Trophoblast Glycoprotein (TPGB/5T4) in Human Placenta: Expression, Regulation, and Presence in Extracellular Microvesicles and Exosomes.

BACKGROUND: Many parallels exist between growth and development of the placenta and that of cancer. One parallel is shared expression of antigens that may have functional importance and may be recognized by the immune system. Here, we characterize expression and regulation of one such antigen, Trophoblast glycoprotein (TPGB; also called 5T4), in the placenta across gestation, in placentas of preeclamptic (PE) pregnancies, and in purified microvesicles and exosomes. METHODS: Trophoblast glycoprotein expression was analyzed by real-time reverse transcription-polymerase chain reaction (RT-PCR), Western blot, and immunohistochemistry. Regulation of 5T4 in cytotrophoblast cells was examined under either differentiating conditions of epidermal growth factor or under varying oxygen conditions. Microvesicles and exosomes were purified from supernatant of cultured and perfused placentas. RESULTS: Trophoblast glycoprotein expression was prominent at the microvillus surface of syncytiotrophoblast and on the extravillous trophoblast cells, with minimal expression in undifferentiated cytotrophoblasts and normal tissues. Trophoblast glycoprotein expression was elevated in malignant tumors. In cytotrophoblasts, 5T4 was induced by in vitro differentiation, and its messenger RNA (mRNA) was increased under conditions of low oxygen. PE placentas expressed higher 5T4 mRNA than matched control placentas. Trophoblast glycoprotein was prominent within shed placental microvesicles and exosomes. CONCLUSION: Given the potential functional and known immunological importance of 5T4 in cancer, these studies reveal a class of proteins that may influence placental development and/or sensitize the maternal immune system. In extravillous trophoblasts, 5T4 may function in epithelial-to-mesenchymal transition during placentation. The role of syncytiotrophoblast 5T4 is unknown, but its abundance in shed syncytial vesicles may signify route of sensitization of the maternal immune system.

Isolation of syncytiotrophoblast microvesicles and exosomes and their characterisation by multicolour flow cytometry and fluorescence Nanoparticle Tracking Analysis.

The human placenta releases multiple types and sizes of syncytiotrophoblast (STB) extracellular vesicles (EV) into the maternal circulation that exhibit diverse biological activities. The placental perfusion technique enables isolation of these STBEV, but conventional flow cytometry can only be used to phenotype EV down to ∼300 nm in size. Fluorescence Nanoparticle Tracking Analysis (fl-NTA) has the potential to phenotype EV down to ∼50 nm, thereby improving current characterisation techniques. The aims of this study were to prepare microvesicle and exosome enriched fractions from human placental perfusate (n=8) and improve fl-NTA STBEV detection. Differential centrifugation and filtration effectively removed contaminating red blood cells from fresh placental perfusates and pelleted a STB microvesicle (STBMV) fraction (10,000×g pellet - 10KP; NTA modal size 395±12 nm), enriched for the STB marker placental alkaline phosphatase (PLAP) and a STB exosome (STBEX) fraction (150,000×g pellet - 150KP; NTA modal size 147±6 nm), enriched for PLAP and exosome markers Alix and CD63. The PLAP positivity of 'standard' 10KP and 150KP pools (four samples/pool), determined by immunobead depletion, was used to optimise fl-NTA camera settings. Individual 10KP and 150KP samples (n=8) were 54.5±5.7% (range 17.8-66.9%) and 30.6±5.6% (range 3.3-51.7%) PLAP positive, respectively. We have developed a reliable method for enriching STBMV and STBEX from placental perfusate. We also standardised fl-NTA settings and improved measurement of PLAP positive EV in STBMV. However, fl-NTA is not as sensitive as anti-PLAP Dynabead capture for STBEX detection, possibly due to STBEX having lower surface expression of PLAP. These important developments will facilitate more detailed studies of the role of STBMV and STBEX in normal and pathological pregnancies.

Interleukin-1 family cytokines and their regulatory proteins in normal pregnancy and pre-eclampsia.

