The lipid transporters ABCA1 and ABCG1 are differentially expressed in syncytiotrophoblasts of preeclamptic and IUGR placentas

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Placental glucose transporter (Glut)-1 expression in preeclampsia

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Sonographic Assessment of the Umbilical Cord

The umbilical cord is not an inert structure, suspended between the fetus and placenta, but it plays an active role and it is involved in several processes afflicting the feto-placental unit. Its study has to be regarding not only its morphology and morphometry, and the impendance of blood flow by Doppler waveform analysis, but it includes also an analysis of the coiling type and the amount of the Wharton Jelly. The umbilical cord has been considered like an important and huge source of informations, useful to assess the well-being of the fetus and the outcome of pregnancy. The standardization of ultrasound techniques is the first step to speak the same language and make the study of this structure a fundamental part of well-being fetus assessment. This article is carefully focused on morphologic, morphometric and functional ultrasound examination of umbilical cord and suggests that any anomaly detected should provide an indication for an intense fetal follow-up, useful for early helpful therapy, preventing serious complication for the pregnancy.

PP141. The lipid transporters ABCA1 and ABCG1 are differentially expressed in preeclamptic and IUGR placentas

INTRODUCTION The ATP-binding cassette (ABC) transporter A1 (ABCA1) and ABCG1 are highly expressed in the placenta in various compartments, including the villous syncytiotrophoblast (V-STB) and foetal endothelial cells. Among other not yet characterized functions, they play a role in the foeto-maternal transport of cholesterol and other lipophilic molecules. In humans, preliminary data suggest expressional changes of ABCA1 and ABCG1 in pathologic gestation, particularly under hypoxic conditions, but a systematic expression analysis in common human pregnancy diseases has never been performed. OBJECTIVES The aim of the present study was to characterize ABCA1 and ABCG1 expression in a large series of pathologic placentas, in particular from preeclampsia (PE) and intrauterine growth restriction (IUGR) which are associated with placental hypoxia. METHODS Placentas from 152 pathological pregnancies, including PE and/or HELLP (n=24) and IUGR (n=21), and 20 normal control placentas were assessed for their ABCA1 and ABCG1 mRNA and protein expression with quantitative RT-PCR and semi-quantitative immunohistochemical analysis, respectively. RESULTS ABCA1 protein expression in the V-STB was significantly less extensive in PE compared with normal controls (<10% of V-STB stained for ABCA1 in 58% PE placentas vs. 25% controls; p=0.035). Conversely, it was significantly more wide-spread in IUGR (>75% of V-STB stained in 57% IUGR placentas vs. 15% controls; p=0.009). Moreover, there was an insignificant trend for increased ABCA1 expression in fetal endothelial cells of stem villi in PE (p=0.0588). ABCA1 staining levels in V-STB were significantly associated with placental histopathological features related with hypoxia: they were decreased in placentas exhibiting syncytial knotting (p=0.033) and decidual vasculopathy (p=0.0437) and increased in low weight placentas (p=0.015). The significant and specific alterations in ABCA1 protein expression found at a specific cellular level were not paralleled by changes in ABCA1 mRNA abundance of total placental tissue. ABCG1 staining was universally extensive in the V-STB of normal placentas, always affecting more than 90% of V-STB surface. In comparison, ABCG1 staining of the V-STB was generally often reduced in pregnancy diseases. In particular, less than 90% of V-STB exhibited ABCG1 staining in 26% of PE placentas (p=0.022) and 35% of IUGR placentas (p=0.003). Similarly to ABCA1, ABCG1 mRNA expression in total placental tissue was not significantly different between controls and PE or IUGR. CONCLUSION ABCA1 and ABCG1 proteins are differentially expressed, with either down- or up-regulation, in the V-STB of placentas exhibiting features of chronic hypoxia, such as in PE and IUGR. This suggests that other factors in addition to hypoxia regulate the expression of placental lipid transporters. The specific changes on a cellular level were masked when only total tissue mRNA was analysed underlining the importance of cell specific expression analysis. The potential effects of decreased placental ABCA1 and ABCG1 expression on foetal nutrition and development remain to be elucidated.

