Syncytiotrophoblast Microparticles Research Articles

A Study on the Role of Urinary Congophilia in Early Detection of Preeclampsia

Abstract Introduction Preeclampsia and eclampsia are important causes of maternal morbidity. Preeclapmtic women secrete misfolded proteins in the urine. Buhimschi et al had developed a new test for diagnosis of preeclampsia. This test is based on staining of misfolded protein with Congo red dye. Misfolded proteins are derived from syncytiotrophoblast microparticles (STBMs). These STBM are membrane bound vesicles and contain misfolded proteins. In preeclampsia, glomeruli of kidneys are disrupted and these damaged protein reach the urine. Aim and Objective This study aimed to investigate the role of urinary congophilia in early prediction of preeclampsia. Materials and Methods This test was done in 250 pregnant women attending the Gynaecological Outpatient Department. Urine sample of early morning was taken and test was done in the Department of Biochemistry. The included pregnant women were of gestational age between 14 and 18 weeks. The staining of urine with Congo red dye was done and washed with methanol. The retention of dye was interpreted with naked eye. The more retention of dye, the more chances of developing preeclampsia later. The patients were followed-up till delivery. The patents who developed preeclampsia later part of pregnancy were recorded. Mean arterial pressure (MAP) and past history and body mass index were also recorded. Results Out of 250 patients, 30 developed preeclampsia later. A total of 34 patients were having positive urinary congophilia and only 20 patients developed preeclampsia later. MAP more than 90 mm Hg is abnormal but 66.7% of patients who developed preeclampsia had MAP >90 mm Hg. In 16.7% of patients, who developed preeclampsia later, had positive past history of hypertension. In 66.7% of patients, who were positive for urinary congophilia, later developed preeclampsia. Conclusion Preeclampsia and eclampsia are important causes of maternal mortality and morbidity. So, early detection can prevent complications and timely management. Urinary congophilia is one of such test which can help in early prediction of preeclampsia. If it is combined with past maternal history and MAP, it gives more good results. The detection rate is much higher if signs and symptoms of preeclampsia are noticed timely.

The Role of Neutrophils and Their Extracellular Traps in the Synergy of Pre-eclampsia and HIV Infection.

In our innate immune system, neutrophils are the first cells to sense signals of infection and to proceed to kill the invading pathogen. This is mediated by their production of neutrophil extracellular traps (NETS) to entrap pathogenic micro-organisms, preventing their amplification and dissemination. Pre-eclampsia (PE) is the leading cause of global maternal mortality, yet to date, there is no cure nor a gold-standard diagnostic strategy. The purpose of this review is to discover the role of neutrophils in PE as early identification markers. Additionally, this review aims to explore the role of neutrophils in HIV-infected pregnancies with PE as a source of synergy. Recent findings demonstrate an elevation of neutrophils and neutrophil extracellular traps (NETs) in PE placentae. This is due to their activation by excessive release of syncytiotrophoblast microparticles (STBM). There is also an elevation of NETs in HIV-infected placentae-where histone H3 entraps HIV by binding to its glycoprotein envelope. Additionally, histones H1 and H2A inhibit HIV infection. It is interesting to note that women with both PE and HIV infection have supressed NETs. This review focuses on the role of neutrophils in the synergy of PE and HIV infection. It is plausible that the deregulation of NETs in the synergy of pre-eclamptic HIV-infected women is strategic for the entrapment of the HIV-1 virus. Finally, it is plausible that neutrophils and NETS may act as early biomarkers of PE development. Graphical abstract.