Maternal systemic inflammation is a feature of pre-eclampsia, a condition in pregnancy characterized by hypertension and proteinuria. Pre-eclampsia is caused by the placenta; many placental factors contribute to the syndrome's progression, and proinflammatory cytokines have been identified previously as one such mediator. The interleukin (IL)-1 family of cytokines are key regulators of the inflammatory network, and two naturally occurring regulatory molecules for IL-1 family cytokines, IL-1RA and sST2, have been found previously to be elevated in maternal blood from women with pre-eclampsia. Here we investigate more recently identified IL-1 family cytokines and regulatory molecules, IL-1RAcP, IL-37, IL-18BP, IL-36α/ß/γ/Ra and IL-38 in pre-eclampsia. Pregnant women have more circulating IL-18BP and IL-36Ra than non-pregnant women, and sIL-1RAcP is elevated from women with pre-eclampsia compared to normal pregnancies. The placenta expresses all the molecules, and IL-37 and IL-18BP are up-regulated significantly in pre-eclampsia placentas compared to those from normal pregnancies. Together, these changes contribute to the required inhibition of maternal systemic cytotoxic immunity in normal pregnancy; however, in pre-eclampsia the same profile is not seen. Interestingly, the increased circulating levels of sIL-1RAcP and increased placental IL-18BP and IL-37, the latter of which we show to be induced by hypoxic damage to the placenta, are all factors which are anti-inflammatory. While the placenta is often held responsible for the damage and clinical symptoms of pre-eclampsia by the research community, here we show that the pre-eclampsia placenta is also trying to prevent inflammatory damage to the mother.

Particle size distribution of exosomes and microvesicles determined by transmission electron microscopy, flow cytometry, nanoparticle tracking analysis, and resistive pulse sensing.

BACKGROUND: Enumeration of extracellular vesicles has clinical potential as a biomarker for disease. In biological samples, the smallest and largest vesicles typically differ 25-fold in size, 300,000-fold in concentration, 20,000-fold in volume, and 10,000,000-fold in scattered light. Because of this heterogeneity, the currently employed techniques detect concentrations ranging from 10(4) to 10(12) vesicles mL(-1) . OBJECTIVES: To investigate whether the large variation in the detected concentration of vesicles is caused by the minimum detectable vesicle size of five widely used techniques. METHODS: The size and concentration of vesicles and reference beads were measured with transmission electron microscopy (TEM), a conventional flow cytometer, a flow cytometer dedicated to detecting submicrometer particles, nanoparticle tracking analysis (NTA), and resistive pulse sensing (RPS). RESULTS: Each technique gave a different size distribution and a different concentration for the same vesicle sample. CONCLUSION: Differences between the detected vesicle concentrations are primarily caused by differences between the minimum detectable vesicle sizes. The minimum detectable vesicle sizes were 70-90 nm for NTA, 70-100 nm for RPS, 150-190 nm for dedicated flow cytometry, and 270-600 nm for conventional flow cytometry. TEM could detect the smallest vesicles present, albeit after adhesion on a surface. Dedicated flow cytometry was most accurate in determining the size of reference beads, but is expected to be less accurate on vesicles, owing to heterogeneity of the refractive index of vesicles. Nevertheless, dedicated flow cytometry is relatively fast and allows multiplex fluorescence detection, making it most applicable to clinical research.

IFPA Senior Award Lecture: making sense of pre-eclampsia - two placental causes of preeclampsia?