Differential expression of the receptor tyrosine kinase EphB4 and its ligand Ephrin-B2 during human placental development

Normal placentation involves the development of an utero-placental circulation following the migration of the extravillous cytotrophoblasts into the decidua and invasion of the spiral arteries, which are thereby transformed into large vessels of low resistance. Given the documented role of the receptor tyrosine kinase EphB4 and its ligand ephrin-B2 in the establishment of the embryonal vascular network, we hypothesized that these molecules are also instrumental in the development of the human placenta. Monitoring the expression during placental development revealed that in first trimester and term placentae both molecules are equally expressed at the RNA level. In contrast, the protein levels were significantly reduced during gestation. Immunohistochemistry revealed a distinct localization of the EphB4 and ephrin-B2 proteins. EphB4 was predominantly expressed in the villous syncytial trophoblast layer and in a subset of intravillous capillaries. Prominent expression was also observed in the extravillous cytotrophoblast giant cells. In contrast, ephrin-B2 expression was detected in the villous cytotrophoblast and syncytial trophoblast cell layers, as well as initially in all intravillous capillaries. Strong expression was also observed in extravillous anchoring cytotrophoblast cells. Hypoxia is a major inducer of placental development. In vitro studies employing trophoblast-derived cell lines revealed that predominantly ephrin-B2 expression is induced by hypoxia, however, in an Hif-1alpha independent manner. These experiments suggest that EphB4 and ephrin-B2 are instrumental in the establishment of a functional placental structure and of the utero-placental circulation.

First trimester markers for pre-eclampsia: placental vs. non-placental protein serum levels

BACKGROUND/AIM: Parallel investigation, in a matched case-control study, of the association of different first-trimester markers with the risk of subsequent pre-eclampsia (PE). METHOD: The levels of different first trimester serum markers and fetal nuchal translucency thickness were compared between 52 cases of PE and 104 control women by non-parametric two-group comparisons and by calculating matched odds ratios. RESULTS: In univariable analysis increased concentrations of inhibin A and activin A were associated with subsequent PE (p < 0.02). Multivariable conditional logistic regression models revealed an association between increased risk of PE and increased inhibin A and translucency thickness and respectively reduced pregnancy-associated plasma protein A (PAPP-A) and placental lactogen . However, these associations varied with the gestational age at sample collection. For blood samples taken in pregnancy weeks 12 and 13 only, increased levels of activin A, inhibin A and nuchal translucency thickness, and lower levels of placenta growth factor and PAPP-A were associated with an increased risk of PE. CONCLUSIONS: Members of the inhibin family and to some extent PAPP-A and placental growth factor are superior to other serum markers, and the predictive value of these depends on the gestational age at blood sampling. The availability of a single, early pregnancy 'miracle' serum marker for PE risk assessment seems unlikely in the near future.

C-reactive protein production in term human placental tissue

OBJECTIVE: C-reactive protein (CRP) is a marker of systemic inflammation. Recently, it has been shown that CRP is present in amniotic fluid and fetal urine, and that elevated levels are associated with adverse pregnancy outcome. However, the precise source of amniotic fluid CRP, its regulation, and function during pregnancy is still a matter of debate. The present in vivo and in vitro studies were designed to investigate the production of CRP in human placental tissues. MATERIAL AND METHODS: Ten paired blood samples from peripheral maternal vein (MV), umbilical cord artery (UA) and umbilical vein (UV) were collected from women with elective caesarean sections at term. The placental protein accumulation capacity of hCG, hPL, leptin and CRP was compared with the dual in vitro perfusion method of an isolated cotyledon of human term placentae and quantified by ELISA. Values for accumulation (release) were calculated as total accumulation of maternal and fetal circuits normalized for tissue weight and duration of perfusion. For gene expression, RNA was extracted from placental tissue and reverse transcribed. RT-PCR and real-time PCR were performed using specific primers. RESULTS: The median (range) CRP level was significantly different between UA and UV [50.1 ng/ml (12.1-684.6) vs. 61 ng/ml (16.9-708.1)]. The median (range) difference between UV and UA was 9.3 ng/ml (2.2-31.6). A significant correlation was found between MV CRP and both UA and UV CRP levels. Median (range) MV CRP levels [2649 ng/ml (260.1-8299)] were 61.2 (6.5-96.8) fold higher than in the fetus. In vitro, the total accumulation rates (mean+/-SD) were 31+/-13 (mU/g/min, hCG), 1.16+/-0.19 (microg/g/min, hPL), 4.71+/-1.91 (ng/g/min, CRP), and 259+/-118 (pg/g/min, leptin). mRNA for hCG, hPL and leptin was detectable using conventional RT-PCR, while CRP mRNA could only be demonstrated by applying real-time RT-PCR. In the perfused tissue the transcript levels for the four proteins were comparable to those detected in the native control tissue. CONCLUSIONS: Our results demonstrate that the human placenta produces and releases CRP mainly into the maternal circulation similarly to other analyzed placental proteins under in vitro conditions. Further studies are needed to explore the exact role of placental CRP during pregnancy.