Syncytial nuclear aggregates as markers for villous maturation in placentas from preterm birth

The syncytiotrophoblast is the multinucleated syncytial layer lining the placental villi. In some areas nuclei form aggregates termed true knots by the process of trophoblast turnover. Thereby, the underlying cytotrophoblast cells proliferate, differentiate and fuse with the overlying synctiotrophoblast cells which have no own generative potency. The nuclei in the syncytiotrophoblast are undergoing apoptosis and form true knots which are shed into the maternal circulation as syncytiotrophoblast microparticles (STBMs). This process ensures that the diffusion distance between maternal and foetal blood is decreased to nourish the growing foetus. Excessive formation of true knots has been reported in placentas of pregnancies complicated by pre-eclampsia. This is a complication of pregnancy characterized by increased maternal blood pressure and induced preterm labour is the only treatment. Pregnancies complicated by chorioamnionitis, a severe inflammation of the foetal membranes due to a bacterial infection, are also at increased risk of preterm labour. The aim of this study is to establish an objective scoring system for placental villous maturation based on true knots, STBMs and syncytial knotting frequency. Our assumption is that the frequency of syncytial knots in placentas complicated by pre-eclampsia are increased. True knots, STBMs and syncytial knotting are assessed by histological examination of placental tissue using a light microscope. In pre-eclamptic placentas the highest numbers of true knots, syncytial knotting and STBMs compared to an idiopathic preterm control group were found. Placentas complicated by chorioamnionitis show a slight decrease of true knots, syncytial knotting and STBMs compared to the idiopathic preterm group and a significant decrease compared to pre-eclamptic placentas. The increase of true knots and syncytial knotting could be a sign for an increased trophoblast turnover during pre-eclampsia. This implies enhanced proliferation, differentiation and apoptosis of the placental epithelium. Chorioamnionitis placentas display lower number of true knots indicating a lower trophoblast turnover which may be part of another anomaly of the placental development. For our scoring system are true knots, STBMs and syncytial knotting convincing markers for analysis of villous maturation in pre-eclampsia. In case of chorioamnionitis other markers may be more useful.

RhoB/ROCK mediates oxygen-glucose deprivation-stimulated syncytiotrophoblast microparticle shedding in preeclampsia.

Increased circulating syncytiotrophoblast microparticles (STBMs) are often associated with preeclampsia (PE) but the molecular mechanisms regulating STBM shedding remain elusive. Experimental evidence has shown that actin plays a key role in STBM shedding and that Rho/ROCK is important in regulating actin rearrangement. To investigate the role of RhoB/ROCK-regulated actin arrangement in STBM shedding in PE, chorionic villous explants were prepared from placenta of patients with normotensive or PE pregnancies and BeWo cells were fused to imitate syncytiotrophoblasts. The oxygen-glucose deprivation (OGD) conditions were applied to imitate the pathophysiology of PE in vitro. The results showed that RhoB and ROCK were activated in the preeclamptic placenta, accompanied by increased actin polymerization and decreased outgrowing microvilli. In villous tissue cultures or BeWo cells, OGD activated RhoB, ROCK1 and ROCK2 and promoted STBM shedding and actin stress fibers formation. In BeWo cells, RhoB overexpression activated ROCK1 and ROCK2, leading to F-actin redistribution and STBM shedding and the OGD-induced actin polymerization and STBM shedding could be reversed by RhoB or ROCK knockdown. These results reveal that RhoB and ROCK play a key role in PE by targeting STBM shedding through actin rearrangement and that RhoB/ROCK intervention may be a potential therapeutic strategy for PE.

HLA class II is aberrantly expressed on circulating syncytiotrophoblast microparticles in early onset pre-eclampsia

