Rapid fusion and syncytium formation of heterologous cells upon expression of the FGFRL1 receptor

The fusion of mammalian cells into syncytia is a developmental process that is tightly restricted to a limited subset of cells. Besides gamete and placental trophoblast fusion, only macrophages and myogenic stem cells fuse into multinucleated syncytia. In contrast to viral cell fusion, which is mediated by fusogenic glycoproteins that actively merge membranes, mammalian cell fusion is poorly understood at the molecular level. A variety of mammalian transmembrane proteins, among them many of the immunoglobulin superfamily, have been implicated in cell-cell fusion, but none has been shown to actively fuse cells in vitro. Here we report that the FGFRL1 receptor, which is up-regulated during the differentiation of myoblasts into myotubes, fuses cultured cells into large, multinucleated syncytia. We used luciferase and GFP-based reporter assays to confirm cytoplasmic mixing and to identify the fusion inducing domain of FGFRL1. These assays revealed that Ig-like domain III and the transmembrane domain are both necessary and sufficient to rapidly fuse CHO cells into multinucleated syncytia comprising several hundred nuclei. Moreover, FGFRL1 also fused HEK293 and HeLa cells with untransfected CHO cells. Our data show that FGFRL1 is the first mammalian protein that is capable of inducing syncytium formation of heterologous cells in vitro.

Generation of an osteogenic graft from human placenta and placenta-derived mesenchymal stem cells

The objective of the study was to determine the feasibility of generating a biodegradable, stem cell-loaded osteogenic composite graft from human placenta. Initially, a scaffold from human chorion membrane was produced. Human placenta mesenchymal stem cells (MSCs) derived from either first-trimester chorionic villi or term chorion membrane were differentiated osteogenically on this scaffold. Outgrowth, adherence, and osteogenic differentiation of cells were assessed by immunohistochemistry (IHC), scanning electron microscopy, protein expression, and real-time polymerase chain reaction (RT-PCR). Our results showed that a cell-free extracellular matrix scaffold can be generated from human chorion. Seeded MSCs densely adhered to that scaffold and were osteogenically differentiated. Calcium and alkaline phosphatase were detected in the cell-scaffold constructs as a proof of mineralization and findings were confirmed by IHC and RT-PCR results. This study shows for the first time that generation of an osteogenic composite graft using placental tissue is feasible. It might allow therapeutic application of autologous or allogeneic grafts in congenital skeletal defects by means of a composite graft.

Techniques for assessing cardiac output and fetal cardiac function

Fetal echocardiography was initially used to diagnose structural heart disease, but recent interest has focused on functional assessment. Effects of extracardiac conditions on the cardiac function such as volume overload (in the recipient in twin-twin transfusion syndrome), a hyperdynamic circulation (arterio-venous malformation), cardiac compression (diaphragmatic hernia, lung tumours) and increased placental resistance (intrauterine growth restriction and placental insufficiency) can be studied by ultrasound and may guide decisions for intervention or delivery. A variety of functional tests can be used, but there is no single clinical standard. For some specific conditions, however, certain tests have shown diagnostic value.

RNA degradation differentially affects quantitative mRNA measurements of endogenous reference genes in human placenta

It has been highlighted that RNA quality and appropriate reference gene selection is crucial for the interpretation of RT-qPCR results in human placental samples. In this context we investigated the effect of RNA degradation on the mRNA abundance of seven frequently used reference genes in 119 human placental samples. Combining RNA integrity measurements, RT-qPCR analysis and mathematical modeling we found major differences regarding the effect of RNA degradation on the measured expression levels between the different reference genes. Furthermore, we demonstrated that a modified RNA extraction method significantly improved RNA quality and consequently increased transcript levels of all reference genes.