Incomplete spiral artery remodelling is the first of two stages of pre-eclampsia, typically of early onset. The second stage comprises dysregulated uteroplacental perfusion and placental oxidative stress. Oxidatively stressed syncytiotrophoblast (STB) over-secretes proteins that perturb maternal angiogenic balance and are considered to be pre-eclampsia biomarkers. We propose that, in addition and more fundamentally, these STB-derived proteins are biomarkers of a cellular (STB) stress response, which typically involves up-regulation of some proteins and down-regulation of others (positive and negative stress proteins respectively). Soluble vascular growth factor receptor-1 (sVEGFR-1) and reduced growth factor (PlGF) then exemplify positive and negative STB stress response proteins in the maternal circulation. Uncomplicated term pregnancy is associated with increasing sVEGFR-1 and decreasing PlGF, which can be interpreted as evidence of increasing STB stress. STB pathology, at or after term (for example focal STB necrosis) demonstrates this stress, with or without pre-eclampsia. We review the evidence that when placental growth reaches its limits at term, terminal villi become over-crowded with diminished intervillous pore size impeding intervillous perfusion with increasing intervillous hypoxia and STB stress. This type of STB stress has no antecedent pathology, so the fetuses are well-grown, as typifies late onset pre-eclampsia, and prediction is less effective than for the early onset syndrome because STB stress is a late event. In summary, abnormal placental perfusion and STB stress contribute to the pathogenesis of early and late onset pre-eclampsia. But the former has an extrinsic cause - poor placentation, whereas the latter has an intrinsic cause, 'microvillous overcrowding', as placental growth reaches its functional limits. This model explains important features of late pre-eclampsia and raises questions of how antecedent medical risk factors such as chronic hypertension affect early and late sub-types of the syndrome. It also implies that all pregnant women may be destined to get pre-eclampsia but spontaneous or induced delivery averts this outcome in most instances.

Microparticle association and heterogeneity of tumor-derived tissue factor in plasma: is it important for coagulation activation?

BACKGROUND: Tumor-derived tissue factor (TF) activates coagulation in vitro and in vivo in an orthotopic model of human pancreatic cancer. Here, we further characterized tumor-derived TF in this model. METHODS: Conditioned medium (CM) of L3.6pl human pancreatic tumor cells and plasma from nude mice bearing L3.6pl tumors were ultracentrifuged, and the pellets were filtered through membranes with different pore sizes. The size distribution of particles was analyzed in CM or plasma fractions with nanoparticle tracking and dynamic light scattering. Human TF antigen and activity were measured in pellets and supernatants with ELISA and clotting or thrombin generation assays, respectively. Human alternatively spliced TF (asTF) was measured with ELISA. Human TF and thrombin-antithrombin complex (TAT) concentrations were assessed in plasma of mice injected with filtered fractions of CM. RESULTS: Particles in both CM and plasma were < 0.4 µm. TF antigen and activity in the CM were mainly associated with microparticles (MP). Approximately 50% of antigen and 20% of activity were associated with particles of < 0.1 µm. Injection of < 0.1 µm particles into mice caused a 30% drop in platelet counts and an increase in TAT levels. In contrast, ~ 90% of TF antigen in tumor-bearing mice plasmas was non-sedimentable, whereas TF activity was exclusively associated with MP. Particles of < 0.1 µm and the supernatants of both CM and plasma gained TF activity after addition of exogenous phospholipids. Although asTF was found in MP-free CM supernatants, it was also present in CM and plasma pellets. CONCLUSIONS: Tumor-derived particles of < 0.1 µm and non-sedimentable TF are or can become procoagulant in the presence of phospholipids, and may contribute to the procoagulant potential of circulating TF.

Review: Does size matter? Placental debris and the pathophysiology of pre-eclampsia.

A variety of 'debris' is shed from the syncytial surface of the human placenta ranging from large deported multinuclear fragments to sub-cellular components. It is increasingly clear that at least some of this material has signalling functions. Many categories of circulating debris are increased in pre-eclampsia, and exhibit proteins that are pro-inflammatory and could contribute to the systemic inflammatory response in normal pregnancy, which is exaggerated in pre-eclampsia. It is now evident that there is a large 'hidden' population of microvesicles and nanovesicles (including exosomes) which are hard to investigate because of their size. We have used a new technology, nanoparticle tracking analysis, to measure the size and concentration of syncytiotrophoblast vesicles prepared by placental perfusion. The vesicles range in size from 50 nm to 1 µm with the majority being <500 nm (which includes both exosomes and microvesicles). We speculate whether changes not only in the numbers, but also in the size (beneficial syncytiotrophoblast exosomes and harmful microvesicles) might be important in the maternal syndrome of pre-eclampsia.

