Abstract Placenta Austrália

Placenta 30 (2009) A.1A.
111
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Placenta
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Abstracts for the Forthcoming International Federation of Placenta Associations Meeting 2009
Abstract Outline - IFPA 2009 Final Keynote Symposium N1 Abstracts Symposium 1: Sex and the Placenta Symposium 2: Prediction of Adverse Pregnancy Outcome Symposium 3: IUGR, the Placenta and Programming Symposium 4: Trophoblast and Endometrial Interactions NIH Senior Researcher Award New Investigator Oral Session 1 New Investigator Oral Session 2 Trophoblast Research Award IFPA Award in Placentology Poster Abstracts (N1N6) (N7N12) (TR1) (L2L3) (P01.01P19.08) (K1K2) (S1S3) (S4S6) (S7S9) (S10S12) A.2 A.3A.4 A.4A.5 A.5A.6 A.7A.8 A.8 A.8A.11 A.11A.14 A.14 A.15 A.16A.111
doi:10.1016/j.placenta.2009.08.001
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Abstracts / Placenta 30 (2009) A.1A.111
K1. MARSUPIALS: PLACENTAL MAMMALS WITH A DIFFERENCE Marilyn B. Renfree, Department of Zoology, The University of Melbourne, Victoria 3010, Australia Viviparity is widespread in the animal kingdom and made possible by the development of a placenta. The placenta is the most varied organ within the Mammalia, but the term placental mammal (aka eutherian mammal) gives the impression that the placenta is found only in that infraclass. There are three major groups of mammals, the egg-laying monotremes and the viviparous marsupials and eutherians. Whilst the eutherian placenta is well understood, the marsupial placenta is often ignored despite the recognition of it by the two great placentologists of the last century, Harland Mossman and Emmanuel C. Amoroso. Both understood that marsupials had a fully functional placenta and emphasized the value of comparative placental physiology. There are many similarities, as well as some differences, in the marsupial embryo and its fetal membranes. In marsupials, the yolk sac forms the denitive chorio-vitelline placenta. Only very few marsupials, such as the bandicoot, have a chorio-allantoic placenta, which supplements the placental functions of the yolk sac. The yolk sac consists of two parts: a bilaminar, non-vascular region (trophoblast and yolk sac endoderm), and a trilaminar, vascular part (trophoblast, yolk sac endoderm and mesoderm). The bilaminar placenta appears to serve primarily in the uptake of uterine secretions, and the trilaminar part may be more important for gas exchange. The degree of this functional differentiation within the yolk sac placenta differs between species in the relative surface area that is attached to the endometrium, in trophoblast thickness, in yolk sac fusion with the luminal epithelium and most markedly in the degree of invasiveness. Implantation, the transition from free oating blastocyst to the implanted embryo, is a critical point in mammalian pregnancy. The extent of implantation and the degree to which the blastocyst becomes closely associated with the maternal endometrial tissues dictates the type of placenta developed. In species in which the blastocyst expands before attachment such as rabbit, dog, ferret and many marsupials, a large area of trophoblast is exposed to the uterine lumen. In the pig, horse, kangaroo and wallaby there are no such special areas and supercial attachment occurs at the unspecialized endometrial surfaces over the entire trophoblastic area. In marsupials, placental physiology has only been extensively studied in the tammar wallaby. Despite the lack of invasion, in the tammar there is nevertheless maternal recognition of pregnancy in response to trophoblast formation. Contrary to popular opinion, the tammar placenta also elaborates hormones: at term it secretes prostaglandin F2a and cortisol, only incipient amounts of progesterone and oestrogen, and it expresses the genes for growth hormone, IGF2, relaxin, prolactin and luteinizing hormone b. As in eutherian mammals, there is genomic imprinting important for placental function. Despite the relatively short gestation and short period of placentation, it is clear that the trophoblast is as important for successful pregnancy in the marsupial as it is in eutherian mammals. Marsupials are certainly placental mammals. However marsupials have an additional trick in their pouches, with the physiologically sophisticated and extended lactation that has allowed them to exchange the umbilical cord for the teat!
K2. EVOLUTION OF EPIGENETIC SILENCING IN MAMMALS Jennifer A. Marshall Graves, Research School of Biological Science, Australian National University, Canberra, Australia; Email: jenny.graves@anu.edu.au In mammals, several clusters of genes display parent-specic silencing (genomic imprinting), and one whole X chromosome is silenced in somatic tissue of females. To understand how these silencing mechanisms evolved and provide clues to how they work, we have investigated the origin and evolution of imprinting and X inactivation by examining orthologous regions in distantly related mammals (marsupials and the egg-laying monotremes). X chromosome inactivation in females is the premier example of epigenetic silencing. In placental mammals, XCI involves DNA methylation and binding with variant and modied histone, under the control of the XIST gene. The marsupial X, equivalent to the ancient region of the human and mouse X, is inactivated in marsupials, but the phenotype and molecular mechanism is very different. Silencing occurs only for the paternal X, and is tissue-specic and incomplete to different extents for different genes. It appears to be a stochastic process, in which alleles of each gene have a certain probability of expression, rather than being expressed at an intermediate level. In monotremes, as in birds, genes on the multiple X chromosomes are partially dosage compensated, again by a stochastic process. This suggests evolution from monoallelic expression of autosomal loci. The molecular mechanism of XCI appears to be quite different in marsupials. XIST is absent from the marsupial genome, no DNA methylation differences are detectable between active and inactive X chromosomes, and immunouoresence shows that although active histone marks are absent from the inactive X (as for placental mammals), inactive marks do not accumulate. This enables us to propose an evolutionary pathway for building up a complex silencing pathway. Genomic imprinting occurs in marsupials as well as placental mammals, but not in monotremes or birds. Thus the rst appearance of imprinting coincides with the evolution of viviparity and placentation. We have identied how imprinted domains (including XIST) were put together from non-imprinted components. Again, ancient mechanisms to control gene expression via epigenetic silencing may have been recruited into the complex imprinting and inactivation systems. Imprinted genes are disproportionately involved in human disease because a mutant allele has no back-up. Diseases involving maternal-fetal interactions might be particularly likely to involve imprinted genes, as I suggested 10 years ago for pre-eclampsia.
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Symposium 1: Sex and the Placenta S1. SEX AND THE HUMAN PLACENTA VL Clifton*, A Osei-Kumah, NA Hodyl, N Scott and MJ Stark, Robinson Institute, University of Adelaide, Adelaide The placenta plays a central role in the development of the fetus by modulating the supply of nutrients and oxygen throughout pregnancy. We have identied that the placenta adapts to the presence of a maternal pathophysiology in a sexually dimorphic manner which results in differences in fetal growth. In pregnancies complicated by asthma or mild preeclampsia we have reported the female fetus reduces her growth in response to these conditions which ensures her survival in the presence of another adverse event that may compromise nutrient or oxygen supply. Conversely, the male fetus continues to grow normally in the presence of maternal asthma or mild preeclampsia but is at risk of a poor outcome in the presence of a second stress. We propose that the sexually dimorphic response of the fetus is derived from differences in placental adaptation to a pathophysiological condition. In the presence of a female fetus and maternal asthma, we have observed global gene changes in the placenta accompanied by signicant alterations in microRNA expression. Downstream of these alterations we have observed differences in protein expression especially in relation to placental cytokines and the glucocorticoid receptor. In the presence of a male fetus there are fewer changes in global placental gene expression and fewer microRNA alterations and we have observed no alterations in placental cytokine expression or glucocorticoid receptor expression. These differential adaptations ensure increased survival of the female fetus and continued growth of the male fetus in adverse conditions.
S2. GLUCOCORTICOIDS AND DRUG TRANSPORTERS: FROM PLACENTA TO FETAL BRIAN Stephen G. Matthews1, Sophie Petropoulos1, William Gibb2, 1Departments of Physiology, Obstetrics and Gynecology and Medicine, University of Toronto, Canada. 2 Departments of Obstetrics and Gynecology and Cellular and Molecular Medicine, University of Ottawa, Canada The fetus may be exposed to a number of therapeutic and non-therapeutic drugs, environmental toxins and other chemicals during pregnancy. Lipohilic compounds can rapidly transfer from the maternal to the fetal compartment to affect developing organs. The fetus is protected from a number of these factors through the expression of drug transporters in the placenta and fetal organs. Multidrug-resistance P-glycoprotein (P-gp), encoded by the ABCB1 gene, and breast cancer resistance protein (BCRP), encoded by the ABCG2 gene, are ATP-dependant extrusion pumps. They have a broad spectrum of substrates that include endogenous factors / hormones (i.e. glucocorticoids), anti-cancer drugs, anti-HIV retroviral drugs as well as herbicides and pesticides. We and others have shown that the effects of these drugs on fetal development are sex-specic and that there is some sex-specicity in the transfer of these substrates from the mother to the fetus. Since one of the primary sites of teratogenesis in the fetus is the brain, we have investigated the expression and regulation of these drug transporters in the placenta and the fetal blood bran barrier, in both male and female fetuses. There are dramatic changes in the expression of these transporters over the second half of gestation in the mouse, guinea-pig and human placenta and fetal blood-brain barrier. While there are no major sex differences in the developmental prole of these drug transporters, there do appear to be sex differences in their regulation by steroids. Dening the processes that regulate the expression of P-gp and BCRP, and therefore drug transport between the mother and fetus, are important in the development of maternal and fetal therapies. Further, determining whether this regulation is specic to the sex of the fetus is critical. Supported by: Canadian Institutes of Health Research (CIHR)
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S3. SEX, DRUGS AND PROGRAMMING OF THE KIDNEY AND PLACENTA Karen Moritz, School of Biomedical Sciences, University of Queensland, Australia The Developmental Origins of Health and Disease hypothesis has caused renewed interest in understanding the factors regulating fetal development. A multitude of prenatal perturbations have now been identied as contributing to the onset of many adults diseases including cardiovascular, metabolic and renal disease. An interesting feature in many animal models of developmental programming is the disparity between males and females in the onset and severity of disease outcomes. Using a variety of animal models (maternal glucocorticoid exposure, maternal low protein diet, uteroplacental insufciency, prenatal alcohol exposure), our studies have identied alterations in kidney development as being a common feature of many of these models. The formation of a low nephron endowment may result in impaired renal function which in turn may underlie the observed disease outcome. However, the same prenatal insult does not always affect males and females to the same degree. For example, the degree of nephron decit and glomerular enlargement in males and females may not be the same despite a similar insult. Even if the decits are similar between the sexes, this does not always result in a similar degree of hypertension or impairments in renal function. Whilst levels of sex hormones after birth are undoubtedly important, there is now growing evidence that the placenta from males and females differ in their responses to insult and this plays a crucial role in regulating fetal growth and development. Most recently, using rodent models, our studies have focused on the molecular changes induced in the placenta and fetal kidney following exposure to short term elevations in maternal glucocorticoids or low doses of ethanol throughout pregnancy. We have shown that changes in expression of receptors of the renin-angiotensin system along with genes involved in the process of branching morphogenesis and uid balance occur in the developing kidney and placenta. These are often sex specic which may in part explain the observed sex differences in disease outcomes.
Symposium 2: Prediction of Adverse Pregnancy Outcome S4. MATERNAL PLASMA NUCLEIC ACIDS AS A TOOL FOR PRENATAL DIAGNOSIS AND MONITORING Dennis Lo, The Chinese University of Hong Kong, Hong Kong The discovery of cell-free fetal nucleic acids in maternal plasma in 1997 has opened up new possibilities for non-invasive diagnosis and monitoring. It has been demonstrated by many groups that the concentrations of circulating fetal nucleic acids are elevated in a number of pregnancy-associated pathologies, most notably preeclampsia. Early work in this area had focused on the use of Y chromosomal markers, which could only be used if the fetus was male. More recent work has resulted in a number of sex- and polymorphism-independent fetal nucleic acid markers, including DNA methylation markers, mRNA and miRNA markers. In addition, exciting new advances in analytical technologies for circulating fetal nucleic acids have been made. One of these involves the use of single molecule counting approaches such as digital PCR and next-generation DNA sequencing. These single molecule approaches offer unprecedented precision in the quantitative analysis of circulating fetal nucleic acids and will be expected to accelerate the clinical applications in this eld.