Air pollution during pregnancy and neonatal outcome: a review

There is increasing evidence of the adverse impact of prenatal exposure to air pollution. This is of particular interest, as exposure during pregnancy--a crucial time span of important biological development--may have long-term implications. The aims of this review are to show current epidemiological evidence of known effects of prenatal exposure to air pollution and present possible mechanisms behind this process. Harmful effects of exposure to air pollution during pregnancy have been shown for different birth outcomes: higher infant mortality, lower birth weight, impaired lung development, increased later respiratory morbidity, and early alterations in immune development. Although results on lower birth weight are somewhat controversial, evidence for higher infant mortality is consistent in studies published worldwide. Possible mechanisms include direct toxicity of particles due to particle translocation across tissue barriers or particle penetration across cellular membranes. The induction of specific processes or interaction with immune cells in either the pregnant mother or the fetus may be possible consequences. Indirect effects could be oxidative stress and inflammation with consequent hemodynamic alterations resulting in decreased placental blood flow and reduced transfer of nutrients to the fetus. The early developmental phase of pregnancy is thought to be very important in determining long-term growth and overall health. So-called "tracking" of somatic growth and lung function is believed to have a huge impact on long-term morbidity, especially from a public health perspective. This is particularly important in areas with high levels of outdoor pollution, where it is practically impossible for an individual to avoid exposure. Especially in these areas, good evidence for the association between prenatal exposure to air pollution and infant mortality exists, clearly indicating the need for more stringent measures to reduce exposure to air pollution.

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.

Aldosterone deficiency adversely affects pregnancy outcome in mice

Circulating aldosterone levels are increased in human pregnancy. Inadequately low aldosterone levels as present in preeclampsia, a life-threatening disease for both mother and child, are discussed to be involved in its pathogenesis or severity. Moreover, inactivating polymorphisms in the aldosterone synthase gene have been detected in preeclamptic women. Here, we used aldosterone synthase-deficient (AS(-/-)) mice to test whether the absence of aldosterone is sufficient to impair pregnancy or even to cause preeclampsia. AS(-/-) and AS(+/+) females were mated with AS(+/+) and AS(-/-) males, respectively, always generating AS(+/-) offspring. With maternal aldosterone deficiency in AS(-/-) mice, systolic blood pressure was low before and further reduced during pregnancy with no increase in proteinuria. Yet, AS(-/-) had smaller litters due to loss of fetuses as indicated by a high number of necrotic placentas with massive lymphocyte infiltrations at gestational day 18. Surviving fetuses and their placentas from AS(-/-) females were smaller. High-salt diet before and during pregnancy increased systolic blood pressure only before pregnancy in both genotypes and abolished the difference in blood pressure during late pregnancy. Litter size from AS(-/-) was slightly improved and the differences in placental and fetal weights between AS(+/+) and AS(-/-) mothers disappeared. Overall, an increased placental efficiency was observed in both groups paralleled by a normalization of elevated HIF1α levels in the AS(-/-) placentas. Our results demonstrate that aldosterone deficiency has profound adverse effects on placental function. High dietary salt intake improved placental function. In this animal model, aldosterone deficiency did not cause preeclampsia.

Serum markers for predicting pre-eclampsia

Pre-eclampsia, a pregnancy-specific disorder, contributes substantially to perinatal morbidity and mortality of both, mother and newborn. An increasing number of biochemical agents were evaluated as markers for predicting pre-eclampsia. None of them has been proved to be of clinical value yet. Much effort has been put into assessing novel potential markers and their combination with other screening methods such as Doppler sonography. The purpose of this review is to reflect the current knowledge of serum markers for predicting pre-eclampsia. So far, the most promising serum markers are placental protein 13 (PP-13), as well as soluble fms-like tyrosine kinase-1 (sFlt-1), placental growth factor (PIGF) and soluble endoglin (sEng). These markers allow screening at a relatively early stage and, most importantly, show relatively high predictive values and improved diagnostic performance if combined with first trimester Doppler sonography. Large-scale prospective studies, assessing these markers, are important to justify their clinical use in view of early intervention to prevent pre-eclampsia in the future.