Placental mesenchymal stem cells as potential autologous graft for pre- and perinatal neuroregeneration

OBJECTIVE: Mesenchymal stem cells (MSCs) have a broad differentiation potential. We aimed to determine if MSCs are present in fetal membranes and placental tissue and to assess their potential to differentiate into neurogenic and mesodermal lineages. STUDY DESIGN: MSCs isolated from first and third trimester chorion and amnion and first trimester chorionic villi and characterized morphologically and by flourescence-activated cell sorting analysis. Their ability to mature under different culture conditions into various cells of mesodermal and neuroectodermal cell lines was assessed by immuno- and cytochemical staining. RESULTS: Independent of gestational age, cells isolated from fetal membranes and placenta showed typical MSC phenotype (positive for CD166, CD105, CD90, CD73, CD49e, CD44, CD29, CD13, MHC I; negative for CD14, CD34, CD45, MHC II) and were able to differentiate into mesodermal cells expressing cell markers/cytologic staining consistent with mature chondroblasts, osteoblasts, adipocytes, or myocytes and into neuronal cells presenting markers of various stages of maturation. The differentiation pattern was mainly dependent on cell type. CONCLUSION: Mesenchymal cells from chorion, amnion, and villous stroma can be differentiated into neurogenic, chondrogenic, osteogenic, adipogenic, and myogenic lineage. Placental tissue obtained during prenatal chorionic villous sampling or at delivery might be an ideal source for autologous stem cell graft for peripartum neuroregeneration and other clinical issues.

Multiple thromboembolic events in fetofetal transfusion syndrome in triplets contributing to the understanding of pathogenesis of hydranencephaly in combination with polymicrogyria

Over the last 180 years, several theories concerning the origin of hydranencephaly have been proposed with an emphasis on infectious, aplastic, and vascular etiologies. In this report, we present a case of triplets with fetofetal transfusion syndrome of which 2 fetuses (1 and 2) developed almost similar hydranencephaly, whereas the third exhibited the features of a fetus papyraceus (3). In the monochorial triamniotic placenta, multiple arteriovenous anastomoses were detected, representing a probable route for the transmission of thrombi originating from fetus 3 causing visceral lesions in fetus 2. Hydranencephaly was histologically characterized by necrosis, macrophage invasion, and endothelial proliferation. In addition, polymicrogyria was seen in fetuses 1 and 2. The combination of multiple visceral thromboembolic events and the death of fetus 3 approximately in the 11th week of gestation suggested a vascular thrombotic pathogenesis of hydranencephaly. Polymicrogyria can be considered as postmigratory laminar necrosis. Our findings contribute to the pathogenetic understanding of combined hydranencephaly and polymicrogyria.

Proteome analysis of human plasma and amniotic fluid by Off-Gel isoelectric focusing followed by nano-LC-MS/MS

This paper presents a comparative proteomic analysis of human maternal plasma and amniotic fluid (AF) samples from the same patient at term of pregnancy in order to find specific AF proteins as markers of premature rupture of membranes, a complication frequently observed during pregnancy. Maternal plasma and the corresponding AF were immunodepleted in order to remove the six most abundant proteins before the systematic analysis of their protein composition. The protein samples were then fractionated by IEF Off-Gel electrophoresis (OGE), digested and analyzed with nano-LC-MS/MS separation, revealing a total of 73 and 69 proteins identified in maternal plasma and AF samples, respectively. The proteins identified in AF have been compared to those identified in the mother plasma as well as to the reference human plasma protein list reported by Anderson et al. (Mol. Cell. Proteomics 2004, 3, 311-326). This comparison showed that 26 proteins were exclusively present in AF and not in plasma among which 10 have already been described to be placenta or pregnancy specific. As a further validation of the method, plasma proteins fractionated by OGE and analysed by nano-LC-MS/MS have been compared to the Swiss 2-D PAGE reference map by reconstructing a map that matches 2-D gel and OGE experimental data. This representation shows that 36 of 49 reference proteins could be identified in both data sets, and that isoform shifts in pI are well conserved in the OGE data sets.

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.