S5. ONE-CARBON METABOLISM GENETIC POLYMORPHISMS AND ADVERSE PREGNANCY OUTCOMES Denise Furness, Gus Dekker, Dee McCormack, Rachael Nowak, Steven Thompson, Claire Roberts. University of Adelaide, Australia Introduction: The aim of this project was to test genetic polymorphisms involved in one-carbon metabolism and vitamin-B12 transport for potential associations with adverse pregnancy outcomes. Methods: In a prospective observational study, DNA was obtained from 586 nulliparous Caucasian couples without fertility treatment. DNA was genotyped for MTHFR C677T, MTHFR A1298C, MTR A2756G, MTRR A66G, MTHFD1 G1958A and TCN2 C766G polymorphisms. Chi-square analysis was used to compare genotype frequencies with pregnancy outcomes. Pregnancies were classied as healthy (n261), preeclampsia (PE, n38), gestational hypertension (GHT, n32), small-for-gestational-age (SGA, n60) and PE+SGA (n22). Associations between maternal, paternal and neonatal genotypes with customised birthweight centiles and placental weight were determined using ANOVA with SIDAK post-hoc analyses. Results: All genotypes were in Hardy-Weinberg equilibrium. The maternal MTR 2756 G allele was associated with decreased placental weight (-87g, P0.040). Both paternal and neonatal MTR 2756 G alleles were associated with lower birthweight (-12%, P0.028 and -10%, P0.039, respectively) while the latter was also associated with PE+SGA (P >0.000). Neonatal MTRR GG genotype was associated with GHT and PE with SGA (P0.033, P0.011). Neonatal MTHFD1 GG genotype was twice as frequent in PE and GHT (P0.037; P0.019) and neonatal TCN2 GG genotype doubled in SGA (P0.042) compared with healthy pregnancies. Discussion: Our study shows that MTR A2756G, MTHFD1 G1958A and TCN2 polymorphisms are related to adverse pregnancy outcomes. MTR with vitamin-B12 as a cofactor, catalyses the methylation of homocysteine to methionine. Formation of methionine through this pathway represents an important component for synthesis of phospholipids, proteins, myelin, catecholamines, DNA, RNA and S-adenosyl methionine. TCN2 encodes the vitamin-B12 transport protein and MTHFD1 catalyses the conversion of one-carbon derivatives of tetrahydrofolate, which are substrates for methionine, thymine and purine synthesis which are all important for healthy placental and fetal development. Future studies will investigate the role of these polymorphisms in the placenta.
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S6. LOW BIRTHWEIGHT IS PRECEDED BY ALTERED IMMUNE CYTOKINE PROFILES ACROSS VERY EARLY PREGNANCY S. Tong*1,2, Y. Sucintri Thio1, M. Permezel1, C. Russell1, H. Georgiou1. 1 University Department of Obstetrics and Gynaecology, Mercy Hospital for Women, Australia. 2Centre for Womens Health Research, Monash Institute of Medical Research, Australia Introduction: It is hypothesised that errors in immune-led implantation cause major diseases of pregnancy, notably low birthweight and preeclampsia. Evidence for this comes from in vitro human (Hiby et al. J Exp Med 2004) and in vivo animal experiments (Hanna et al Nature Med 2006). Critically, there is no clinical evidence temporally linking the immune system around the time of implantation with the development of serious diseases of later pregnancy. We looked for evidence of differential immune system proles at early pregnancy preceding birth of a small for gestational age (SGA; birthweight <10th centile). Methods: We prospectively collected maternal serum at 7-11 weeks gestation. After recruiting 756 participants, we selected 60 cases where the delivery outcome was a SGA baby and 71 controls (median birthweight 50th centile [range 30th-80th]). We used a multiplex protein bead array to quantify 27 pro and anti-inammatory cytokines, chemokines and growth factors. Results: Pregnancies complicated by SGA were preceded by profound derangements in cytokine proles. Of 21 detectable cytokines/chemokines, 14 varied signicantly (p 0.004 all comparisons) among those destined to birth a SGA baby 30 weeks later, compared to controls. Besides interferon-g (IFN-g) which was raised, all other inammatory cytokines that varied decreased with SGA, including major pro (Interleukin (IL)-2, IL7, IL-12p70) and anti-inammatory (IL-1 receptor antagonist, IL-4, IL-10, IL-13) cytokines. Combining four cytokines/chemokines (IFN-g, IL-7, IL-1 receptor antagonist, and Eotaxin) predicts the occurrence of an SGA baby with 60% sensitivity, 90% specicity and a negative predictive value of 95%. Conclusion: The immune system may be involved in pathogenic mechanisms occurring as early as the middle of the rst trimester causing at least half of SGA deliveries. Early pregnancy immunodulation might be a novel therapeutic approach to decrease the immediate and lifelong health burden that affects babies born at low birthweight.
Symposium 3: IUGR, the placenta and programming S7. REACTIVE OXYGEN AND NITROGEN ADAPTATION OF THE PLACENTA
SPECIES
AND
FUNCTIONAL
Leslie Myatt, Center for Pregnancy and Newborn Research, University of Texas Health Science Center San Antonio, USA The type and amount of nutrients transferred, together with the type, intensity and timing of the hormonal signals it generates to inuence both maternal and fetal systems, allows the placenta to control fetal growth and development. The placenta is in a constant state of growth and differentiation throughout gestation and changes its functional capacity by vascular growth and by trophoblast proliferation and differentiation. Both the vasculature and trophoblast produce a variety of reactive oxygen and nitrogen species including superoxide and nitric oxide that regulate their function. In addition interaction of NO and superoxide may produce a more potent powerful pro-oxidant, peroxynitrite. Production of these mediators, and hence placental patterns of growth and differentiation, may be altered by exposure to varying oxygen concentrations ranging from hypoxia to hyperoxia. The high metabolic demands of the placenta mean that pregnancy per se is a state of oxidative stress. This is further exacerbated in conditions such a preeclampsia and IUGR or with obesity or diabetes where many markers of oxidative stress and inammation are increased. The reactive oxygen and nitrogen species per se have effects as second messengers in the placenta, but may also regulate activity of other molecules, including proteins, lipids and DNA, via covalent modication. Superoxide produced from mitcochondria, and from enzymes including xanthine oxidase and NADPH oxidase can give carbonylation of lysine, arginine, proline and threonine residues on placental proteins which will alter function. Peroxynitrite may cause nitration of specic tyrosine residues in proteins, a covalent modication that can cause either, loss of, gain of or no change in protein function, Hence the extent of oxidative and nitrative stress at the tissue or cellular level may affect placental function and hence fetal growth and development by covalent modication of enzymes, receptors transporters and structural proteins.
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S8. PLACENTAL PROGRAMMING OF METABOLIC HEALTH OF PROGENY Julie A Owens, Kathy Gatford, Miles DeBlasio, Jeffrey Robinson, School of Paediatrics and Reproductive Health, Faculty of Health Sciences and Research Centre for Early Origins of Health and Disease, The Robinson Institute, University of Adelaide, Adelaide, SA, Australia Placental insufciency is a common pregnancy complication and major cause of fetal growth restriction and reduced size at birth for gestational age. Low birth weight consistently predicts an increased risk of adult diabetes, accounting for a substantial proportion of its population prevalence. In humans, this appears to be mediated through early onset defects in insulin secretion, which then combines with onset of peripheral insulin resistance in early adult life to initiate progression of impaired glucose tolerance and diabetes. Experimental placental restriction has been induced in sheep and rodents and the life course and tissue/cellular and molecular basis of its programming of insulin action and diabetes more directly characterised. In both species, placental restriction induces early onset defects in insulin secretion, followed by later onset of insulin resistance in progeny. In sheep, placental restriction impairs insulin secretion from before birth, in part due to reduced b cell mass. This persists into adult life, despite increased pancreatic expression of PDX-1 and other factors that help restore b cell mass after birth, with impairment of intrinsic b cell function, in association with reduced expression of calcium channels a major and early defect following placental restriction. Placental restriction in sheep also does not impair insulin sensitivity before birth, with whole body insulin resistance only evident by one month of age, following catch-up growth and in association with central obesity. As in young human adults of low birth weight, this occurs with reduced skeletal muscle expression of key insulin signalling molecules, including that of IRS-1, subunits of PI3kianse, AKT and targets such as GLUT4. Therefore the timing of onset of placentally programmed defects in insulin secretion and sensitivity varies, with molecular targets and windows of opportunity for intervention now being identied.
S9. ADAPTATION IN PLACENTAL NUTRIENT SUPPLY TO MEET FETAL GROWTH DEMAND: IMPLICATIONS FOR PROGRAMMING Colin P. Sibley, University of Manchester, UK Fetal growth is absolutely dependent on supply of nutrients from mother via the placenta. A variety of human and mouse data together suggest now that placental capacity to supply nutrient is adaptable in relation to fetal demand. (1) In normal human pregnancy the activity, per mg membrane protein, of the System A amino acid transporter in the microvillous plasma membrane (MVM) of the placental syncytiotrophoblast is inversely related to the birthweight and size of baby. (2) In mice with a deletion of the placental transcript of the insulin-like growth factor 2 (igf2) gene, placental weight is reduced compared to wild type mice at e16 (term e20) but fetal weight is not reduced at this time, perhaps because System A expression and activity, per g placenta, is higher in the knockout conceptuses. (3) In the same knockout mice fetal calcium accretion is reduced compared to wild type at e17, but returns to normal by e19 apparently through an increased activity/expression of placental calcium transport mechanisms. (4) Coan et al (J. Physiol 586, 4567-4576, 2008) examined natural inter-litter variation in placental transfer capacity in normal mice; they found that there are gestation dependent morphological and functional adaptations in the smallest placentas enabling them to maintain fetal size as compared to larger placentas. The variable nature of the placental transfer adaptations in different situations, as found in these different studies, will variably affect the mix of nutrient transferred and so potentially variably programme fetal homeostatic mechanisms. Grant support from Tommys [Lets Talk Baby] Charity, The Medical Research Council and The Wellcome Trust is gratefully acknowledged, as are the contributions of many colleagues in the Manchester Maternal and Fetal Health Research Group.
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Symposium 4: Trophoblast and endometrial interactions S10. A FUNCTIONAL ROLE FOR UTERINE NATURAL KILLER (uNK) CELLS IN EARLY PREGNANCY HUMAN DECIDUAS Gendie Lash, University of Newcastle upon Tyne, UK Uterine natural killer (uNK) cells are the major leucocyte in early pregnancy decidua, comprising 70% of decidual leucocytes and 20-30% of all decidual cells. They are phenotypically distinct from pheripheral blood NK cells in that they are CD56+CD16-. They have been proposed to play a crucial role in early pregnancy development, although this role is only just becoming clear. Using a well characterised matrigel invasion assay with placental vellous explants and uNK cell supernatants taken from either 8-10 or 12-14 weeks gestational age we have demonstrated that only uNK supernatants from 12-14 weeks gestational age has a stimulatory effect on EVT invasion. In contrast, total decidual cell isolates supernatants from both 8-10 and 12-14 weeks gestational age stimulate EVT invasion. In addition, the effect of uNK supernatants from 12-14 weeks gestational age could be partially blocked by neutralisng antibody to IL-8, an abundant cytokine whose production increases from 8-10 to 12-14 weeks gestational age. The uNK supernatant effect on EVT invasion as associated with an increase in protease activity and a decrease in EVT apoptosis. EVT are naturally highly invasive although their invasive ability decreased after 10 weeks gestational age. Our results suggest that later in gestation when EVT invasion is still required, but to a lesser degree than in very early pregnancy uNK cells aid in facilitating this process. One of the other roles proposed for uNK cells in early pregnancy is in mediating the non-trophoblast component of spiral artery remodelling. Early in the remodelling process, in the absence of EVT, there is separation of the vascular smooth muscle cells, dilation of the vessel and some vascular permeability. uNK cells are a major source of several key angiognic growth factors, production of which decreases with increasing gestational age, that may play a role in these early stages of remodelling. In addition, in early pregnant decidua uNK cells are localised around spiral arteries. Using a chorionic plate artery model we have shown that uNK supernantants can facilitate similar precessess in vitro. The exact mechanisms of these actions are currently under investigation in our laboratory. Overall, we would suggest that uNK cells are involved in at least two key processes of early pregnancy that change with gestational age. Firstly, in early spiral artery remodelling and ten in mediating EVT invasion. The trigger for this switch in function is unclear. It is interesting to note that early in pregnancy uNK cells are a major producer of angiogenic growth factors whose production decreases with increasing gestational age whereas in contrast the uNK cell production of cytokines increases with gestational age.