Dual in vitro perfusion of an isolated cotyledon as a model to study the implication of changes in the third trimester placenta on preeclampsia

In the current study perfusions of an isolated cotyledon of term placenta using standard medium were compared to medium containing xanthine plus xanthine oxidase (X+XO), which generates reactive oxygen species (ROS). A time-dependant increase in the levels of different cytokines (TNF-alpha, IL-1ss, IL-6, IL-8 and IL-10) was observed between 1 and 7h with more than 90% of the total recovered from the maternal compartment with no significant difference between the 2 groups. For 8-iso-PGF2alpha 90% of the total was found in the fetal compartment and a significantly higher total release was seen in the X+XO group. Microparticles (MPs) isolated from the maternal circuit were identified by flow cytometry as trophoblastic sheddings, whereas MPs from the fetal circuit were predominantly derived from endothelial cells. More than 90% of the total of MPs was found in the maternal circuit. The absolute amount of the total as well as the maternal fraction were significantly higher in the X+XO group. Immunohistochemistry (IHC) of the perfused tissue revealed staining for IL-1beta of villous stroma cells, which became clearly more pronounced in experiments with X+XO. Western blot of tissue homogenate revealed 2 isoforms of IL-1beta at 17 and 31kD. In X+XO experiments there was a tendency for increased expression of antioxidant enzymes in the tissue. Western blot of MPs from the maternal circuit showed increased expression of antioxidant enzymes in the X+XO group and for IL-1beta only the 17kD band was detected. In vitro reperfusion of human placental tissue results in mild tissue injury suggestive of oxidative stress. In view of the increased generation of ROS in perfused tissue with further increase under the influence of X+XO, the overall manifestation of oxidative stress remained rather mild. Preservation of antioxidant capacity of human placental tissue could be a sign of integrity of structure and function being maintained in vitro by dual perfusion of an isolated cotyledon. The observed changes resemble findings seen in placentae from preeclampsia.

Differential release of plasminogen activator inhibitors (PAIs) during dual perfusion of human placenta: implications in preeclampsia

Plasminogen activator inhibitors (PAIs) play critical roles in regulating cellular invasion and fibrinolysis. An increase in the ratio of PAI-1/PAI-2 in placenta and maternal serum is suggested to result in excessive intervillous fibrin deposition and placental infarction in pregnancies complicated by preeclampsia (PE) and intrauterine growth restriction (IUGR). In the current study we used dual (maternal and fetal) perfusion of human term placentas to examine the release of PAIs to the intervillous space. ELISA revealed a significant time-dependent increase in total PAI-1 levels in maternal perfusate (MP) between 1 and 7h of perfusion. Conversely, PAI-2 levels decreased resulting in a 3-fold increase in the PAI-1/PAI-2 ratio in MP. Levels of PAI-1, but not PAI-2, in placental tissue extracts increased during perfusion. In perfusions carried out with xanthine and xanthine oxidase (X + XO), compounds used to generate reactive oxygen species (ROS), no time-dependent increase in total PAI-1 levels was observed. In addition, X + XO treatment promoted a 3-fold reduction in active PAI-1 levels in MP, indicating that ROS decrease PAI-1 release to MP. The finding of a time-dependent change in patterns of PAI expression and response to ROS indicates the utility of dual perfusion as a model to dissect mechanism(s) promoting aberrant fibrinolysis in pregnancies complicated by PE and IUGR.

Oral misoprostol for third stage of labor: a randomized placebo-controlled trial

OBJECTIVE: To investigate whether orally administered misoprostol during the third stage of labor is efficient in reducing postpartum blood loss. METHODS: In a double-masked trial, during vaginal delivery women were randomly assigned to receive a single oral dose of misoprostol (600 microg) or placebo in third stage of labor, immediately after cord clamping. The third stage of labor was managed routinely by early cord clamping and controlled cord traction; oxytocin was administered only if blood loss seemed more than usual. Blood loss was estimated by the delivering physician and differences in hematocrit were measured before and after delivery. RESULTS: Mean (+/- standard error of the mean) estimated blood loss (345 +/- 19.5 mL versus 417 +/- 25.9 mL, P = .031) and hematocrit difference (4.5 +/- 0.9% versus 7.9 +/- 1.2%, P = .014) were significantly lower in women who received misoprostol than those who received placebo. Fewer women in the misoprostol group had postpartum hemorrhage (blood loss of at least 500 mL), but that difference was not statistically significant (7% versus 15%, P = .43). Additional oxytocin before or after placental separation was used less often in the misoprostol group (16% versus 38%, P = .047). There were no differences in the length of third stage of labor (8 +/- 0.9 minutes versus 9 +/- 1 minutes, P = .947). There were no differences in pain during third stage of labor, postpartum fever, or diarrhea, but shivering was more frequent in the misoprostol group. CONCLUSION: Oral misoprostol administered in the third stage of labor reduced postpartum blood loss and might be effective in reducing incidence of postpartum hemorrhage.