s / Placenta 36 (2015) A1eA60 A7 NI3.3. HLA CLASS II IS ABERRANTLY EXPRESSED ON CIRCULATING SYNCYTIOTROPHOBLAST MICROPARTICLES IN EARLY ONSET PREECLAMPSIA Chiara Tersigni , Christopher Redman , Dionne Tannetta , Rebecca Dragovic , Sylvia Shahjahan , Nicoletta Di Simone , Ian Sargent , Manu Vatish . Department of Obstetrics and Gynaecology, Universit a Cattolica del Sacro Cuore, Policlinico A. Gemelli, 00168, Rome, Italy; Nuffield Department of Obstetrics and Gynaecology, University of Oxford, Women’s Centre, John Radcliffe Hospital, OX3 9DU, Oxford, UK Objectives: The lack of expression of major histocompatibility complex (MHC) class II in trophoblast tissues is believed to be one of the escape mechanisms of the fetus from maternal immune system. Since an exaggerate systemic inflammatory response is known to be a feature of pre-eclampsia (PET), aim of this study was to investigate the possible abnormal expression of MHC class II molecules, namely HLA-DR, in circulating syncytiotrophoblast microparticles (STBMs) from preeclamptic patients. Methods: STBMs obtained by dual placental perfusion after caesarean section from 9 women with early onset ( 34 weeks of gestation) PET (EOPET), 7 women with late onset (>34 weeks of gestation) PET (LOPET) and 12 women with uncomplicated pregnancies were analysed for HLADR and the syncytiotrophoblast-specific placental alkaline phosphatase (PLAP) expression by flow cytometry. Results: STBMs from women with EOPET showed a significant expression of HLA-DR coupled with PLAP compared to controls (P1⁄40.03). No significant difference was found between either controls and LOPET (P1⁄40.24) or EOPET and LOPET (P1⁄40.35) (Figure 1). Figure 1. Percentage of STBMs double positivity for HLA-DR and PLAP obtained from uncomplicated pregnancies (controls), women with EOPET or LOPET. A significant aberrant expression of HLA-DR was found in EOPET women compared to controls (*P1⁄40.03). Conclusion: To our knowledge, this is the first observation of an aberrant expression of HLA-DR in STBMs from women with EOPET. This finding might lead the way to the identification of a novel immunological mechanism representing a co-cause or a consequence of the exaggerate pro-inflammatory status that is a feature of PET. More studies are needed to confirm this observation in a larger cohort of patients and to define the possible functional role of aberrant HLA-DR expressed on STBMs spread in maternal circulation in PET. NI3.4. AN INTEGRATED TRANSCRIPTIONAL, EPIGENETIC, AND CLINICAL ANALYSIS OF PREECLAMPTIC PLACENTAS Katherine Leavey , Samantha Wilson , Shannon Bainbridge , Wendy Robinson , Brian Cox . University of Toronto, Toronto, Ontario, Canada; University of British Columbia, Vancouver, British Columbia, Canada; University of Ottawa, Ottawa, Ontario, Canada Objectives: Preeclampsia (PE) is a complex, multi-system disorder of pregnancy, demonstrating a high degree of variability in observed maternal symptoms and fetal outcomes. We hypothesize that this heterogeneity, leading to a lack of robust predictive biomarkers and effective treatments for this disorder, is due to the existence of multiple molecular forms of PE. While transcriptional analysis of placenta samples can group and classify patients, the integration of epigenetic information should reveal gene regulatory mechanisms behind the pathology. Methods: To address our hypothesis, microarray gene expression data was collected for 330 placentas, including 157 highly annotated samples drawn from the RCWIH BioBank. This combined data set was then subjected to unsupervised model-based clustering and correlative analysis with the available clinical covariates. Additionally, 48 BioBank samples were further assessed by methylation arrays, and genes differentially methylated and/or expressed between clusters were investigated by pathway enrichment analysis. Results: Clustering of the transcriptional data revealed four subclasses of PE samples, including a group with relatively healthy placentas, normal birth weights, and term deliveries, indicating a possible maternal origin of the pathology. An identified “canonical” PE subclass, with clinical presentation of low placental weights and early deliveries, was associated with hypo-methylation and increased expression of genes related to secretion, metabolism, and response to hypoxia, including known PE markers ENG, INHA, and LEP. The third PE group demonstrated severe fetal growth restriction and hypo-methylation of genes involved in immune response, while the fourth subclass formed due to chromosomal abnormalities, confirmed by aCGH analysis, leading to cohesive changes in gene expression but not in methylation status. Conclusion: Overall, our integrated molecular and clinical analysis of preeclamptic placentas has identified multiple subtypes of this disorder, fitting with our hypothesis. Hopefully, this research will eventually lead to the development of robust biomarkers and personalized treatments for all PE subclasses. NI3.5. INTEGRATIVE ANALYSIS OF PLACENTAL TRANSCRIPTOME ORGANIZATION REVEALS HIGHLY CONSERVED REGULATORY PROGRAMS AND POINTS TOWARD A PREECLAMPSIA GENE CLUSTER Sam Buckberry , Tina Bianco-Miotto , Stephen Bent , Gustaaf Dekker , Claire Roberts . 1 The Robinson Research Institute, The University of Adelaide, SA, Australia; 2 School of Agriculture, Food & Wine, The University of Adelaide, SA, Australia; 3 Lyell McEwin Hospital, SA,