S11. TROPHOBLAST-VASCULAR CELL INTERACTIONS IN EARLY PREGNANCY: HOW TO REMODEL A UTERINE ARTERY Lynda Harris, University of Manchester, UK During the rst twenty weeks of pregnancy, extravillous trophoblasts (EVT) colonise the decidua and remodel the uterine spiral arteries as far as the rst third of the myometrium. This process leads to an irreversible vasodilatation, ensuring that maximal blood ow is delivered to the materno-fetal interface at an optimal velocity for nutrient exchange. There is accumulating evidence that subtle changes in vascular structure precede EVT colonisation; however, full physiological transformation is only achieved in the presence of trophoblast and requires vascular cell loss and extracellular matrix breakdown. Following extensive analysis of decidual spiral arteries between 8 and 12 weeks of gestation, we have observed that endothelial cell hypertrophy and SMC disorganisation occur in the absence of trophoblast, and instead correlate with the perivascular and intramural accumulation of uterine natural killer (uNK) cells. We hypothesise that these initial changes in vascular structure aid trophoblast entry into the arterial wall. Using a combination of monolayer co-cultures, immunohistochemical analysis of rst trimester decidua and excised spiral arteries perfused with rst trimester trophoblasts, we have shown that trophoblasts have the capacity to induce vascular cell apoptosis, mediate elastin breakdown and upregulate protease expression in vascular smooth muscle cells (SMC). Trophoblasts express and secrete the apoptotic cytokine Fas ligand, and induce apoptosis of vascular endothelial cells and SMC in vitro. A Fas ligand function-blocking antibody signicantly inhibited apoptosis when added to co-cultures, and reduced cleaved PARP expression and terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) in spiral artery segments perfused with trophoblasts or trophoblast-conditioned medium. EVT also express surface-associated TNF-alpha-related apoptosis-inducing ligand (TRAIL), which can induce apoptosis of spiral artery SMC via ligation of TRAIL receptor-1 or -2. This mechanism requires intercellular contact, reinforcing the importance of intramural incorporation of trophoblast in the remodelling process. In order to effect a permanent increase in the calibre of the spiral arteries, the internal elastic lamina and medial elastin bres must be degraded. We have shown that rst trimester trophoblasts utilise matrix metalloproteinases (MMP) to mediate elastin breakdown, and that a specic MMP-12 inhibitor reduces elastase activity by >50%. Furthermore, vascular SMC also exhibit elastase activity, and spiral artery SMC upregulate MMP-12 expression in response to trophoblast-derived factors. These data support a model in which the actions of uNK cells facilitate colonization of the spiral arteries by EVT. SMC and EVT act cooperatively to effect local degradation of elastin and other matrix components, whilst a subset of SMC respond to trophoblast with an elevated rate of apoptosis. These ndings illuminate the complex steps involved in achieving vascular transformation while maintaining vascular integrity in vivo.
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S12. CYTOKINES REGULATE TROPHOBLAST-ENDOMETRIAL INTERACTIONS IN THE ESTABLISHMENT OF PREGNANCY Eva Dimitriadis, Prince Henrys Institute of Medical Research, Australia Embryo implantation involves the initial adhesion of the trophectoderm to the endometrial surface epithelium followed by the migration and invasion of the trophoblast through the maternal decidua remodeling the endometrial arteries to sequester blood to the developing placenta. These highly regulated processes are critical for pregnancy success inadequacies in which can have far reaching consequences including early miscarriage, preeclampsia, preterm birth and intrauterine growth restriction. It is likely that abnormalities in the very early stages of trophoblast invasion contribute to the development of such pregnancy complications. Due to the great difculty in obtaining very early implantation sites very little is known about how the trophoblast interacts with the decidua to facilitate the process of trophoblast invasion. Numerous cytokines are produced at the human maternal-fetal interface including interleukin (IL) 11, leukemia inhibitory factor (LIF) and activin A. IL-11 and LIF, two IL6 family member cytokines appear to have distinct roles in trophoblast invasion shown using both ex vivo and in vitro models. The TGF beta superfamily member cytokine, activin A similarly regulates trophoblast function. The role, interactions and mechanisms of action of these cytokines will be presented. The data suggests that these locally produced cytokines have important roles in the very early stages of placentation. NIH Senior Researcher Award THE PLACENTA IS A PROGRAMMING AGENT FOR CARDIOVASCULAR DISEASE K. Thornburg1,2,3, P.F. OTierney1 & S. Louey1,2. Heart Research Center1, Departments of Medicine (Cardiovascular Medicine)2, and Physiology and Pharmacology3, Oregon Health and Science University, USA. Cardiovascular disease remains the number one killer in western nations in spite of declines in death rates following improvements in clinical care; disease rates are rapidly increasing in developing countries. Cardiac deaths are primarily due to coronary artery occlusion, progressive heart failure or sudden death from brillation. It has been 20 years since David Barker and colleagues showed that slow rates of prenatal growth predict mortality due to ischemic heart disease. Thus, undoubtedly fetal undergrowth and its associated cardiovascular diseases are due to placental inadequacies. This conclusion is supported by a number of studies linking placental characteristics with various adult diseases. Martyn and colleagues discovered a U shaped relationship between placental-to-fetal weight ratio and heart disease. This is powerful evidence that placental growth regulating processes initiate vulnerabilities for later heart disease in offspring. Recent unpublished evidence from Finland indicates that placental morphological characteristics predict risks for coronary artery disease, heart failure, hypertension and several cancers. The level of risk imparted by placental shape is sex dependent. Further, maternal diet and body composition strongly inuence placental growth, levels of inammation, nutrient transport capacity and oxidative stress with subsequent effects on offspring health. We have used several animal models to determine the placental roots of vulnerability for heart disease. Drs. Shaut and Stadler have shown that abnormal endothelial development in the placenta is associated with undergrown myocardial walls in the embryo. As shown in our laboratory and several others, placental insufciency leads to depressed maturation and proliferation of working myocytes in the fetal sheep heart. Together these models suggest that the ultimate tness of the heart for a life of hard work is determined by hemodynamic, growth factor, and oxygen/nutrient cues before birth, all of which are inuenced, if not regulated by the placenta.
New Investigator Oral Session 1 N1. MATERNAL DIETS HIGH IN SUGAR AND FAT IMPAIR LABYRINTHINE DEVELOPMENT AND AMINO ACID TRANSFER IN THE MOUSE PLACENTA Amanda N Sferruzzi-Perri, Owen R Vaughan, Graham J Burton, Abigail L Fowden, Centre for Trophoblast Research, University of Cambridge, UK Women of reproductive age are consuming increasing amounts of dietary sugar and fat. In mice, high sugar-high fat (HSHF) diets during pregnancy have adverse consequences postnatally but little is known about their effects in utero. This study compared fetal and placental growth in mice fed diets of differing fat and sugar content. Methods: C57BL6/J female mice were fed diets of natural plant material (N, energy content from fat 11%, sugar 3%) or of processed ingredients high in sugar and animal fat, HSHF1 (energy; fat 22%, sugar 20%) or HSHF2 (energy; fat 30%, sugar 36%) throughout pregnancy. On day (D) 19 of pregnancy, placental transport of 14C-methyl aminoisobutyric acid (MeAIB) was measured in vivo under anaesthesia, and placental histology assessed. Differences between diets were determined by linear mixed model repeated measures ANOVA with Sidak post hoc test and considered signicant if P<0.05. Results: Maternal energy intake per day remained constant irrespective of diet. There was no effect of diet on litter size or viability. Fetal weight was similar while placental weight was 11% lower on both HSHF than N diets (Figure). The fetal:placental weight ratio was 10-14% higher in HSHF relative to the N groups (P<0.001). Compared to the N group, labyrinthine volume was reduced by 12-16% on the HSHF diets (P<0.04). These changes were accompanied by a 25-35% reduction in placental MeAIB transfer in mothers fed the HSHF diets (Figure).
Figure. Effect of maternal High sugar-high fat (HSHF) diets on feto-placental growth and placental transport.
Conclusions: The source of energy in the maternal diet has an important role in feto-placental development. Feeding HSHF diets during mouse pregnancy reduced placental growth and amino acid transport without affecting fetal growth near term. Other compensatory mechanisms must therefore exist to maintain fetal growth in response to HSHF diets. The efciency of other nutrient transfer systems in the small HSHF placenta and maternal metabolic proles, are currently being investigated.
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N2. ACTIVIN A - AN EARLY INDUCER OF DECIDUALIZATION - SYNERGISES WITH IL11 TO PROMOTE PROGESTERONE INDUCED DECIDUALIZATION Ellen Menkhorst*, Lois A. Salamonsen, Jin Zhang, Craig A. Harrison, Jun Gu and Evdokia Dimitriadis, Prince Henrys Institute, Australia Decidualization of endometrial stromal cells (ESC) is a critical requirement for the formation of a functional placenta. The mechanisms of human (H)ESC decidualization are poorly understood although locally produced factors such as interleukin (IL)11 and activin (A)A regulate decidualization. We hypothesised that IL11 and AA interact to progress decidualization. Treatment with 0.5mM cAMP or 10-8mol/l estradiol 17b plus 10-7mol/l progesterone (P) for 13 days, decidualized HESC. Decidualizing HESC were treated with IL11 (100ng/ml), AA (50ng/ml), IL11+AA or control and media collected every 2 days to measure prolactin (PRL; a decidual marker), IL11 and AA secretion. Non-decidualized HESC were treated with IL11, AA, or AA plus inhibitor (follistatin [200ng/ml], or SB542431, a TGFb1 receptor [Alk5] inhibitor [10mM]) or control and IL11/AA secretion or phosphorylation (p) of the AA signalling component, SMAD2, measured. cAMP-induced HESC decidualization was biphasic, peaking on day (D) 8 and D13 whereas P-induced decidualization was linear, peaking on D13. cAMP enhanced AA and P reduced IL11 secretion. Treatment with IL11 or AA signicantly enhanced P-induced decidualization on D10 (AA) and D13 (IL11 and AA; P<0.001). IL11+AA signicantly promoted P-induced decidualization above IL11 or AA treatment alone on D7 and D10 (P<0.05). AA induced pSMAD2 (P<0.005) and IL11 secretion (8.25.1 vs 1.90.7pg/ 106cells; P<0.05), which were blocked by inclusion of the AA inhibitors (P<0.005). This study demonstrates that IL11 and AA synergized to promote P- but not cAMP-induced decidualization. This suggests that the two in vitro decidualization stimuli regulate decidualization via different mechanisms and demonstrates the importance in choosing an appropriate decidualization stimulus to study the decidualization process. AA increased IL11 secretion via pSMAD2 in non-decidualized HESC, suggesting AA is an early inducer of HESC decidualization. This data demonstrates that two locally produced cytokines interact to regulate decidual formation, an essential component in the development of a functional placenta.
N3. ADENOSINE A2B INCREASES L-ARGININE TRANSPORT AND NITRIC OXIDE SYNTHESIS IN HUMAN PLACENTAL MICROVASCULAR ENDOTHELIAL CELLS FROM PREECLAMPTIC PREGNANCIES C. Escudero*1,2, L. Myatt3, P. Casanello,1 L. Sobrevia1. 1Ponticia Universidad Catolica de Chile, Chile. 2Universidad del Bo-Bo, Chile. 3University of Texas Health Science Center, USA This study aimed to characterize the role of adenosine receptor A2A and A2B on L-arginine and nitric oxide (NO) synthesis in human placental microvascular endothelial cells (hPMEC). Methods: hPMEC from normal and preeclamptic pregnancies were isolated using positive immunoselection with CD31-magnetics beads. Kinetic parameters for L-arginine transport, mRNA for human cationic amino acid transporter 1 (hCAT1) and 2B (hCAT2B), activity and expression of inducible NO synthase (iNOS), nitrite and nitrotyrosine formation were determined. Adenosine receptor involvement on L-arginine transport and NOS activity was analyzed using agonists (CGS-21680 for A2A, NECA general adenosine receptors) and the antagonists (ZM-241385 for A2A and A2B, MRS-1754 for A2B). Results: Preeclamptic derived hPMEC exhibits higher NOS activity (w4fold), nitrite formation (w6-fold) and nitrotyrosine formation (w2-fold), associated with higher maximal velocity (Vmax) for L-arginine transport (w2-fold) but lower iNOS protein abundance (w50%), mRNA level (w40%) and iNOS promoter activity (w50%) compared with cells from normal pregnancies. In addition, preeclampsia was associated with higher hCAT2B mRNA level (w3-fold), without signicant changes in hCAT1 mRNA. LLysine trans-stimulation of L-arginine transport was found in cells from normal pregnancies (w4-fold), but no signicant alterations were detected in preeclampsia. CGS-21680 restored L-arginine transport and NOS activity in preeclampsia to values in normal pregnancies. NECA did not alter Larginine transport or NOS activity in preeclampsia, however increased (w2-fold) these parameters in cells from normal pregnancies. MRS-1754 reduced L-arginine transport (w50 and 30%) and NOS activity (w70 and 50%) in cells from preeclamptic and normal pregnancies, respectively. ZM241385 reduced NOS activity (w75 and 55 %), but did not alter L-arginine transport in cells from normal and preeclamptic pregnancies, respectively. Conclusion: Stimulation of adenosine A2B receptor, rather than A2A, could be responsible for elevated NO synthesis and L-arginine transport via hCAT-2B in hPMEC from preeclampsia. This phenomenon could be a mechanism attempting to re-establish to normal values the reduced placental vascular blood ow characteristic of preeclampsia. Supported by FONDECYT 1070865/1080534, CONICYT 24071039. C. Escu dero holds PhD-MECESUP and Ponticia Universidad Catolica de Chile fellowships.