Marked disturbance of calcium homeostasis in mice with targeted disruption of the Trpv6 calcium channel gene

We report the phenotype of mice with targeted disruption of the Trpv6 (Trpv6 KO) epithelial calcium channel. The mice exhibit disordered Ca(2+) homeostasis, including defective intestinal Ca(2+) absorption, increased urinary Ca(2+) excretion, decreased BMD, deficient weight gain, and reduced fertility. Although our Trpv6 KO affects the closely adjacent EphB6 gene, the phenotype reported here is not related to EphB6 dysfunction. INTRODUCTIOn: The mechanisms underlying intestinal Ca(2+) absorption are crucial for overall Ca(2+) homeostasis, because diet is the only source of all new Ca(2+) in the body. Trpv6 encodes a Ca(2+)-permeable cation channel responsible for vitamin D-dependent intestinal Ca(2+) absorption. Trpv6 is expressed in the intestine and also in the skin, placenta, kidney, and exocrine organs. MATERIALS AND METHODS: To determine the in vivo function of TRPV6, we generated mice with targeted disruption of the Trpv6 (Trpv6 KO) gene. RESULTS: Trpv6 KO mice are viable but exhibit disordered Ca(2+) homeostasis, including a 60% decrease in intestinal Ca(2+) absorption, deficient weight gain, decreased BMD, and reduced fertility. When kept on a regular (1% Ca(2+)) diet, Trpv6 KO mice have deficient intestinal Ca(2+) absorption, despite elevated levels of serum PTH (3.8-fold) and 1,25-dihydroxyvitamin D (2.4-fold). They also have decreased urinary osmolality and increased Ca(2+) excretion. Their serum Ca(2+) is normal, but when challenged with a low (0.25%) Ca(2+) diet, Trpv6 KO mice fail to further increase serum PTH and vitamin D, ultimately developing hypocalcemia. Trpv6 KO mice have normal urinary deoxypyridinoline excretion, although exhibiting a 9.3% reduction in femoral mineral density at 2 months of age, which is not restored by treatment for 1 month with a high (2%) Ca(2+) "rescue" diet. In addition to their deranged Ca(2+) homeostasis, the skin of Trpv6 KO mice has fewer and thinner layers of stratum corneum, decreased total Ca(2+) content, and loss of the normal Ca(2+) gradient. Twenty percent of all Trpv6 KO animals develop alopecia and dermatitis. CONCLUSIONS: Trpv6 KO mice exhibit an array of abnormalities in multiple tissues/organs. At least some of these are caused by tissue-specific mechanisms. In addition, the kidneys and bones of Trpv6 KO mice do not respond to their elevated levels of PTH and 1,25-dihydroxyvitamin D. These data indicate that the TRPV6 channel plays an important role in Ca(2+) homeostasis and in other tissues not directly involved in this process.

The cytochrome P450 aromatase lacking exon 5 is associated with a phenotype of nonclassic aromatase deficiency and is also present in normal human steroidogenic tissues

OBJECTIVE: The previously described c655G>A mutation of the human cytochrome P450 aromatase gene (P450aro, CYP19) results in aberrant splicing due to disruption of a donor splice site. To explain the phenotype of partial aromatase deficiency observed in a female patient described with this mutation, molecular consequences of the c655G>A mutation were investigated. DESIGN: To investigate whether the c655G>A mutation causes an aberrant spliced mRNA lacking exon 5 (-Ex5), P450aro RNA was analysed from the patient's lymphocytes by reverse transcription polymerase chain reaction (RT-PCR) and by splicing assays performed in Y1 cells transfected with a P450aro -Ex5 expression vector. Aromatase activity of the c655G>A mutant was predicted by three dimensional (3D) protein modelling studies and analysed in transiently transfected Y1 cells. Exon 5 might be predicted as a poorly defined exon suggesting a susceptibility to both splicing mutations and physiological alternative splicing events. Therefore, expression of the -Ex5 mRNA was also assessed as a possibly naturally occurring alternative splicing transcript in normal human steroidogenic tissues. PATIENTS: An aromatase deficient girl was born with ambiguous genitalia. Elevated serum LH, FSH and androgens, as well as cystic ovaries, were found during prepuberty. At the age of 8.4 years, spontaneous breast development and a 194.6 pmol/l serum oestradiol level was observed. RESULTS: The -Ex5 mRNA was found in lymphocytes of the P450aro deficient girl and her father, who was a carrier of the mutation. Mutant minigene expression resulted in complete exon 5 skipping. As expected from 3D protein modelling, -Ex5 cDNA expression in Y1 cells resulted in loss of P450aro activity. In addition, the -Ex5 mRNA was present in placenta, prepubertal testis and adrenal tissues. CONCLUSIONS: Alternative splicing of exon 5 of the CYP19 gene occurs in the wild type (WT) as well as in the c655G>A mutant. We speculate that for the WT it might function as a regulatory mechanism for aromatization, whereas for the mutant a relative prevalence of the shorter over the full-length protein might explain the phenotype of partial aromatase deficiency.