Placental stress and pre-eclampsia: a revised view.

In pre-eclampsia, poor placentation causes both oxidative and endoplasmic reticulum stress of the placenta. It is believed placental hypoxia stimulates excessive production of soluble fms-like tyrosine kinase 1 (sFlt-1), which binds and deactivates circulating vascular endothelial growth factor (VEGF). When maternal endothelium is deprived of VEGF it becomes dysfunctional hence leading to the clinical syndrome of the mother. In this paper the previous claim that poor placentation may predispose more to placental oxidative stress than hypoxia is reiterated. We show why pre-eclampsia is not only an endothelial disease, but also a disorder of systemic inflammation. We question that hypoxia is the only or indeed the main stimulus to release of sFlt-1; and emphasise the role of inflammatory mechanisms. Hypoxia cannot be assumed simply because hypoxia-inducible transcription factors (HIF) are upregulated. Concurrent assessments of nuclear factor-kappaB (NF-kappaB), a transcription factor for inflammatory responses are desirable to obtain a more complete picture. We point out that the pre-eclampsia placenta is the source of bioactive circulating factors other than sFlt-1 in concentrations that are much higher than in normal pregnancy. These may also contribute to the final inflammatory syndrome. We propose a modified version of the two-stage model for pre-eclampsia.

Conditioned medium from human decidual stromal cells has a concentration-dependent effect on trophoblast cell invasion.

The regulation of trophoblast cell invasion is a crucial aspect of implantation and placental development. Evidence indicates that the uterine microenvironment exerts important influence over trophoblast cell invasion. However, the precise effect of decidual cells on trophoblast cell invasion remained unidentified. In the present study, a cell line representative of normal human trophoblast (B6Tert) was used to examine the effect of decidual stromal cell conditioned media (DSCM) on trophoblast cell invasion. In vitro assay showed the concentration-dependent effect of DSCM on B6Tert cell invasion. RT-PCR and gelatin zymography demonstrated that DSCM evidently produced an effect on the mRNA expression and proenzyme production of MMP-2 in a dose-dependent manner, but exerted no effect on mRNA expression and proenzyme production of MMP-9. The data indicates that the decidual microenvironment may exert the key control for trophoblast cell invasion mainly through influencing MMP-2 expression.

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.

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 microM) and E (50 microM) 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 96h 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.

Circulating microparticles in normal pregnancy and pre-eclampsia.

Cellular particles may be larger shed microparticles (>or=100 nm, MPs) that are the products of cell activation or necrosis. There are also smaller endocytic nanoparticles (<100 nm), called exosomes, which are internal vesicles of late endosomes or multivesicular bodies and are released into the extracellular milieu upon fusion of the multivesicular body with the cell surface. Both MPs and exosomes can be detected in the circulations of non-pregnant and pregnant women. In the former MPs are increased in conditions associated with systemic inflammation such as sepsis or metabolic syndrome. During normal pregnancy MPs are increased and they increase further with pre-eclampsia. They include not only MPs derived from platelets, endothelium and various leukocytes but also syncytiotrophoblast derived MPs (often called STBMs). STBMs interact with both immune and endothelial cells and may contribute to the systemic inflammation of both normal and pre-eclamptic pregnancies. However inhibitory activity has also been ascribed to trophoblast derived exosomes. In vitro, they down-regulate T cell activity, a T cell change that has been repeatedly observed, ex vivo, during normal pregnancy.

Microparticles and immunomodulation in pregnancy and pre-eclampsia.