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N4. FATTY ACIDS UPREGULATE THEIR OWN STORAGE INTO LIPID DROPLETS AND PROMOTE CELL AGGREGATION AND CYTOKINE PRODUCTION INDEPENDENT OF GLUCOSE IN HUMAN CYTOTROPHOBLASTS A.N. Pathmaperuma*1, P. Mana1, S.N. Cheung1, K. Kugathas1, M.E. Koina2, V. Delghingaro-Augusto4, D.A. Ellwood3, J.E. Dahlstrom2, and C.J. Nolan1, 1 Diabetes and Endocrinology Research Unit, 2Anatomical Pathology and 3 Fetal Medicine Unit, Australian National University Medical School at The Canberra Hospital, Australia. 4Montreal Diabetes Research Center, University of Montreal, Canada Aims: The diabetic pregnancy is characterized by maternal hyperglycaemia and dyslipidaemia, such that placental trophoblast cells are exposed to both. The objective was to determine the effects of hyperglycaemia, elevated non-esteried fatty acids (NEFA) and their interaction on trophoblast cell metabolism and function. Methods: Trophoblasts were isolated from normal term human placentas and established in culture for 16 h prior to experiments. Glucose utilization, fatty acid oxidation and fatty acid esterication were determined using radiolabelled metabolic tracer methodology at various glucose and NEFA concentrations. Lipid droplet formation, cell aggregation, viability, proliferation and apoptosis and the secretion of hormones and proinammatory cytokines were also assessed. Results: Glucose utilization via glycolysis was near maximal at the low physiological glucose concentration of 4 mM; whereas NEFA esterication into triacylglycerol (TG) and diacylglycerol increased linearly with increasing NEFA concentrations without evidence of plateau. Hyperglycaemia caused intracellular glycogen accumulation, but had no other effects on trophoblast metabolism or function. Culture of trophoblasts in 0.25 mM NEFA for 24 h, however, upregulated fatty acid esterication processes, inhibited fatty acid oxidation, inhibited glycerol release (a marker of lipolysis) and promoted lipid droplet formation, all consistent with upregulation of fatty acid storage and buffering capacity. NEFA also promoted trophoblast aggregation and TNFa, IL-1b, IL-6 and IL-10 production without effect on cell viability, proliferation, apoptosis or hormone secretion. Conclusion: NEFA have effects on trophoblast metabolism and function, independent of glucose, that may have protective as well as pathophysiological roles in diabetic pregnancy.
N5. SHED PLACENTAL ANTIGENS ARE CROSS-PRESENTED BY MATERNAL DENDRITIC CELLS IN PREGNANCY L. Moldenhauer*1, D. Thring2, J. Hayball2,3 and S. Robertson1. 1Research Centre for Reproductive Health, University of Adelaide, Australia; 2Hanson Institute, Australia; 3Sansom Institute, University of South Australia, Australia The events through which the maternal T cell repertoire establishes tolerance to paternally-derived antigens expressed by the placenta is unclear. To investigate this, we utilised a transgenic Act-mOVA male mouse expressing ovalbumin (OVA) ubiquitously as a model paternal antigen. OVA is inherited by the conceptus and expressed by placental tissue. OVA-specic CD8+ OT-I T cells were adoptively transferred to ActmOVA mated B6 females during pregnancy or post-partum. Extensive T cell activation and proliferation was evident in all lymphoid tissue and the spleen from day 7.5 pc until 20 days post-partum, indicating systemic presentation of placental OVA antigen. No activation of OT-I T cells was detected when bm1 mice, carrying a mutation in MHC class I H-2Kb, were mated with Act-mOVA males. This indicates that OVA-antigen presentation depends upon processing by maternal antigen-presenting cells and cannot be presented by placental cells. Similarly, female mice with a mutation in the gene encoding transporter associated with antigen processing (TAP) failed to present placental OVA. When in vitro activated, cytotoxic OT-I T cells were adoptively transferred to pregnant females carrying OVA+ foetuses, fatal death occurred. This indicates that placental cells express MHC class IOVA peptide complexes that can be targeted by cytotoxic OT-I T cells. We conclude that placental antigens are processed and presented by professional, bone marrow-derived antigen presenting cells of maternal origin, presumably dendritic cells, via a TAP-dependent MHC class I pathway. While placental MHC class I-OVA complexes are expressed by placental cells and can be targeted in T cell-mediated cytotoxicity, placental cells do not present OVA antigen to elicit T cell activation, likely due to the absence of placental cell expression of appropriate co-stimulatory molecules.
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N6. THE EFFECT OF MALARIA INFECTION ON TERM PLACENTAL 11 BETA HSD2 EXPRESSION Alexandra Umbers*, Caroline Clapham, Stephen Rogerson. University of Melbourne, Adelaide Introduction: Malaria is responsible for almost 1 million deaths per year, with pregnant women being particularly at risk of infection. Primigravid mothers are at greater risk of infection and severe disease and have elevated serum cortisol levels compared to multigravidae. The fetus is at risk of adverse birth outcomes including intrauterine growth restriction, low birth weight (LBW), preterm delivery, still birth and neonatal malaria. During infection Plasmodium falciparum infected erythrocytes concentrate in the intervillous space and are often accompanied with inammatory cells and elevated pro-inammatory cytokines, both of which are strongly associated with LBW. The biological mechanisms that lead to adverse fetal outcomes are not known, but placental insufciency may underlie the pathogenesis. Placental 11-b hydroxy steroid dehydrogenase 2 (11-bHSD2) converts maternal cortisol to inactive cortisone, limiting transplacental transfer and fetal exposure to the growth restricting properties of cortisol. Because there is a well recognized relationship between elevated maternal cortisol and inammation on 11bHSD2 function (and consequently birth weight) in other disorders of pregnancy, we assessed the relationship between malaria infection and 11bHSD2 mRNA and protein levels. Method: mRNA and protein were measured using real time PCR and western blotting in placental biopsies from a cohort of 90 primigravid Malawian women with and without malaria infection. Results: Mean 11bHSD2 mRNA levels were reduced 40% compared to uninfected controls. Moreover, 11bHSD2 mRNA was reduced 25% in LBW cases and 44% in mothers with more severe disease but differences did not reach signicance. Protein analysis on 46 samples revealed a signicant decrease (68%) (p 0.001) compared to controls. Furthermore, expression was reduced 54% (p0.15) in both LBW, and in 55% (p0.06) of mothers with severe disease. Discussion: This study reveals a signicant reduction in 11bHSD2 and highlights the potential role for excess fetal glucocorticoid exposure in the pathogenesis of fetal growth restriction observed in malaria during pregnancy.
New Investigator Oral Session 2 N7. EFFECTS OF MATERNAL DEXAMETHASONE TREATMENT ON AMINO ACID CLEARANCE AND DIFFUSIONAL EXCHANGE CAPACITY OF THE MOUSE PLACENTA DEPEND ON GESTATIONAL AGE OR Vaughan*, AN Sferruzzi-Perri, PM Coan, GJ Burton & AL Fowden. Centre for Trophoblast Research, Department of Physiology, Development and Neuroscience, University of Cambridge, UK Introduction: Synthetic glucocorticoids, like dexamethasone (dex), are given to pregnant women threatened with pre-term delivery to improve neonatal viability. In experimental animals, this treatment restricts fetoplacental growth but often increases fetal:placental weight ratio1. However, the effects of this treatment on placental phenotype remain unknown. Hence, this study examined the morphology and amino acid transfer capacity of the mouse placenta after maternal dex administration in the last trimester. Methods: Ad libitum fed pregnant C57/BL/6J mice were injected with dex (200ng/g, s.c., n24) or saline (n23) for 5 days before measurement of unidirectional materno-fetal clearance of 14C-methyl-aminoisobutyric acid (MeAIB) under terminal anaesthesia at day (D) 16 (n18) or 19 of pregnancy (n29; term 21days)2. Placental labyrinthine morphology was assessed by stereology3. Differences between treatments were determined by linear mixed model repeated measures ANOVA with Bonferroni post hoc test and considered signicant when P<0.05. Results: On D16, placental but not fetal weight was signicantly less in dex than saline treated mice (Table 1). The fetal:placental weight ratio was greater in dex (4.4 0.1) than saline treated animals (3.9 0.1) (P<0.001). Fetal capillary volume, interhaemal membrane (IM) theoretical diffusion capacity and MeAIB clearance were signicantly higher in dex than saline treated placentae at D16 (Table 1). On D19, both placental and fetal weights were lower in dex than saline treated animals. Labyrinthine growth was proportionally restricted in dex animals (Table 1). Neither labyrinthine architecture nor MeAIB clearance differed with treatment at D19 (Table 1). Table 1 Fetal and placental weights (n8-15), placental morphology (n4-5) and MeAIB clearance (n7-12) of dex and saline treated mice at D16 and D19. *, P<0.05 vs control
D16 Saline Fetal Weight (mg) Placental Weight (mg) Component Volume (mm3) Labyrinthine zone Maternal blood space Fetal capillary Labyrinthine trophoblast IM Mean Surface Area (cm2) IM Harmonic Mean Thickness (mm) Theoretical Diffusion Capacity (mm2min-1.kPa) MeAIB Clearance (ml.min-1.g-1) 394 5 101 1 D16 Dex D19 Saline D19 Dex
398 6 91 1*
1141 11 1106 11* 86 1 78 1* 48 1 7.5 1.5 7.1 1.4 33 3 14.9 1.9 3.8 0.4 7.1 1.3 44 1* 8.7 0.7 6.2 0.5 29 1 15.6 1.1 3.5 0.1 7.8 0.8
43 2 41 1 4.6 0.5 5.1 0.1 2.9 0.2 3.9 0.3* 36 2 33 0.1 7.6 0.8 10.9 0.1* 5.5 0.5 4.5 0.1* 2.4 0.2 4.2 0.1*
38.0 2.0 52.1 1.7* 143.4 7.1 139.1 6.3
Discussion: The effect of maternal dex treatment upon placental phenotype depends on gestational age. The small dex treated placenta adapts to maintain fetal growth by altering amino acid transfer and labyrinthine morphology at mid but not late gestation. References 1. Fowden, AL. et al (2008) J Neuroendocrinol 20: 439. 2. Sibley, CP. et al (2004) PNAS 101: 8204. 3. Coan, PM. et al (2004) Biol Reprod 70: 1806.