Acetaminophen application during murine pregnancy impairs pathways of maternal adaptation to pregnancy

the escape mechanisms of the semiallogeneic fetus from maternal immune system recognition and rejection. Since an exaggerate systemic inflammatory response is known to be a feature of pre-eclampsia (PET), aim of this study was to investigate the possible abnormal expression of MHC class II molecules, namely HLA-DR, in circulating syncytiotrophoblast microparticles (STBMs) from pre-eclamptic patients. Methods: STBMs obtained by dual placental perfusion after caesarean section from 9 women with early onset (≤34 weeks of gestation) PET (EOPET), 7 women with late onset (>34 weeks of gestation) PET (LOPET) and 12 women with uncomplicated pregnancies were analysed for HLA-DR and the syncytiotrophoblastspecific placental alkaline phosphatase (PLAP) expression by flow cytometry. Results: STBMs from women with EOPET showed a significant expression of HLA-DR coupled with PLAP compared to controls (P=0.03). No significant difference was found between either controls and LOPET (P=0.24) or EOPET and LOPET (P=0.35) (Fig. 1). Conclusion: To our knowledge, this is the first observation of an aberrant expression of HLA-DR in STBMs fromwomenwith EOPET. This finding might lead the way to the identification of a novel immunological mechanism representing a co-cause or a consequence of the exaggerate pro-inflammatory status that is a feature of PET. More studies are needed to confirm this observation in a larger cohort of patients and to define the possible functional role of aberrant HLA-DR expressed on STBMs spread in maternal circulation in PET.

HLA class II is aberrantly expressed on circulating syncytiotrophoblast microparticles in early onset preeclampsia

HLA class II is aberrantly expressed on circulating syncytiotrophoblast microparticles in early onset preeclampsia

Hypoxia changes the coagulation capacities of syncytiotrophoblast microparticles (STBM)

Hypoxia changes the coagulation capacities of syncytiotrophoblast microparticles (STBM)

Placenta-derived exosomes and syncytiotrophoblast microparticles and their role in human reproduction: immune modulation for pregnancy success.

The syncytiotrophoblast (STB) of human placenta constitutively produces and secretes extracellular vesicles of different size, morphology and function that enter the maternal circulation, and participate in the maternal-fetal cross-talk during pregnancy. Syncytiotrophoblast-derived microvesicles/microparticles (STBM) are larger microvesicles (0.2-2 μm) shed by the apical plasma membrane of the STB as a result of cell activation and turnover. Simultaneously with the STBM shedding, the STB produces and secretes exosomes--nanosized (30-100/150 nm) membrane-bound microvesicles that originate from the endosomal compartment. They convey cell-cell contact 'by proxy' transporting signals/packages of information between donor and recipient cells locally or/and at a distance. STBM and exosomes, delivered directly in the maternal blood surrounding the chorionic villi of the placenta, have contrasting biological functions. While the exosomes are immunosuppressive down regulating maternal immunity in pluripotent ways, the main effects of STBM on the maternal immune system are pro-inflammatory, immune activating, and pro-coagulant. Since both STBM and exosomes are present in the maternal circulation throughout normal pregnancy logical questions are what is the net effect of these vesicles on the maternal immune system and is this effect beneficial or detrimental to pregnancy. In this review, the current knowledge about placenta-derived extracellular vesicles with a main focus on exosomes is summarized and discussed. In a concluding remark, a hypothetical proposal on how STBM and exosomes might interact in pregnancy is discussed and a way to evaluate this interaction is suggested.