Cellular microparticles are ubiquitously shed from cell membranes or secreted as endocytic vesicles called exosomes. Shed microparticles are >/=100nm in size and are generated during apoptosis or necrosis. In contrast, exosomes are smaller (<100nm), express more limited protein content and are released from late endosomes. Both membrane particles and exosomes can be detected in the circulation in non-pregnant and pregnant women. In the former, they are increased in conditions associated with systemic inflammation such as sepsis or metabolic syndrome. During pregnancy, they are also associated with pre-eclampsia and include not only particles derived from platelets, endothelium and various leukocytes but also syncytiotrophoblast-derived microparticles. Syncytiotrophoblast membrane microparticles (often called STBMs) interact with both immune and endothelial cells. They may contribute to the systemic inflammatory response of both normal and pre-eclamptic pregnancies, although inhibitory activity has also been described. Moreover, trophoblast-derived exosomes may contribute to or cause the downregulation of T cell activity that has been repeatedly observed during pregnancy. Deletion of activate T cells which express Fas ligand by Fas-expressing exosomes derived from trophoblast may contribute to immunoregulation necessary for normal pregnancy.

NK cells and pre-eclampsia.

The immunological interaction between the mother and fetus has classically been thought of as one between paternal antigen and maternal T cells. However, the MHC antigen expression on human trophoblast and the immune cell populations present in the decidua suggest that this interaction primarily involves decidual NK cells rather than T cells, and this is supported by new functional studies. It is becoming apparent also that the maternal systemic immune response in pregnancy (Th1/Th2 shift) primarily involves NK cells. Aberrant NK cell activation both locally in the decidua and systemically in the maternal blood may be the cause of pre-eclampsia.

Caveolin-1 and lipid rafts in confluent BeWo trophoblasts: evidence for Rock-1 association with caveolin-1.

Lipid rafts are detergent-insoluble, low-density membrane domains that are rich in cholesterol and sphingolipids; caveolae are a subdomain of the biochemically defined glycolipid raft whose expression is associated with the protein caveolin-1. This protein associates with numerous signalling molecules, regulating their activity by holding them inactive. Human villous cytotrophoblasts contain caveolin-1, but levels reduce greatly during their differentiation into syncytiotrophoblast. Since caveolin-1 is a known regulator of apoptosis and trophoblast syncytialisation involves the apoptotic cascade, we hypothesised that cytotrophoblast caveolin-1 may also play a role in regulating fusion events involved in syncytium formation. The BeWo choriocarcinoma cell line has previously proved valuable for studying trophoblast syncytialisation, hence the present work was carried out to determine whether BeWo cells could be used as a model for the exploration of caveolin-1's role in regulating the syncytialisation process. Undifferentiated BeWo cells were found to express caveolin-1 in similar amounts to villous cytotrophoblasts isolated from term placenta. Lipid raft fractions prepared from these BeWo cells at confluence contained the raft-associated proteins caveolin-1 and -2, flotillin-1 and -2, stomatin and the heterotrimeric G protein, Galphaq. Confocal immunofluorescence studies revealed that caveolin-1 is internalized to the mitochondria, but not to the Golgi or endoplasmic reticulum, in subconfluent BeWo and that the protein relocates to the plasma membrane upon confluence, an observation confirmed by caveolin-1 and cytochrome c Western blotting of lipid raft fractions and mitochondria purified from confluent and subconfluent cells. Western blotting and immunofluorescence experiments comparing undifferentiated cells and those induced to differentiate using the cAMP analogue, dibutyryl cAMP, showed that BeWo syncytialisation was accompanied by a reduction in caveolin-1 levels, similar to the situation in primary villous cytotrophoblasts. Confluent, undifferentiated BeWo cultures were then used to investigate the cellular localisation of Rock-1, a protein which promotes cytoskeletal re-organisation important for syncytialisation and apoptosis. Its association with caveolin-1 was evidenced by the demonstration that the 160kDa proenzyme form of Rock-1 co-immunoprecipitates with caveolin-1 and vice versa, as well as by the co-localisation of the two proteins at the plasma membrane, as shown in immunofluorescence studies. A proportion of the total cell Rock-1 content was found in BeWo lipid raft fractions, confirming its membrane presence in confluent cells. This close association of plasmalemmal caveolin-1 with Rock-1 protein raises the possibility that caveolin-1 may regulate Rock-1 in these trophoblasts. We conclude that cell-cell contact is required for BeWo trophoblast to exhibit plasmalemmal caveolin-1; BeWo cells at confluence offer a useful model for the study of trophoblast raft behaviour during syncytialisation and for the exploration of the potential Rock-1-regulating role of caveolin-1 in this process.