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N8. GENOME-WIDE DNA METHYLATION ANALYSIS OF FIRST TRIMESTER HUMAN PLACENTA Boris Novakovic*1,2, Katrina Bell1, Hong Kiat Ng1, Andrew Sharkey3, Ashley Moffet3, Ursula Manuelpillai4, Eva Dimitriadis5, Jeff Craig1,2, Richard Saffery1,2. 1Developmental Epigenetics, Murdoch Childrens Research Institute, Australia, 2Department of Paediatrics, University of Melbourne, Australia, 3Department of Pathology, University of Cambridge, United Kingdom, 4Monash Institute of Medical Research, Monash University, Australia, 5Prince Henrys Institute of Medical Research, Australia Epigenetic modications, such as DNA methylation, play a major role in controlling gene expression in tissues, and as such are involved in many aspects of differentiation, development and function. The human placenta is a unique organ in that it only exists for 9 months and displays many similarities to human tumours, such as growth in a low oxygen environment, cell migration, and invasion of surrounding tissue and escape from immune detection. Not surprisingly therefore, the placenta also displays a unique epigenetic prole with many similarities to the DNA methylation prole of cancers including low global DNA methylation levels and hypermethylation of specic tumour-suppressor gene promoter regions. Our group, as well as others, has previously shown placental-specic methylation of tumour suppressor genes, Ras-signalling inhibitors, Wntsignalling inhibitors and genes regulating Vitamin D homeostasis. These specic methylation patterns are likely to contribute to the development and function of the placenta; however, they are only a small part of the overall placental epigenome. In order to determine the extent of placentaspecic methylation, an Illumina Innium Human Methylation27 BeadChip array was used to detect genome wide methylation levels in rst trimester (8 and 12 week) human placental tissue. Gene methylation status was then compared to three somatic tissues, and genes were classied into one of four categories: (i) methylated in all tissues, (ii) specically methylated in the placenta, (iii) hypomethylated in all tissues, (iv) hypomethylated specically in the placenta. Class (ii) genes were further validated using the Sequenom Epityper quantitative methylation platform. This conrmed placenta-specic promoter methylation of genes involved in several pathways, including multiple transcription factors, regulation of apoptosis, oxidative-stress response, and cancer progression. We believe that disruption of these methylation patterns may contribute to poor placental development and pregnancy-associated diseases such as preeclampsia and IUGR.
N9. ANTIOXIDANTS REDUCE IMPACT OF ACTIVIN A INDUCED OXIDATIVE STRESS IN HUVECs Rebecca Lim, Stephen Mandang, Pavitra Delpachitra, Rutu Acharya, Sebastian Hobson, Euan Wallace, Department of Obstetrics and Gynaecology, Monash University, Australia Background: The precise links between disturbed placental function and maternal syndrome preeclampsia (PE) remain unclear. Maternal serum levels of activin A increase 10-fold in PE and are increased months before clinical onset of signs and symptoms. We previously reported placental oxidative stress as the likely cause of increased circulating activin. More recently, we have been exploring its possible systemic effects. Aims: Determine the role of activin A in the induction of endothelial dysfunction, specically exploring endothelial cell oxidative stress, mechanisms of oxidative stress induction and efcacy of targeted antioxidants. Methods: HUVECs were cultured in the presence of activin A (50ng/mL) or preeclamptic serum (10%) prior to treatment with follistatin 288 (FS; 600ng/mL), tempol, superoxide dismutase or NOX inhibitor apocynin. Oxidative stress was assessed by ROS production and lipid peroxidation product, 8-isoprostane. Changes in NADPH oxidases (NOX) gene expression were studied as likely mechanisms of oxidative stress. Endothelial dysfunction was assessed by ZO-1 expression and endothelial permeability. Results: Activin A induces lipid peroxidation determined by increased 8-isoprostane levels in a dose-dependent manner (*p<0.05) negated by FS. Compared to normal pregnancy serum, PE-serum increased ROS and 8-isoprostane levels mitigated by FS. Similar trends were observed when HUVECs were cultured in media containing PE serum, or PE serum and FS. ROS production was reduced in Activin treated HUVECs following exposure to Tempol, SOD and Apocynin. Following 2hrs stimulation with Activin, gene expression of ZO-1 was signicantly reduced by approximately 50% (**p<0.01), ET-1 expression increased >5-fold (*p<0.01), Nox2 and Nox4 expression increased ~4-fold (*p<0.05) and 1.6-fold (*p<0.05) respectively while Nox1 and 5 remained unchanged compared to untreated HUVECs. Effects were mitigated by addition of Tempol, SOD or Apocynin and related to endothelial integrity as determined by transendothelial permeability and resistances assays. Conclusions: Activin A directly induces oxidative stress in HUVECs, most likely through NOX activation and induction, thereby inducing increased permeability. The reduction in bioavailability of activin through addition of FS or reduction of ROS production through administration of antioxidants or NOX inhibitors may offer therapeutic potential for the treatment of PE.
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N10. THE REGULATORY ROLE TROPHOBLAST INVASION
OF
AP2a
IN
HUMAN
EXTRAVILLOUS
N11. SEX-SPECIFIC CYTOKINE RESPONSES OF PLACENTAE FROM PREGNANCIES COMPLICATED BY ASTHMA FOLLOWING IN-VITRO LIPOPOLYSACCHARIDE STIMULATION N. Hodyl*1, N. Scott2, A. Osei-Kumah1, M. Stark1 & V. Clifton1. 1Robinson Institute, University of Adelaide, Australia; 2The University of Newcastle, Australia Introduction: Asthma affects 12% of pregnant women in Australia and results in reduced female fetal growth. We have previously identied sexspecic alterations in placental cortisol metabolism and cytokine mRNA expression with maternal asthma. We propose that activation of proinammatory pathways in the placenta is therefore dependent on fetal sex. Hypotheses: We hypothesise that potentiated levels of pro-inammatory cytokines will be evident in placentae of females compared to males following lipopolysachharide (LPS) stimulation in-vitro, and be further increased in the presence of asthma. Additionally, females will exhibit greater cortisol inhibition of cytokine responses than males. Methods: Placentae were collected from asthmatic (n12) and nonasthmatic women (n10). Placental explants were stimulated with LPS (1ng/ml) in the presence and absence of cortisol. Supernatant was collected at 2 and 24 hours, and cytokine concentrations (tumour necrosis factor (TNF) a, IL (interleukin) 1b, IL6, IL8, IL10) measured by ELISA (Luminex). Results: In control placentae post LPS stimulation, equivalent cytokines were observed in both sexes. In the presence of asthma, female placentae produced greater IL1 and IL10 than male placentae (p<0.05). Only in the presence of a female, TNFa, IL-1b and IL6 were increased in the asthma group at 2 hours (p<0.05), yet decreased at 24 hours relative to controls (p<0.05). Cortisol inhibited placental responses equivalently in placentae from both sexes at 2 hours. Conclusions: Increased pro-inammatory cytokine production in placentae was demonstrated only in the presence of a female fetus and maternal asthma. This may contribute to the reduction in growth that we have observed in female fetuses in the presence of asthma. As cortisol inhibition of cytokine production was observed in both male and female placentae, the differential inammatory responses between the sexes may be due to other regulatory pathways, and is the focus of our ongoing work.
K. Biadasiewicz*, S. Sonderegger, S. Haider, P. Haslinger, M. Knoer. Department of Obstetrics and Gynecology, University of Vienna, Austria Background: The AP2 family of transcription factors consists of ve isoforms: a-d. They are involved in important developmental and biological processes such as differentiation, proliferation, migration and invasion. In the placenta, AP2a and AP2g are predominantly produced in villous trophoblasts. In particular, AP2a was shown to regulate transcription of the chorionic gonadotrophin subunits a and b. In immunohistochemical and gene chip analyses, we previously noticed that AP2a is also present in extravillous trophoblasts (EVTs), suggesting that it could be critical for trophoblast invasion. Objective: To investigate the role of AP2a in controlling gene expression and invasion of extravillous trophoblast cell models. Methods: Stable knock-down of AP2a was performed in the cell line SGHPL-5 after infection with a murine stem cell virus (MSCV) based vector harbouring shRNAmir against AP2a. As a control SGHPL-5 cell pools containing a non-targeting shRNAmir were established. Primary EVTs were isolated from rst trimester placentae and cultivated on Matrigel coated dishes. Knock-down in EVTs was done with AP2a siRNA oligos (96h). Western blot analyses and real-time PCR were used to investigate potential AP2a target genes. Proliferation was determined by counting cumulative cell numbers (4days). Invasion assays (24h) were done using Matrigel coated transwells. Both functional assays were performed in the absence or presence of EGF (5ng/ml). Results: Western blot data indicate that the extent of AP2a gene silencing was approximately 70% in both the SGHPL-5 cell pool and EVTs compared to non-targeting controls. Western blot and real-time PCR analyses revealed upregulation of TIMP3 but downregulation of MMP2, MMP9, PAI1, PAI-2, TIMP1, TIMP2, as well as the active form of uPA in the stable AP2a knock-down pool. Interestingly, the uPA pro-enzyme accumulated in SGHPL-5 knock-down cells suggesting that AP2a may affect uPA processing enzyme(s). So far, downregulation of MMP2 was also noticed upon AP2a gene silencing in EVTs. EGF-dependent invasion of the SGHPL-5 AP2a shRNAmir cell pool was signicantly decreased compared to controls, proliferation was largely unaffected. Conclusions: AP2a controls expression of proteases and inhibitors critically involved in EVT differentiation. Transwell assays with AP2a knock-down cells suggest that the factor could be important for growth factor-dependent trophoblast invasion.
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N12. GENOMIC INVESTIGATION NETWORK
Trophoblast Research Award OF A CONSERVED PLACENTAL GENE TR1. IMMUNE REGULATION BY CD4+CD25BRIGHT REGULATORY T CELLS AT THE FETAL-MATERNAL INTERFACE DURING HUMAN PREGNANCY Tamara Tilburgs1,2,3, Sicco A. Scherjon2, Barbara J. van der Mast2, Geert Haasnoot1, Minke Versteeg-v.d.Voort-Maarschalk1, Dave L. Roelen1, and Frans H. J. Claas1. 1Department of Immunohematology and Blood Transfusion and 2Department of Obstetrics, Leiden University Medical Center, Netherlands, 3Current work address: Department of Molecular and Cellular Biology, Harvard University, USA During pregnancy maternal lymphocytes at the fetal-maternal interface play a key role in the immune acceptance of the allogeneic fetus. Recently, CD4+CD25bright regulatory T cells have shown to be concentrated in decidual tissue where they are able to suppress fetus-specic and nonspecic responses. HLA-C is the only polymorphic classical histocompatibility antigen expressed by fetal trophoblasts at the fetal-maternal interface. Thus far no evidence has been provided that decidual T cells specically recognize and respond to fetal HLA antigens at the fetalmaternal interface. In this study, we show that pregnancies containing an HLA-C mismatched child induce an increased percentage of CD4+CD25dim activated T cells in decidual tissue. In addition, HLA-C mismatched pregnancies exhibit a decidual lymphocyte response to fetal cells and contain functional CD4+CD25bright regulatory T cells in decidual tissue, whereas HLA-C matched pregnancies do not. This suggests that decidual T cells specically recognize fetal HLA-C at the fetal-maternal interface but are prevented from initiating a destructive immune response in uncomplicated pregnancies.
E.B. Chuong*, D. Leuenberger, G. Bejerano, J.C. Baker, Department of Genetics, School of Medicine, Stanford University, USA The placenta exhibits striking structural and physiological diversity across mammalian taxa, and this variation is reected in the evolution of many species-specic placental genes. These apparent differences challenge research on pregnancy because the mouse model is presumed insufcient to represent human placentation. However, a growing number of key genes including cdx2, hand1, and gcm1 have been observed to be fundamental for placentation in all mammals, and our lab recently reported a signicant enrichment of conserved genes involved early placental development (Knox and Baker, 2008). Therefore, we hypothesize that all eutherian mammals share a core genetic network that is conserved and essential for placental development. To investigate the placental gene network, we are utilizing next-generation sequencing to assay genome-wide functional data over a timecourse of mouse placental development. We have begun carrying out highthroughput transcript tag quantication (RNA-Seq) to generate a comprehensive transcriptome of placental development, and in parallel we are performing genome-wide chromatin immunoprecipitation experiments (ChIP-Seq) to assay chromatin-level changes. Together these data will provide a comprehensive picture of how placental genes are regulated at a global level. We have also developed computational methods to predict the specic cis-regulatory elements that dene the placental gene network, where we focus particularly on elements conserved across all eutherians. These elements may have emerged during the evolution of the ancestral placenta and remained conserved due to their importance in placental development. To test the ability of these elements to confer placental expression in vivo, we have established a lentiviral transfection reporter assay that is specic to the trophoblast lineage in mouse. Elucidation of the mammalian core placental gene network at the genomic level will provide novel insight into placental development, enable more focused translational research using animal placenta models, and shed light on the evolution of mammalian live birth.