Extracellular Nucleic Acids in Maternal Circulation as Potential Biomarkers for Placental Insufficiency

Since the placenta is being continuously remodeled during normal placental development, extracellular nucleic acids of both fetal and placental origin, packed into either trophoblast-derived apoptotic bodies or shedding syncytiotrophoblast microparticles, may be detected in maternal circulation during the course of normal gestation. Placental-insufficiency-related pregnancy complications have been shown to be associated with excessive placental trophoblast apoptosis and shedding of placenta debris. Recent advances in the field are reviewed with a focus on the diagnostic potential of particular molecular biomarkers and their eventual implementation in the currently used predictive and diagnostic algorithms for placental-insufficiency-related pregnancy complications.

The Functions of Microparticles in Pre-Eclampsia

Pre-eclampsia (P-EC), a heterogenic multisystem disorder characterized by hypertension and proteinuria, usually develops in the second half of pregnancy. The incidence is 2 to 5%, and P-EC is therefore a major cause of maternal and perinatal morbidity and mortality. Although the exact etiology is unknown, placental factors released into the maternal circulation lead to systemic maternal inflammation and endothelial dysfunction. Growing evidence indicates that placenta-derived microparticles, best known as syncytiotrophoblast microparticles (STBM), are important among these factors. This review provides an overview of the presence and function(s) of STBM and other cell-derived microparticles and exosomes.

Placental Microparticles, DNA, and RNA in Preeclampsia

Preeclampsia is a common disorder of the second half of pregnancy that complicates 2% to 7% of all pregnancies worldwide and remains a major cause of maternal and fetal morbidity and mortality. Although the origin of the disease is still elusive, population-based studies have suggested that it might implicate genetic, immunologic, or physiologic factors. On the other hand, there is no doubt that the placenta plays an important role in its development. In preeclampsia, the shedding of placenta debris, such as syncytiotrophoblast microparticles (STBMs) and DNA and messenger RNA molecules, into the maternal peripheral blood is increased. The analysis of this material may give new insight into placentation and the underlying etiology of this disorder, as well as yield new tracks of research for the understanding of the molecular mechanisms, leading to the generation of the clinical symptoms.

Protein composition of microparticles shed from human placenta during placental perfusion: Potential role in angiogenesis and fibrinolysis in preeclampsia

Shedding of syncytiotrophoblast microparticles (MPs) from placenta to maternal blood occurs in normal pregnancy and is enhanced during preeclampsia (PE). The syncytiotrophoblast synthesizes plasminogen activator inhibitors (PAIs) which regulate fibrinolysis, as well as soluble forms of the fms-like tyrosine kinase (sFlt-1) and endoglin, which exert anti-angiogenic actions. An increase in the ratio of PAI-1/PAI-2 and elevated levels of sFlt-1 and sEng in maternal serum are linked to placental damage and maternal endothelial cell dysfunction in PE. The goal of the current study was to determine whether MPs released to maternal perfusate during dual perfusion contain these factors associated with placental pathophysiology in PE. Initially, high levels of alkaline phosphatase activity and Annexin V binding were found in MPs isolated by sequential centrifugation of maternal perfusates at 10,000 and 150,000×g(10 K and 150 K MPs), indicating their plasma membrane origin. ELISA revealed the presence of these factors at the following relative levels: Eng>PAI-2⋙PAI-1>sFlt-1. Based on comparisons of their concentration in perfusates, MPs, and MP-free 150 K supernatants, we determined that MPs constitute a significant portion of Eng released by placenta. Flow cytometric analysis of 10 K MPs supported the levels of expression found by ELISA and indicated that Eng and PAI-2 were almost exclusively localized to the surface of MPs, a site with biological potential. These results indicate that MPs shed from the syncytial surface express factors which may alter the fibrinolytic and angiogenic balance at the maternal–fetal interface and play a role in the pathophysiology of PE.

