these conditions is moderate at best. We investigated whether automated image analysis of placental villi and syncytial knots could improve standardization in diagnosing these conditions.MethodsPlacentas of antepartum fetal death at or near term were diagnosed as normal, delayed or accelerated villous maturation. Histologic sections of 5 cases per group were photographed at ×10 magnification. Automated image analysis of villi and syncytial knots was performed, using ImageJ public domain software. Analysis of hundreds of histologic images was carried out within minutes on a personal computer, using macro commands.ResultsCompared to normal placentas, villi from delayed maturation were larger and fewer, with fewer and smaller syncytial knots. Villi from accelerated maturation were smaller. The data were further analyzed according to horizontal placental zones and groups of villous size.DiscussionNormal placentas can be discriminated from placentas of delayed or accelerated villous maturation using automated image analysis. Automated image analysis of villi and syncytial knots is not equivalent to interpretation by the human eye. Each method has advantages and disadvantages in assessing the 2-dimensional histologic sections representing the complex, 3-dimensional villous tree. Image analysis of placentas provides quantitative data that might help in standardizing and grading of placentas for diagnostic and research purposes.     

Detection of class I MHC mRNA in subpopulations of first trimester cytotrophoblast cells by in situ hybridization

Class I MHC mRNA has been identified in both villous and extravillous cytotrophoblast cells in first trimester placentas by in situ hybridization. In this report, we expand those observations to additional morphologically and anatomically distinct subpopulations of trophoblast cells in early placentas using the same experimental approach. In the transition zone of first trimester placental villi, where cytotrophoblast cells are proliferating to form new villi or to migrate into adjacent tissue, both cytotrophoblast cells beneath the uninterrupted syncytium and the proliferating cytotrophoblast cells contained class I mRNA whereas a layer of cytotrophoblast cells proximal to the villus core did not contain class I mRNA. In the placental bed, migrating cytotrophoblast cells contained high levels of class I mRNA as determined by the intensity of staining. In contrast, multinucleated giant trophoblast cells contained little specific message. Alterations in levels of class I mRNA seem therefore to be associated with trophoblast proliferation, migration and differentiation.     

Immunocytochemical Localization of Endothelin-1 in Human Placenta from Normal and Preeclamptic Pregnancies

Objective. The aim of this study was to examine the distribution of endothelin-1 (ET-1) in the human placenta at different gestational ages and to determine whether differences in ET-1 immunoreactivity occurred in preeclamptic compared with uncomplicated pregnancies.

Methods. Localization of ET-1 was investigated by the immunoperoxidase technique in first-trimester, second-trimester, and term human placentas from normal pregnancies and in placentas from preeclamptic pregnancies.

Results. In normal placentas from all gestational ages studied, endothelin-1 immunoreactivity (ET-1 IR) was specifically detected in the endothelium of the fetal vessels and in the syncytiotrophoblast. ET-1 IR was also expressed by the villous cytotrophoblast of first- and second-trimester normal placentas. The extravillous cytotrophoblast of the basal and chorionic plates also exhibited ET-1 IR, but with varying degrees of intensity. In preeclamptic placentas, the expression of ET-1 IR was uneven with a negative staining in all placentas from pregnancies between the 29th and 32nd weeks of gestation. The expression of ET-1 IR was most intense in some syncytiotrophoblast tissue in the terminal villi after the 33rd week of gestation. In placentas from preeclamptic pregnancies between the 35th and the 36th weeks of gestation, strong ET-1 IR expression was evident in the endothelium of fetal vessels and in the syncytiotrophoblast. Regardless of gestational age, ET-1 IR was also observed in the extravillous cytotrophoblast of the basal and chorionic plates of preeclamptic placentas.

Conclusion. This study demonstrates that ET-1 IR is widely distributed in the human placenta and provides further evidence to support the concept that ET-1 plays an important role as a modulator of vascular tone in the uteroplacental and fetoplacental units and may participate in the pathogenesis of preeclampsia.     

Stereologic examination of placentas from mothers who smoke during pregnancy

OBJECTIVE: Structural changes in the placenta might contribute to the lower birth weight seen among infants born to mothers who smoke cigarettes. In this study, a morphologic examination and a stereologic quantitation of placentas from mothers who smoked cigarettes and who did not smoke cigarettes during pregnancy were performed. STUDY DESIGN: Twenty-five placentas from mothers who did not smoke cigarettes, 15 placentas from mothers who smoked 5 to 10 cigarettes per day, 16 placentas from mothers who smoked 11 to 20 cigarettes per day, and 16 placentas from mothers who smoked >20 cigarettes per day were delivered at term after normal pregnancies and were fixed by dual perfusion. The volume and the surface area of villi, the trophoblast volume, and the volume and the surface area and length of villous capillaries were estimated. A measurement of the concentration of cadmium in serum was used to assess the validity of information concerning smoking habits. RESULTS: No differences were shown in the total volume of placenta between the groups. The estimated volume and surface area and the calculated lengths for villous capillaries were significantly reduced in all 3 groups of smokers. A significant increase of the trophoblast volume was observed in the mothers who smoked cigarettes. CONCLUSION: Cigarette smoking during pregnancy influences the placental vasculature. The reduced dimensions of fetal capillaries in villi may affect the placental blood flow, and the diminished area for exchange of gases and nutrients between the mother and the fetus will increase the risk of fetal undernourishment.