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IFPA Award in Placentology L2. THE ROLE OF APOPTOSIS IN THE TURNOVER OF VILLOUS TROPHOBLAST Berthold Huppertz, Medical University of Graz, Austria Only about 15 years ago apoptosis has been attributed to the development of the human placenta. Even today a few scientists still ignore the overwhelming load of data that unambiguously show that programmed cell death plays an essential role in placental growth and differentiation, especially in the villous trophoblast. Focussing on the apoptosis cascade within the villous trophoblast my group has detected the link between differentiation of cytotrophoblasts and syncytiotrophoblast on one hand and the progress of apoptosis on the other hand. The progress of differentiation of the villous trophoblast can be traced by following specic activities of proteins that are normally described to become activated during the apoptosis cascade. Proteases such as caspase 8 but not calpains are used to trigger specic differentiation processes, and thus their activity is tightly regulated in terms of space and time. Dysregulation of processes such as proliferation, differentiation and apoptosis in the villous trophoblast may end up in pregnancy pathologies such as intrauterine growth restriction, preeclampsia or even spontaneous abortion. Especially preeclampsia seems to be closely related to the maintenance of the villous trophoblast. Under normal conditions apoptotic corpuscular fragments are released from the syncytiotrophoblast (called syncytial knots), which are engulfed in the maternal lungs. During preeclampsia this release shifts towards a release of non-apoptotic subcellular fragments (even necrotic or aponecrotic), which is supposed to induce the systemic inammatory response of the mother typical for preeclampsia. Today there is an intense search for markers to facilitate the early prediction of preeclampsia already during the rst trimester of pregnancy. First very promising candidates such as placental protein 13 (PP13) are now being launched for a general testing of pregnant women. I hope that this is the rst step towards the identication of the origin of preeclampsia and thus the rst step towards strategies to prevent this syndrome.
L3. COMPLICATED INTERACTIONS BETWEEN GENES AND ENVIRONMENT IN PLACENTATION AND PREGNANCY OUTCOME Claire T. Roberts. University of Adelaide, Australia
THE
Fetal programming can often be attributed to sub-optimal, but potentially modiable, maternal factors such as smoking and poor nutrition. Much of the research in this eld points to factors that cause intrauterine growth restriction (IUGR) and the long term consequences for offspring health. It is not greatly appreciated, however, that other complications that may occur with, or independently of, IUGR predispose offspring, and their mothers, to poor health. Elevated maternal BMI increases the risk for most pregnancy complications but it is clear that defects in placental invasion and function predispose to many adverse pregnancy outcomes. Our new data show that paternal obesity is associated with IUGR, independent of maternal BMI. We have also identied polymorphisms in a number of genes that regulate how the placenta invades the decidua and differentiates, and how the mother adapts to pregnancy, that are associated with poor outcomes. Excitingly, many of these are polymorphisms in the paternal genome. One might reasonably expect that these would be found in imprinted genes expressed only from the paternal allele. However, we have also found several non-imprinted genes in which paternal genotype has a signicant inuence on pregnancy outcome both on maternal and infant disease states, but also on fetal and placental growth parameters. Furthermore, these genes interact with the maternal environment including diet and smoking to profoundly affect maternal and infant health. Consequently we now propose a complicated model of the control of optimal placental and fetal growth and pregnancy outcome that includes important genetic contributions from both parents to placental genotype that regulate conceptus growth and function. Importantly, paternal genotype can inuence placental gene expression and the myriad of placental hormones and growth factors secreted into the maternal circulation that modulate maternal adaptation to pregnancy and, in susceptible women, these interact with maternal genotype, BMI and lifestyle to cause poor maternal and infant health.
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[P01.01]. HDL: A NOVEL FETAL SIGNAL REGULATING PLACENTAL FUNCTION Manuela Augsten*1, Birgit Ebner3, Angela Chemelli4, Uwe Lang1, Gernot Desoye1, Christian Wadsack1. 1Clinic of Obstetrics and Gynaecology, Medical University Graz, Austria, 2Institute of Medical and Chemical Laboratory Diagnostics, Medical Univeristy Graz, Austria, 3Center of Medical Research, Medical University Graz, Austria, 4Institute of Chemistry, University of Graz, Austria Introduction: Endothelial cells lining placental vessels are exposed to lipoproteins in fetal blood which differs from maternal circulation. The predominant fetal lipoprotein is high density lipoprotein (HDL), which contains more apolipoprotein E (ApoE) than adult HDL. We hypothesize that ApoE has specic functions in the placenta. The aim was to identify genes regulated by ApoE-rich HDL in primary endothelial cells isolated from placental arteries (ECA) by incubating them with fetal HDL or reconstituted HDL-particles, following microarray analyses. Methods: Fetal HDL was isolated from cord plasma by ultrazentrifugation. Reconstituted HDL-particles were prepared of L-a-phosphatidylcholine, cholesterol and ApoE. Both were characterized by lipid electrophoresis and light scattering. ECA (n5) were incubated with HDL or reconstituted HDLparticles for 16h versus untreated. RNA was controlled on BioAnalyzer. cDNA was hybridised onto microarrays (ABI) representing 29,098 human genes. Results: The discoidal Apo-E rich particles had a size of 12.5nm in contrast to fetal HDL (17.5nm). Among 11.224 genes in the ECA transcriptome, 73 genes were differentially expressed (p<0.05) when cells were exposed to HDL compared to untreated cells. 16 genes had a fold change <0.5 and 5 genes a fold change >2. When ECA were treated with reconstituted HDL-particles 1380 (12%) genes were regulated (p<0.05) versus untreated cells. Thereof 157 genes were downregulated <0.5 fold and 359 genes upregulated >2 fold. Genes regulated by fetal HDL were clustered in lipid metabolism and steroid biosynthesis, whereas those regulated by reconstituted HDLparticles participate in amino acid metabolism and stress response. 32 (0.3%) genes were commonly regulated in both treatment groups. Their functional clusters predominantly altered related to cell signalling (upregulated) and developmental processes (downregulated). Discussion: Fetal HDL alters gene expression in ECA. Some of these changes are induced by ApoE. HDL was identied as a novel fetal signal that may regulate placental functions. (supported by grants 10053 to GD, 10896 to UH and 11165 to CW, OENB Jubilee Fund) Keywords: primary placental endothelial cells, fetal high density lipoprotein (HDL), reconstituted HDL particle, gene expression
[P01.02]. LPA PROMOTES MIGRATION OF HUMAN EXTRAVILLOUS TROPHOBLAST CELL LINE, HTR-8/SVneo T Kotani*, S Sumigama, E Yamamoto, H Hayakawa, Y Mano, F Kikkawa. Nagoya Graduate University School of Medicine, Japan Introduction: Successful pregnancy depends on the appropriate invasion of extravillous trophoblast (EVT) into the maternal decidua. Lysophosphatidic acid (LPA) is lysophospholipid mediators of diverse cellular processes exerted through a group of G protein-coupled receptors. It has been reported to be involved in cell proliferation, cell survival, cell differentiation regulation of gap junctions, stimulation of the serum response element, induction of inward ion currents, cell morphological changes. LPA is important for blastocyst implantation in mice and sheep. In humans, serum lysophospholipase D activity increases during pregnancy and LPA are involved in angiogenesis of rst trimester decidua. However, the role of LPA in EVT function remains unknown. Therefore, we investigated the expression of LPA receptors and function of LPA in EVT, using human extravillous trophoblast cell line, HTR-8/SVneo. Methods and Results: RT-PCR analysis reveals that mRNA of LPA1 and LPA3 receptors were identied in HTR-8/SVneo and primary EVT cells. Immunoreactive LPA1 and LPA3 receptors were localized to EVT cells in rst trimester implantation sites. LPA signicantly stimulated invasion of HTR8/SVneo cells. Moreover, LPA induced the phosphorylation of p42/44 MAPK in HTR-8/SVneo cells, which play roles in the stimulation of EVT motility. Conclusions: Our results suggested that LPA might stimulate human EVT invasion with phosphorylation of p42/44 MAPK directly in placentation. Keywords: LPA, EVT, invasion, p42/44 MAPK [P01.03]. PLACENTAL UPTAKE OF POSTPRANDIAL VITAMIN A Lesley Wassef1, Elena Giordano1,2, Loredana Quadro*1. 1Rutgers University, United States, 2University of Cagliari, Italy Introduction: The developing mammalian embryo obtains vitamin A (VA) from the maternal bloodstream. Two majors circulating VA forms are: retinol (ROH) secreted from the liver stores bound to retinol-binding protein (RBP), in the fast state; retinyl ester (RE) within chylomicron lipoproteins of intestinal origin, in the fed state. Both forms are used by the embryo to meet its VA needs (1). Intestinal chylomicrons are secreted into the bloodstream and hydrolyzed by lipoprotein lipase (LPL) into smaller chylomicron remnants-RE. Although the liver takes up the majority of them, 25% is taken up by extrahepatic tissues. LPL catalyzes the hydrolysis of chylomicron-RE and the amount of postprandial RE taken up by extrahepatic tissues correlates with LPL activity. LPL is expressed in placenta (2). We investigated whether LPL facilitates the uptake of postprandial VA in this tissue. Methods and Results: We showed that whole body lipase inhibition by administration of P-407, followed by gavage of 3H-ROH of pregnant wildtype females, reduces the amount of postprandial VA taken up by the developing tissues. Mice lacking LPL and overexpressesing human LPL only in muscle (MCKhL/LPL-/-) still produce chylomicron remnants RE, but do not express neither mouse nor human LPL in placenta (3). MCKhL/LPL-/mice were crossed with mice lacking RBP (RBP-/-), which cannot mobilize efciently their VA stores and rely on dietary VA to support normal embryogenesis. MCKhLPL/LPL-/-RBP-/- mice were maintained on different regimens of dietary VA during pregnancy (VA-sufcient, -decient and -excess diet) and sacriced at 14.5 dpc. MCKhLPL/LPL-/-RBP-/- embryos were morphologically similar to RBP-/- embryos bred under similar maternal dietary regimens. Also, steady state levels of maternal serum and embryonic VA levels in the MCKhLPL/LPL-/-RBP-/- strain were similar to those of the RBP-/- control strain. Discussion: To date, our results suggest that systemic, but not placentalspecic inhibition of LPL activity, affects uptake of postprandial VA in this tissue. References 1. Quadro L., et al. Mol Aspect Med 24, 421-430 (2003). 2. van Bennekum et al., J Lipid Res 40, 565-574 (1999). 3. Weinstock, P. H., J Clin Invest 96, 2555-2568 (1995). Keywords: vitamin A, retinyl ester, lipoprotein lipase
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[P01.04]. A NOVEL ROLE OF CERAMIDE, SPHINGOSINE AND SPHINGOSINE1-PHOSPHATE IN TROPHOBLAST DIFFERENTIATION A.T. Singh*, A Dharmarajan, J.A. Keelan. University of Western Australia, Australia Introduction: Differentiation and fusion of placental trophoblasts throughout pregnancy is a unique process, resulting in formation of a multinucleated cell membrane (syncytium). Critical mechanisms underlying and regulating this fusion process remain unclear. Sphingolipids such as ceramide and sphingosine-1-phosphate have extensively been associated with regulation of multiple biological activities, including apoptosis, differentiation and proliferation. However, their role in trophoblast differentiation has not yet been dened. We have previously demonstrated a correlation between trophoblast differentiation and ceramide levels in transporter studies. Given that sphingolipids are key players in early and late stages of apoptosis, which is an essential component of trophoblast differentiation, we hypothesized that they might regulate trophoblast differentiation. Methods: Cultured cytotrophoblasts, isolated from term human placentas by enzyme digestion, were allowed to differentiate over 7 days in culture. Cell fusion commenced around day 2 of culture and was complete by day 4, according to hCG secretion levels. Results: Signicant changes in intracellular levels of ceramide and sphingosine during differentiation were detected by LC-MS/MS. Expression of enzymes of sphingolipid biosynthesis/metabolism also altered signicantly during differentiation and correlated with levels of their substrates/products. Exogenous administration of a sphingosine kinase inhibitor (which regulates S1P levels) signicantly suppressed trophoblast differentiation, whereas modulation of ceramide or sphingomyelin levels upregulated it. Conclusion: These ndings are consistent with the hypothesis that sphingolipid metabolism is involved in trophoblast differentiation and syncytialisation. Dysregulation of ceramide-sphingosine metabolism by cellular stress modulators might have adverse effects on trophoblast differentiation and thereby placental function. Keywords: trophoblast differentiation, ceramide, sphingosine, sphingosine-1-phosphate