Study on apoptosis of placental syncytiotrophoblast in patients with severe preeclampsia

to investigate apoptosis of placental syncytiotrophoblast in patients with early-onset and late-onset severe preeclampsia. from November 2008 to May 2009, 15 cases with early-onset severe preeclampsia and 15 cases with late-onset severe preeclampsia matched with 10 women with normal pregnancies as control at Sixth Affiliated Hospital of Shanghai Jiaotong University were enrolled in this study. Enzyme-linked immunosorbent assay was used to measure syncytiotrophoblast microparticles (STBM) levels in peripheral venous plasma. Caspase-3 were measured by western blotting. (1) STBM: the level of STBM of (71 ± 21) microg/L in early-onset group were significantly higher than (42 ± 30) microg/L in late-onset group and (26 ± 11) microg/L in control group (P < 0.05). The level of STBM in late-onset group and control group did not reach statistical difference (P > 0.05). (2) Caspase-3: the level of caspase-3 protein were 0.85 ± 0.61 in early-onset group and 0.77 ± 0.46 in late-onset group, which were all significantly higher than 0.32 ± 0.15 in control group (P < 0.05). However, the level of caspase-3 protein in early-onset group did not show remarkable difference compared with that in late-onset group (P > 0.05). STBM are shed into the maternal circulation in higher amounts in early-onset preeclampsia, which indicated that early-onset and late-onset severe preeclampsia may have different etiology and pathogenesis.

Flow cytometric analysis of circulating microparticles in plasma

Microparticles, which include exosomes, micro-vesicles, apoptotic bodies and apoptotic microparticles, are small (0.05 - 3 mum in diameter), membranous vesicles that can contain DNA, RNA, miRNA, intracellular proteins and express extracellular surface markers from the parental cells. They can be secreted from intracellular multivesicular bodies or released from the surface of blebbing membranes. Circulating microparticles are abundant in the plasma of normal individuals and can be derived from circulating blood cells such as platelets, red blood cells and leukocytes as well as from tissue sources, such as endothelial and placental tissues. Elevated levels of microparticles are associated with various diseases such as thrombosis (platelet microparticles), congestive heart failure (endothelial microparticles), breast cancer patients (leukocyte microparticles) and women with preeclampsia (syncytiotrophoblast microparticles). Although microparticles can be detected by microscopy, enzyme-linked immunoassays and functional assays, flow cytometry is the preferred method because of the ability to quantitate (fluorescent bead- or flow rate-based method) and because of polychromatic capabilities. However, standardization of pre-analytical and analytical modus operandi for isolating, enumerating and fluorescent labeling of microparticles remains a challenge. The primary focus of this article is to review the preliminary steps required to optimally study circulating in vivo microparticles which include: 1) centrifugation speed used, 2) quantitation of microparticles before antibody labeling, 3) levels of fluorescence intensity of antibody-labeled microparticles, 4) polychromatic flow cytometric analysis of microparticle sub-populations and 5) use of polyclonal antibodies designed for Western blotting for flow cytometry. These studies determine a roadmap to develop microparticles as biomarkers for a variety of conditions.

Prevention of Preeclampsia: Is it Still a Disappointment?