Urinary iodine concentration during pregnancy in an area of unstable dietary iodine intake in Switzerland

We prospectively investigated urinary iodine concentration (UIC) in pregnant women and in female, non-pregnant controls in the canton of Berne, Switzerland, in 1992. Mean UIC of pregnant women [205 +/- 151 microg iodine/g creatinine (microg l/g Cr); no. = 153] steadily decreased from the first (236 +/- 180 microg l/g Cr; no. = 31) to the third trimester (183 +/- 111 microg l/g Cr, p < 0.0001; no. = 66) and differed significantly from that of the control group (91 +/- 37 microg l/g Cr, p < 0.0001; no. = 119). UIC increased 2.6-fold from levels indicating mild iodine deficiency in controls to the first trimester, demonstrating that high UIC during early gestation does not necessarily reflect a sufficient iodine supply to the overall population. Pregnancy is accompanied by important alterations in the regulation of thyroid function and iodine metabolism. Increased renal iodine clearance during pregnancy may explain increased UIC during early gestation, whereas increased thyroidal iodine clearance as well as the iodine shift from the maternal circulation to the growing fetal-placental unit, which both tend to lower the circulating serum levels of inorganic iodide, probably are the causes of the continuous decrease of UIC over the course of pregnancy. Mean UIC in our control group, as well as in one parallel and several consecutive investigations in the same region in the 1990s, was found to be below the actually recommended threshold, indicating a new tendency towards mild to moderate iodine deficiency. As salt is the main source of dietary iodine in Switzerland, its iodine concentration was therefore increased nationwide in 1998 for the fourth time, following increases in 1922, 1965 and 1980.

Regulation of fetal growth: consequences and impact of being born small

The first trimester of pregnancy is the time during which organogenesis takes place and tissue patterns and organ systems are established. In the second trimester the fetus undergoes major cellular adaptation and an increase in body size, and in the third trimester organ systems mature ready for extrauterine life. In addition, during that very last period of intrauterine life there is a significant increase in body weight. In contrast to the postnatal endocrine control of growth, where the principal hormones directly influencing growth are growth hormone (GH) and the insulin-like growth factors (IGFs) via the GH-IGF axis, fetal growth throughout gestation is constrained by maternal factors and placental function and is coordinated by growth factors. In general, growth disorders only become apparent postnatally, but they may well be related to fetal life. Thus, fetal growth always needs to be considered in the overall picture of human growth as well as in its metabolic development.

A novel enzyme immunoassay specific for ABCA1 protein quantification in human tissues and cells

ATP-binding cassette transporter A1 (ABCA1) mediates the transport of cholesterol and phospholipids from cells to lipid-poor HDL and maintains cellular lipid homeostasis. Impaired ABCA1 function plays a role in lipid disorders, cardiovascular disease, atherosclerosis, and metabolic disorders. Despite the clinical importance of ABCA1, no method is available for quantifying ABCA1 protein. We developed a sensitive indirect competitive ELISA for measuring ABCA1 protein in human tissues using a commercial ABCA1 peptide and a polyclonal anti-ABCA1 antibody. The ELISA has a detection limit of 8 ng/well (0.08 mg/l) with a working range of 9-1000 ng/well (0.09-10 mg/l). Intra- and interassay coefficient of variations (CVs) were 6.4% and 9.6%, respectively. Good linearity (r = 0.97-0.99) was recorded in serial dilutions of human arterial and placental crude membrane preparations, and fibroblast lysates. The ELISA measurements for ABCA1 quantification in reference arterial tissues corresponded well with immunoblot analysis. The assay performance and clinical utility was evaluated with arterial tissues obtained from 15 controls and 44 patients with atherosclerotic plaques. ABCA1 protein concentrations in tissue lysates were significantly lower in patients (n = 24) as compared with controls (n = 5; 9.37 +/- 0.82 vs. 17.03 +/- 4.25 microg/g tissue; P < 0.01). The novel ELISA enables the quantification of ABCA1 protein in human tissues and confirms previous semiquantitative immunoblot results.