[P02.03]. HEME OXYGENASE-1 GENE PLACENTA OF PREECLAMPSIA
EXPRESSION
IN
UMBILICAL
CORD,
Jong yun Hwang*, Ji yeon Lee, Sung Hun Na, Hyang ah Lee, Dongheon Lee. School of Medicine, Kangwon National University, Republic of Korea Background: Heme oxygenase (HO) is an important enzyme to degrade from heme to carbon monoxide, biliverdin and ferritin. HO and its products are important oxidants to prevent tissue damage from oxidative stress. HO has three isoform enzymes. Especially, HO-1 is an inducible form of the enzyme that is induced by heme, cytokines, endotoxins, hyperthermia and hypoxia. HO and its products play signicant role in placentation, placental angiogenensis and antioxidant protection from oxidative stress. Preeclampsia (PET) is a hypertensive disorder in pregnancy. It is hypothesized that the cause of PET is disturbance of spiral artery modication of trophoblast in the early placentation. I hypothesized that the deciency of HO and its catalytic products may be associated with PET and then adequate HO and its products may prevent the PET. Objects: The aim of this study was to evaluate the difference of the HO-1 gene expression on placenta and umbilical cord between PET and normal pregnancy. Methods: In this study, we designed Case-control study including seven women with PET. The umbilical cord and placenta tissue samples were obtained from PET patients and normal delivery. We quantied HO-1 gene expression on tissue samples by RT-PCR, real time PCR, immnunohistochemistry. Results: In our study, we observed a signicant down-regulation of HO-1 gene expression in umbilical cord and placenta when compared to normal placenta and umbilical cord. Especially, HO-1 gene expression in the placenta decreased lower than it in umbilical cord. Conclusion: The present study demonstrates that the down-regulation of HO-1 gene expression in placenta and umbilical cord is associated with preeclampsia. Source of funding: This work was supported by the Korea Research Foundation Grant funded by the Government(MOEHRD, Basic Research Promotion Fund) (KRF2008-331-E00163) Keywords: heme oxygenase-1, preeclampsia, placenta, umbilical cord
[P02.02]. THE IMPRINTED AMINO ACID TRANSPORTER SNAT4 CONTROLS FETAL GROWTH BY REGULATING UPTAKE OF AMINO ACIDS REQUIRED FOR PLACENTAL GROWTH E Angiolini1,2, R Smith1, K Hoelle2, I Sandovici2, H-W Yung2, G Konfortova1. 1 The Babraham Institute, United Kingdom, 2The University of Cambridge, United Kingdom Imprinted genes are major regulators of fetal growth by controlling the provision of maternal resources to the fetus. Slc38a4, which encodes the System A member SNAT4, is an imprinted gene abundantly expressed in the placenta. Here, we show that a deletion of the predominant imprinted Slc38a4 transcript in mice leads to placental and fetal growth restriction from mid-gestation. SNAT4 is essential for placental cell proliferation during the peak phase of placental expansion and for volume regulation of cells with important endocrine functions during late gestation. Unexpectedly, we found little evidence that SNAT4 mediates the transfer of amino acids to the fetus through the placenta. We propose that SNAT4 plays an essential role in fetal growth by regulating amino-acid supply to key placental cell-types and is a major determinant in the allocation of maternal resources within the placenta. Keywords: Genomic Imprinting, System A amino-acid transporters, Placental growth, Fetal growth
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[P02.04]. THE DIFFERENT GENE EXPRESSION OF HO-1 AND iNOS IN PLACENTA OF INTRAUTERINE GROWTH RESTRICTION Jong yun Hwang*, Ji yeon Lee, Sung hun Na, Tae gu Ahn, Dong heon Lee, Young Myeong Kim. Kangwon National University, Republic of Korea Background: Heme Oxygenase (HO) and nitric oxide synthase (NOS) have similarities in some aspects; antioxidant function, generating gaseous molecule (carbon monoxide, nitric oxide), similar isoform. HO and NOS play signicant role in placentation, placental angiogenensis and antioxidant protection from oxidative stress in normal pregnancy. Some researcher suggested that HO and NOS may be regulated by mutual isoform enzymes. However, the exact comprehension for the compensatory regulation between two enzymes was decient. Intrauterine growth restriction (IUGR) is a growth failure of the fetus. IUGR was related to an inadequacy in the supply of nutrients and oxygen from mother to the fetus through the placenta. The common cause of IUGR is a placental insufciency to result in hypoxia in fetus. We hypothesized that the deciencies of HO, HO-catalytic products, NOS and NOS-catalytic products may result in IUGR and there is a reciprocal compensatory system in two systems. Objects: The aim of this study was to identify the gene expression of HO-1 and iNOS in placenta of IUGR and to evaluate the potential reciprocal compensatory regulation between two systems. Methods: In this study, we designed Case-control study including women with IUGR. The placenta tissue samples were obtained from IUGR patients and normal pregnancy. We quantied gene expression of HO-1 and iNOS on placenta by RT-PCR, real time PCR. Results: In this study, it demonstrates that a signicant down-regulation of HO-1 gene expression in placenta when compared to normal placenta. However, we observed a signicant up-regulation of iNOS gene expression in placenta when compared to normal placenta. Conclusion: The present study demonstrates that there are different responses to placental insufciency in two enzyme systems; the downregulation of HO-1 gene expression and up- regulation of NOS gene expression. We suggest that HO-1 and iNOS system may be regulated by mutual gene expression between two enzymes. Keywords: IUGR, Heme oxygenase, nitric oxide synthase, placenta
[P02.05]. MICRORNA-mRNA NETWORKS IN THE LATE GESTATION MOUSE PLACENTA W Kong*. University of Adelaide, Australia Placental functional development is characterised by dynamic, co-ordinated changes in expression of many regulatory and functional genes and proteins that drive invasion, differentiation and growth. These changes may arise in part from altered expression of microRNA (miRNA) regulatory networks. MiRNAs are short, single-stranded, non-coding RNAs involved in the post-transcriptional repression of gene expression. MiRNAs bind to complementary sites in the 3UTR of target mRNAs to repress or silence translation. This study aims to identify potential miRNA-mRNA regulatory networks that may be involved in the function of the mature mouse placenta. In the current study, microarrays were used to compare miRNA and mRNA gene expression in the labyrinth and junctional zone of the late gestation (day 18) mouse placenta (term w19 days). 13 miRNAs and 601 mRNAs were signicantly differentially expressed between the labyrinth and junctional zone, as selected using Welch t-statistic. The interactions of miRNAs and their putative target mRNAs were modeled as their dependencies with Bayesian networks [1]. This method integrates the prior targeting knowledge and expression proles of miRNAs and mRNAs into Bayesian networks. Putative targets of miRNAs were extracted from freely accessible target databases, miRBase [2], PicTar [3], and TargetScan [4] and assessed by Ingenuity Pathways Analysis. Functional pathways involving the members of Bayesian networks in the junctional zone included, integrin signaling, regulation of actin-based motility, IL-8 signalling (involving genes rhoc, rhod, rhov) and androgen and estrogen metabolism (involving genes hsd17b7, hsd3b4). This could correspond to migration of glycogen cells into the decidua that occurs in late gestation [5]. Signicantly represented pathways found in the labyrinth zone, included amino acid degradation and metabolism, fatty acid metabolism and synthesis and degradation of ketone bodies (involving genes acat1, hsd17b4). This may reect the extensive metabolism of nutrients in the labyrinth zone that occurs in the mature placenta. References 1. Pearl, J., ed. Probabilistic reasoning in intelligent systems: networks of plausible inference. 1988, Morgan Kaufmann Publishers Inc. 2. Grifths-Jones, S., et al., miRBase: microRNA sequences, targets and gene nomenclature. Nucleic Acids Res, 2006. 34(Database issue): p. D140-4. 3. Krek, A., et al., Combinatorial microRNA target predictions. Nat Genet, 2005. 37(5): p. 495-500. 4. Lewis, B.P., Burge, C.B. and Bartel, D.P. Conserved seed pairing, often anked by adenosines, indicates that thousands of human genes are microRNA targets. Cell, 2005. 120(1): p. 15-20. 5. Coan, P.M., et al., Origin and characteristics of glycogen cells in the developing murine placenta. Dev Dyn, 2006. 235(12): p. 3280-94. Keywords: MicroRNA, mouse, placenta, networks
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[P02.07]. E2-EPF UBIQUITIN CARRIER PROTEIN (UCP) MEDIATES HIF-1 INDUCED GENE EXPRESSION IN CHORIOCARCINOMA CELL T Sasaki*, H Nishi, J Liang. Tokyo Medical University, Japan Introduction: E2-EPF ubiquitin carrier protein (UCP) is a member of an E2 family of enzymes that catalyzes the ligation of ubiquitin to proteins targeted for destruction by the proteasome. UCP is overexpressed in common human cancers, suggesting its involvement in oncogenesis, and detected coincidently with Hypoxia Inducible Factor (HIF)-1alpha in human primary liver, colon and breast tumors, and metastatic cholangiocarcinoma. UCP targets the von Hippel-Lindau tumor suppressor, pVHL, for ubiquitin-mediated proteolysis in cells, thereby stabilizing HIF-1alpha. We focused on preeclamptic placenta and examined the relationship between UCP and HIF-1alpha expression. Methods: Total RNAs were isolated from 42 normal placentas and 13 preeclamptic placentas. Real-time reverse transcription-polymerase chain reaction (RT-PCR) was performed to evaluate the UCP mRNA expression level. We examined the effect of overexpression of UCP on HIF-1alpha in choriocarcinoma cell lines by western blot and luciferase assays using luciferase constructs containing the Hypoxia Responsive Element (HRE). Results: Real-time RT-PCR analysis revealed that UCP mRNA expression was signicantly lower in the preeclamptic placentas, compared with normal placentas. By western blot analysis, UCP overexpression enhanced the endogenous HIF-1alpha protein expression. Overexpression of UCP also activated the transcriptional activity of the promoter containing the HRE by luciferase assays. Discussion: We found that UCP expression was down-regulated in preeclamptic placentas and its overexpression induced HIF-1alpha expression in choriocarcinoma cell lines. Our results suggest that high HIF-1alpha expression in preeclamptic placentas is regulated by real hypoxic condition, not by UCP expression. Keywords: hypoxia, transcription, preeclampsia [P02.08]. PLACENTAL LACTOGEN FUNCTION IN POST-IMPLANTATION MURINE PREGNANCY S M Rawn*, J C Cross. University of Calgary, Canada Introduction: In rodents, the Placental Lactogen (PL) hormones are produced by the placenta, detectable in the blood of pregnant mothers from mid-gestation and act through the Prl receptor (PrlR). PL knockout mice have not been reported, but is complicated because there are four PL genes. Prl and PrlR mutant female mice are infertile but in PrlR mutants (129SvJ genetic background) their implantation defect can be rescued by exogenous progesterone. Our aim was to infer PL function by comparing the phenotypes of Prl-/- and PrlR-/- mice, as any differences must be due to effects of PLs. Methods: Maternal parameters (blood pressure, blood glucose, metabolites, spleen weight), implantation rates and fetal growth were observed in Prl-/- and PrlR-/- mice supplemented with progesterone. To account for possible genetic background effects, both mutations were maintained in the C57/Bl6 strain. All females were mated to wildtype C57/Bl6 males. Results: 1) In the C57/Bl6 background, whereas Prl-/- mice were able to carry pregnancies to term, none of the PrlR-/- mice have been able to establish a pregnancy to date. 2) Pregnant Prl-/- females had normal blood pressure and blood glucose. 3) In Prl-/- mice, w50% of fetuses detectable at mid-gestation survived to term. 4) All fetuses in Prl-/- mothers had reduced heart rates and crown-rump lengths at mid-gestation and were leaner at term. Discussion: Our major conclusion is that maternal Prl is not required for pregnancy if implantation is rescued with progesterone. The greater severity of the PrlR mutant phenotype implies that PLs, acting through the PrlR, are essential for establishment and/or maintenance of pregnancy. Of interest, embryos in Prl-/- females are developmentally delayed. Whether this is related to Prl deciency or progesterone supplementation is unclear. Unexpectedly, genetic background affects the PrlR mutant phenotype such that it is less severe in 129SvJ compared to the C57/Bl6 strain. Keywords: Placental Lactogen, Prolactin, post-implantation, developmental delay