Preeclampsia is a major cause of maternal mortality worldwide, with many preventive strategies tested. In this review we intend to provide a synthesis of available studies of these strategies that have been tested, including systematic reviews. We will not be performing systematic review of the studies here. Of these strategies tested only low dose acetyl salicylic acid (ASA) and calcium can be considered helpful for prevention at this time. A recent meta-analysis showed a benefit of low dose ASA for both high (RR 0.75, 95% CI 0.66 to 0.85) and moderate risk groups (RR 0.86, 95% CI 0.79 to 0.95). Therefore, low dose ASA in high risk groups may be useful, with a possible smaller benefit in moderate to low risk women. Another meta-analysis looking at calcium shows benefit for both high and low risk groups with an overall reduction in the risk of preeclampsia (RR 0.48, 95% CI 0.33 to 0.69). As a result, prenatal supplementation of calcium may be beneficial for the prevention of preeclampsia. However, vitamins C and E, zinc, fish oil, and magnesium supplementation have been discounted as potential preventive strategies. Nitric oxide, folic acid, and antithrombotics have not been well studied and there is insufficient data for reliable conclusions to be made. Areas of ongoing research that appear promising in the prevention of preeclampsia include modifiable metabolic factors, angiogenic proteins, angiotensin receptor antibodies, and syncytiotrophoblast microparticles. Strategies targeting these areas may provide opportunities for therapeutic interventions.

The Effect of Labour and Placental Separation on the Shedding of Syncytiotrophoblast Microparticles, Cell-free DNA and mRNA in Normal Pregnancy and Pre-eclampsia

The clinical features of the maternal syndrome of pre-eclampsia can be explained by generalised maternal endothelial cell dysfunction, which is a part of a more global maternal systemic inflammatory response. There is growing evidence that these effects are associated with the shedding of cellular debris, including syncytiotrophoblast microparticles (STBM), cell-free DNA and mRNA, from the surface of the placenta (syncytiotrophoblast) into the maternal circulation. The increased shedding of this debris seen in pre-eclampsia is believed to be caused by placental ischaemia, reperfusion and oxidative stress. This study was carried out to determine whether uterine contractions during labour and subsequent placental separation lead to an acute increase in the release of placental debris into the maternal circulation. To assess the effects of labour, samples were taken from 10 normal pregnant (NP) and 10 pre-eclamptic (PE) women at varied time points. Similarly to assess the effects of placental delivery, plasma samples were taken from 10 NP and 10 PE women undergoing elective caesarean section. There was a significant increase in the shedding of STBM in pre-eclampsia which was not seen in normal pregnancy and there was a small rise in STBM levels at placental separation in both normal pregnant and pre-eclamptic women undergoing caesarean section, but the differences were not significant. However, levels of placental cell-free corticotrophin releasing hormone mRNA were significantly increased in labour in both normal pregnancy and pre-eclampsia and were still high 24 h after delivery in the pre-eclamptic women. There was no significant increase in fetal or total DNA in labour, but the overall levels of total DNA (maternal and fetal) was increased in labour in pre-eclampsia compared to normal labour. The enhanced shedding of STBM and CRH mRNA in pre-eclampsia labour may have a role in cases of postpartum worsening of pre-eclampsia.

Phenotype and mRNA Expression of Syncytiotrophoblast Microparticles Isolated from Human Placenta

Fetal and neonatal alloimmune thromboctyopenia due to maternal human platelet antigen (HPA)-1a antibodies affects primigravidas. Immunization must occur early in pregnancy before fetal platelets enter maternal blood via fetomaternal hemorrhage. The HPA-1a antigen is located on platelet glycoprotein (GP)IIIa (CD61, beta3 integrin), which is also present on the placental syncytiotrophoblast (ST) and in direct contact with maternal blood. Since ST debris is shed into maternal blood during pregnancy, this material might be immunogenic in vivo. For experimental purposes, we prepared and characterized ST microparticles (STMPs) in vitro from term placentas. Phenotype analysis by flow cytometry and Western blotting showed that STMP expressed more placental alkaline phosphatase (PLAP) than GPIIIa. Quantitative real-time PCR demonstrated expression of human placental lactogen (HPL), human chorionic gonadotrophin (HCG), and GPIIIa by STMP, in the order HPL > HCG > GPIIIa. PLAP, HPL, and HCG are trophoblast-specific proteins. These STMPs may be a useful model for studying the natural ST debris in plasma of pregnant women.