Inflammation and pre-eclampsia.

Pre-eclampsia is a common and potentially dangerous disorder of human pregnancy. The maternal syndrome of hypertension, proteinuria and oedema is part of a severe systemic inflammatory response that includes leukocyte and endothelial cell activation. Although the origins of pre-eclampsia remain unclear, a major cause is the failure to develop an adequate blood supply to the placenta, leading to placental oxidative stress. This results in the excess release of placental factors, such as syncytiotrophoblast debris or soluble fms-like tyrosine kinase-1 (sFlt-1), the soluble receptor for vascular endothelial growth factor (VEGF), into the maternal circulation, where they trigger an inflammatory response and endothelial dysfunction. Alternatively, pre-eclampsia can develop in the presence of a normal placenta in women that are susceptible to systemic inflammation, such as with chronic cardiovascular disease or diabetes. While clinical management of pre-eclampsia does not currently include anti-inflammatory agents, current research is focusing on ways to reduce inflammation and oxidative stress.

Excess syncytiotrophoblast microparticle shedding is a feature of early-onset pre-eclampsia, but not normotensive intrauterine growth restriction.

RATIONALE: Syncytiotrophoblast microparticles (STBM) are shed into the maternal circulation in higher amounts in pre-eclampsia compared to normal pregnancy and are believed to be the stimulus for the systemic inflammatory response and endothelial cell damage which characterises the maternal syndrome. The excess shedding of STBM may be caused by hypoxia as a result of poor placentation, which is often a feature of pre-eclampsia. Similar placental pathology occurs in some cases of normotensive intrauterine growth restriction (nIUGR), but in the absence of maternal disease. OBJECTIVE: To examine whether the shedding of STBM in nIUGR occurs to the same extent as in pre-eclampsia. METHODS: A prospective case-control study in a tertiary referral centre of: 1) women with early-onset pre-eclampsia (EOPET < 34 week), 2) women with late-onset pre-eclampsia (LOPET > or = 34 week), 3) women with nIUGR), 4) matched normal pregnant women (NPC), and 5) non-pregnant women. An ELISA using the antitrophoblast antibody NDOG2 was used to measure STBM levels in peripheral venous plasma. Non-parametric analyses were conducted with statistical significance set at p < 0.05. RESULTS: STBM levels rise during normal pregnancy. EOPET was associated with increased STBM levels (EOPET (median): 41 ng/ml, n = 15) compared with matched normal pregnancy (16 ng/ml, n = 15; Wilcoxon p = 0.005). LOPET (50 ng/ml, n = 10) and nIUGR (18 ng/ml, n = 8) STBM levels did not differ from matched normal pregnancy (36 ng/ml, n = 15, and 36 ng/ml, n = 8, respectively). Background levels in non-pregnant plasma were 0.49 ng/ml, n = 10. CONCLUSIONS: Increased STBM levels were found in EOPET but not in nIUGR providing further evidence for their role in the pathogenesis of the maternal syndrome.

Does 'soluble' HLA-G really exist? Another twist to the tale.

HLA-G is thought to play a key role in implantation by modulating cytokine secretion to control trophoblast invasion and to maintain a local immunosuppressive state. It differs from other class I molecules in that the gene can be alternatively spliced to produce four membrane-bound (G1, G2, G3 and G4) and three soluble isoforms (G5, G6 and G7). The soluble isoforms have recently attracted attention as their levels may be diagnostic of poor trophoblast invasion in miscarriage or pre-eclampsia and the implantation potential of IVF embryos. Although the expression and function of HLA-G2, G3, G4 and G7 has previously been a matter of debate, until now it has been generally accepted that soluble HLA-G5 and HLA-G6 are both expressed and secreted by trophoblast. However, Blaschitz et al. (2005) have reinvestigated this question and come to the surprising conclusion that they are not. They have shown that trophoblast only expresses the membrane-bound HLA-G1 isoform and not soluble HLA-G5 and -G6. Furthermore, although soluble HLA-G could be found in trophoblast culture supernatants, it appears not to be derived by alternative splicing but by the cleavage of HLA-G1. The source of the soluble HLA-G may not matter from a diagnostic perspective, but these findings, if confirmed, have important implications for our understanding of the biology of HLA-G.

The secretion and effect of inhibin A, activin A and follistatin on first-trimester trophoblasts in vitro.

OBJECTIVE: The objectives of this study were to investigate the effect of activin A and follistatin on first-trimester cytotrophoblast invasion in culture and to study the secretion of inhibin A, activin A and follistatin by these cells in vitro. DESIGN AND METHODS: Cytotrophoblasts were isolated from human placental chorionic villous tissue obtained from 6-8, 8-10 and 10-12 weeks gestation. Cells were cultured for 3 days on cell-culture inserts coated with gelatine for invasion studies and in 24-well culture plates for secretion studies. The effects of activin A (10 ng/ml), follistatin (100 ng/ml), interleukin 1beta (IL-1beta; 10 ng/ml) and epidermal growth factor (EGF; 10 ng/ml) on cytotrophoblast invasion were investigated using a non-radioactive invasion assay. Secretion of inhibin A, activin A and follistatin in the presence of EGF, IL-1beta, activin A and follistatin were measured using in-house ELISAs. RESULTS AND CONCLUSION: Activin A, follistatin and EGF had a significant stimulatory effect on cytotrophoblast invasion from 6-10 weeks gestation. IL-1beta had a significant stimulatory effect at 8-10 weeks and a significant inhibitory effect on invasion at 10-12 weeks gestation. Follistatin also had a significant inhibitory effect on invasion at 10-12 weeks gestation. In the secretion study, activin A secretion at 8-10 weeks was significantly stimulated by IL-1beta and EGF. At 10-12 weeks, follistatin and EGF had a significant inhibitory effect on activin A secretion. Follistatin secretion was significantly increased in the presence of IL-1beta at 6-8 weeks gestation. Inhibin A secretion was not significantly altered by EGF, IL-1beta, activin A and follistatin. These results show that activin A promotes invasion of first-trimester cytotrophoblasts until 10 weeks gestation. There is a difference in the control of secretion of these proteins dependent on the gestation, suggesting that there is a tight regulation in the function of first-trimester trophoblasts depending on the gestational age.

An analysis using DNA microarray of the time course of gene expression during syncytialization of a human placental cell line (BeWo).

Placental trophoblast syncytialization is a unique biological process. We have studied the time course of this process using DNA microarray in a cell model of syncytialization (the cytotrophoblast cell line BeWo following increased intracellular cAMP by forskolin). Total RNA was extracted from BeWo cells and labelled-cRNA target was then hybridized to a specific oligonucleotide probe set containing probes to over 12?000 human transcripts. Detectable levels of signal were found on average for 44 per cent of the total number of genes assayed. The correlation coefficient for the level of expression of independent replicates was #10878;0.99. The mRNA expression profile of specific genes analysed by microarray correlated quantitatively well with that analysed by reverse transcription-polymerase chain reaction and with protein secretion. In the absence of forskolin there are relatively few changes in gene expression (reaching a threshold of two fold); in the presence of forskolin there are a substantial number of changes. By clustering the patterns of altered gene expression at least ten groups could be extracted. Seven of these clusters involved increased gene expression and three decreased expression. Each cluster has been categorized by gene ontology (confining the analysis to genes with 'known' function). Among the genes with increased expression following forskolin treatment were many required for cellular communication (such as placental specific peptide hormones) and metabolism (such as cholesterol side chain cleavage enzyme). Several genes known to be involved in cell adhesion and fusion have markedly changed expression levels very early following forskolin exposure, thus preceding morphological fusion of BeWo cells. Further analysis of this data and expression profiling in general will be able to contribute to understanding the functional basis for the formation of the placental syncytiotrophoblast.