Vitamins C and E Inhibit Apoptosis of Cultured Human Term Placenta Trophoblast

Preeclampsia can be lethal to both mother and baby. The prominent symptoms of this syndrome are hypertension, proteinuria and oedema, resulting from an exaggerated aseptic systemic inflammatory response, triggered by placental factors shed into the maternal circulation. Syncytiotrophoblast microparticles (STBM) are one possible factor, shed when the placenta is exposed to stressors such as hypoxia/reperfusion. These can disrupt mitochondria, triggering apoptosis and necrosis, placental pathologies which are increased in preeclampsia. We tested the effects of antioxidant vitamins C (50 μM) and E (50 μM) on trophoblast in culture, using term villous cytotrophoblast preparations. Following Percoll gradient centrifugation and MHC class I expressing cell depletion of placenta digests, syncytial fragments were removed using anti-placental alkaline phosphatase antibody. This yielded cytotrophoblasts of consistently high purity. EGF (10 ng/ml) stimulated syncytialisation and hCG and progesterone production. However, mitochondrial induced apoptosis (MIA) was evident 96 h post-isolation, as mitochondrial membrane potential loss and caspase 9 and caspase 3 activation. ROCK-1 cleavage and syncytiotrophoblast particle shedding increased concurrently with apoptosis induction. Vitamins blocked MIA and syncytiotrophoblast particle shedding and significantly increased hCG ( p < 0.005) and progesterone ( p < 0.02) concentrations in culture supernatants, reflecting the increased survival rates. Although more cells survived in culture, syncytialisation rate (%) was significantly reduced ( p < 0.005). We conclude that vitamins C and E can significantly reduce mitochondrial damage generated following syncytialisation in vitro. However, further work is required to determine whether antioxidant vitamins interfere with normal fusion processes.

204. Proteomic analysis of the effluent from perfused placental cotyledons identifies proteins associated with pre-eclampsia

Pre-eclampsia (PE) is proposed to involve maternal systemic endothelial cell dysfunction which may be caused by excessive shedding of syncytiotrophoblast microparticles (STBM) from the placenta into the maternal circulation. We have examined STBM harvested by perfusion of placental cotyledons from PE and gestation-matched control (GMC) pregnancies. Following proteomic analysis, the proteins Calreticulin, Chloride intracellular channel 3 (CLIC3), Valosin-containing protein (VCP), Protein disulfide-isomerase A3 (ERp57) and Protein kinase C inhibitor protein 1 (14–3-3) were identified as having increased expression in PE samples. This result suggests that there may be increased rate of release of these proteins from the placenta into the maternal circulation. The aims of this work are to measure the expression of these proteins in placental extracts and maternal plasma, and to compare expression between PE and GMC using western blots and/or ELISA with antibodies specific to each protein. Placental expression was confirmed for all five proteins, however, CLIC3 was the only protein significantly increased in the placenta in PE (17.858µg+1.62, n = 27, t-test P &lt; 0.05) compared with GMC (10.478µg+0.76, n = 25). western blot determined there was a significant increase in calreticulin expression in plasma from PE women compared with GMC (as shown by Gu et al. 2008). Likewise, VCP was also increased in PE plasma (1810+280 density/0.25µl, n = 10) compared with GMC (884+260, n = 9). In contrast, the protein 14–3-3 was decreased in PE plasma (4 out of 27 PE plasma samples tested expressed 14–3-3) compared with GMC (10 out of 27 control plasma samples expressed 14–3-3). One of the western blots determined that there was a significant decrease in 14–3-3 expression in plasma from PE women (199.24+186 density/2.5µl, n = 10, un-paired t-test P &lt; 0.05) compared with GMC (2174.91 ± 723.29, n = 11). Neither ERp57 nor CLIC3 was detected in maternal plasma. Those proteins found to be significantly different in PE compared with GMC may be involved in the pathophysiology of PE. These results demonstrate that proteomic analysis of the effluent from perfused placental cotyledons is a reliable screening method to successfully identify proteins that have altered expression with PE. (1) Gu et al. Mole. Human Repro. April 16 2008