[P02.09]. GLUCOCORTICOIDS REPRESS INDUCTION OF CYP1A1 GENE MEDIATED BY ARYLHYDROCARBON RECEPTOR (AhR) LIGANDS METHYLCHOLANTHRENE (MC) OR 2,3,7,8-TETRACHLOROBENZOP-DIOXIN (TCDD) IN PLACENTAL JEG-3 CELL LINE Lucie Stejskalova*1, Katerina Pospechova1, Lucie Svecova1, Radim Vrzal2, Zdenek Dvorak2, Petr Pavek1. 1Department of Pharmacology and Toxi cology, Faculty of Pharmacy in Hradec Kralove, Charles University in Prague, Czech Republic, 2Department of Cell Biology and Genetics, Faculty of Sciences, Palacky University in Olomouc, Czech Republic CYP1A1, an enzyme of the cytochrome P450 family, is the most important biotransformation enzyme in the placental barrier for which relevant inducible activity has been demonstrated in the placental trophoblast throughout pregnancy. CYP1A1 metabolizes several drugs widely used in pharmacotherapy. At the same time CYP1A1 plays a key role in bioactivation of procarcinogens and proteratogens such as polycyclic aromatic hydrocarbons (PAHs) to form DNA-adducts, which bind to placental and fetal DNA and damage it. Expression of CYP1A1 is transcriptionally regulated through the ligand-activated aryl hydrocarbon receptor (AhR). It has been reported that glucocorticoids (synthetic and endogenous) modulate the induction of CYP1A1 via activated AhR. The aim of this work was to study the cross-talk between arylhydrocarbon receptor and glucocorticoid receptor in transcriptional regulation of CYP1A1 in placental JEG3 cell line exposed to AhR ligands methylcholanthrene (MC) or 2,3,7,8-tetrachlorobenzo-p-dioxin (TCDD) alone and in combination with glucocorticoid dexamethasone. The effect of dexamethasone on ligands-mediated transactivation of CYP1A1 was assessed employing real-time RT-PCR and gene reporter assay in transiently transfected cells with luciferase gene reporter plasmids containing native promoter or responsive sequences of CYP1A1 gene. In addition, nuclear translocation of AhR/ARNT was monitored using Western blotting. Our preliminary results show that 24-h treatment with dexamethasone decrease AhR-mediated transactiovation of CYP1A1 gene in gene reporter assay with both p1A1-luc and pXRE-luc reporter plasmids and that the aromatic hydrocarbon-induced CYP1A1 mRNA was suppressed by dexamethasone. We show that the phenomenon is likely due to decreased nuclear translocation of AhR after treatment with dexamenthasone. Based on our preliminary data, we can suggest cross-talk of GR/AhR pathways in gene regulation of CYP1A1 in placental trophoblast, which is not likely at the level of transcriptional regulation. Acknowledgement This project was supported by the grant number GAUK 170/50/95003. Keywords: arylhydrocarbon receptor, glucocorticoid receptor, cytochrome CYP1A1, TCDD
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[P02.10]. MICRORNA hsa-miR-517a IS POSSIBLY INVOLVED IN TUMOR NECROSIS FACTOR (TNF)-MEDIATED SIGNALING IN HUMAN PLACENTA T Takizawa*, O Ishibashi, T Ishikawa, G Ishikawa, A Katayama, T Takeshita. Nippon Medical School, Japan Objectives: MicroRNAs (miRNAs), small noncoding RNAs, are involved in a variety of biological processes in animals. However, little is known about the role of miRNAs in physiological and pathological processes during pregnancy. Recently, we found that human placental trophoblast cells expressed placenta-specic miRNAs (e.g., hsa-miR-517a). In this study, we performed a proteome analysis by miRNA overexpression, using a trophoblast cell line, to elucidate the biological function(s) of placentaspecic miRNAs. Methods: BeWo cells were transfected with Pre-miR hsa-miR-517a miRNA Precursor Molecules (Applied Biosystems). The transfected cells were harvested 24-72 h after transfection, and subjected to proteome analysis. Cell lysates were subjected to two-dimensional difference gel electrophoresis (2D-DIGE). Proteins in the spots were analyzed using a LC/MS mass spectrometer. Data from mass spectrometry were analyzed using MASCOT software, and proteins hit with signicantly high scores were subjected to IPA (Ingenuity Systems) to create possible networks/pathways involving the proteins. Results: Our proteome analysis by 2D-DIGE resulted in a total of 40 spots with differential intensities between BeWo cells transfected with hsa-miR517a and the negative controls. Subsequent mass spectrometry analysis identied a total of 58 proteins as promising candidates of hsa-miR-517aregulated proteins. Although none of these proteins are included in the in silico targets of hsa-miR-517a, a network-based analysis using IPA suggested that most of the hsa-miR-517a-regulated proteins were mapped to two networks. The rst network is NFKB1- and MAPK-related signaling; the second network TNF-related signaling. Since TNF elicits the activation of both MAPK and NFKB1accompanied by apoptosis induction in many types of cells, the two networks can be merged. Conclusions: A possible function of hsa-miR-517a could be to regulate signal transduction mediated by TNF and/or other death ligands, although more detailed studies are necessary. Our data provide new insights into miRNA biology of the human placenta. Keywords: human placenta, trophoblast, microRNA, TNF [P02.11]. MICRORNA EXPRESSION CHANGES DETECTED BY MICROARRAY WITH FORSKOLIN SYNCYTIALISATION OF BeWo CELLS Y Gao1,3, UW Nilsson1,2, C Whitehead1,2, S Tong*1,2. 1Centre for Womens Health Research, Monash Institute of Medical Research, Australia, 2Centre for Cancer Research, Monash Institute of Medical Research, Australia, 32nd Afliated Hospital, Xian Jiaotong University School of Medicine, China Introduction: Syncytialisation is a process of cytotrophoblast maturation where cells undergo cytoplasmic fusion. The resulting syncytium is a multinuclear structure lining the fetoplacental interface, and plays crucial endocrine, immune quenching and nutrient exchange roles. MicroRNAs (miRNAs) are endogenously produced and non-coding RNAs that bind to the 3-untranslated region of mRNAs, suppressing translation. They represent a new level of genetic control, possibly regulating a third of all genes. miRNAs highly expressed in the placenta, and putative placental specic miRNAs have been reported1,2. We set out to characterise changes in miRNA expression with syncytialisation. Methods: We used cultured BeWo cells (placental derived cells from choriocarcinoma), syncytialised in 100uM of forskolin and left in 37 C for 48 hours (n4 per condition). Syncytialisation was conrmed by increased hCG levels in the supernatant (51,441 1,551 IU/L with forskolin, vs 3,919 246 IU/L for controls) and loss of E-Cadherin staining on immunohistochemistry (Figure 1). Total RNA was extracted using Trizol reagent. RNA purity and quality was conrmed with the Experion system (Bio-Rad). A miRNA array (Exiqon) was performed on all samples, with >500 human miRNAs represented.
Results: Of 1,537 miRNAs on the array, there were 18 human specic miRNAs that showed signicantly different changes in expression (Table 1). Eleven were downregulated. Of these, six were previously shown to be highly expressed in the placenta with three [miR 20, 492, 584] being possibly placental specic (4-6 fold higher than all other tissues screened1,2). Of seven miRNAs that were upregulated, four were previously shown to be expressed in placenta with one being possibly placental specic [miR 23a]1,2. Conclusion: Syncytialisation is associated with rapid changes of placental expressed miRNAs, including miRNAs possibly unique to placenta. miRNAs may play crucial roles in syncytialisation. References 1. Bentwich et al. Nature Genetics 2005;37(7):766-770. 2. Barad et al. Genome Research 2004;14:2486:2494.
Table 1: miRNAs that changed in expression with syncytialisation.

miRNAs not miRNAs reported to specic to be highly expressed placenta in placenta Downregulated miR32 miR175b miR422b miR565 miR654 Upregulated miR455 miR483 miR637 miR20a miR20b miR21 miR32 miR183 miR492 miR23a miR23b miR326 miR513 miRNAs that may be placental specic (ref 1,2) subset of previous column miR-20a miR-492 miR584
miR-23a
Keywords: microRNA, syncytialisation, BeWo cells, microarray
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[P02.12]. THE IMPRINTED GENE Phlda2 REGULATES THE GLYCOGEN CELL POPULATION OF THE MOUSE PLACENTA S J Tunster*1, B Tycko2, R M John1. 1Cardiff University, United Kingdom, 2 Columbia University, United States Introduction: Genomic imprinting is proposed to account for the transition from a relatively primitive non-invasive placenta in marsupials to the more complex and invasive placenta characteristic of eutherian mammals (John and Surani 2000; Kaneko-Ishino et al. 2003). The maternally expressed imprinted gene Phlda2 has previously been shown to regulate placental development, with loss of expression associated with placentomegaly due to a disproportionate expansion of the spongiotrophoblast layer (Frank et al. 2002). Here we further characterise the effect of overexpressing this gene in the mouse placenta. Methods: We have previously briey reported on transgenic mice that carry 3 copies of a BAC transgene spanning the imprinted gene Phlda2 (Salas et al. 2004). Here we present the detailed characterisation of placental development in response to incrementally increasing dosage of Phlda2 on both 129/Sv and mixed 129/Sv x C57BL/6 genetic backgrounds. Results: We directly show that placental stunting in Phlda2 transgenic mice is due to a disproportionate loss of the junctional zone. A defect in the ectoplacental cone is apparent from E10.5 with a loss of the proliferating component. Late in development there is a striking reduction in glycogen cell staining and at E16.5 the glycogen cells fail to migrate to the maternal decidua in a timely manner. Instead, Tpbpa-positive cells progressively mislocalise in the labyrinth layer. Furthermore, embryonic growth is adversely affected from E16.5 with transgenic animals born w13% lighter than wild type. Conclusions: Glycogen cells may represent a source of readily metabolised energy during the nal stages of gestation when the embryo is placing the highest demand on maternal resources (Coan et al. 2006). Over-expression of Phlda2 is associated with a reduced glycogen cell population. Imprinting of Phlda2 may be responsible for the expansion of this cell type thus permitting prolonged gestation and the birth of developmentally mature progeny. Keywords: Imprinting, Glycogen cells, Placenta, IUGR
[P02.13]. BIPARENTAL EXPRESSION OF HUMAN CGB GENES IS REQUIRED FOR SUCCESSFUL IMPLANTATION L Uuskula*1, K Rull1,2, L Nagirnaja1, M Laan1. 1Institute of Molecular and Cell Biology, University of Tartu, Estonia, 2Department of Obstetrics and Gynecology, University of Tartu, Estonia Introduction: Placentally expressed Chorionic Gonadotropin Hormone (hCG), composed of a and b subunits, has an irreplaceable role in the implantation of an embryo in primates. In human, the four genes coding for hCG and luteinizing hormone beta subunits are co-located in the LHB/ CGB gene cluster at 19q13.32. Several genes expressed in placenta and essential for reproduction are known to be imprinted. This study aimed to (i) explore the parental origin of hCG beta transcripts; and (ii) compare DNA methylation status of hCG beta promoters between trophoblastic tissue and blood leukocytes. Chorionic Gonadotropin beta 5 (CGB5) and Chorionic Gonadotropin beta 8 (CGB8) genes were used as a model since these two loci are contributing 2/3 up to 3/4 of total hCG beta mRNA transcripts. Study groups: We analysed trophoblastic material from 23 motheroffspring duos and 9 mother-father-offspring trios including cases of (1) III trimester normal delivery at term (n14), (2) I trimester elective termination of pregnancy (n10) and (3) recurrent (!3) miscarriages (RM) during I trimester of pregnancy (n8). Methods: (1) Parental origin of the transcribed alleles of CGB5 and CGB8 was determined using gene-specic RT-PCR, cloning and sequencing. (2) Methylation status of a CGB5 promoter CpG island was assessed by methylation-specic PCR after bisulphite treatment of the genomic DNA, followed by cloning and sequencing. (3) Large chromosomal rearrangements were detected using Illumina CNV370-Duo array system. Results: (1) Normal successful pregnancy requires biparental expression of the CGB5 and CGB8 genes. (2) Trophoblastic material of some RM cases revealed an allelic imbalance only maternally inherited CGB5 gene alleles were expressed. (3) RM cases with uniparental CGB5 gene expression were characterized by hemimethylated CGB5 promoter as well as abundant chromosomal arrangements across genome. We hypothesize that chromosomal abnormalities (e.g. aneuploidy) may affect epigenetic programming during embryonic development. Keywords: reproduction genetics, chorionic gonadotropin genes, DNA methylation
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[P02.14]. DIFFERENTIALLY METHYLATED REGION IN INTRON 1 OF STOX1 EXPLAINS PARENT-OF-ORIGIN EFFECT SEEN IN PRE-ECLAMPSIA LINKAGE ANALYSIS M van Dijk*, CBM Oudejans. VU University Medical Center, Netherlands Introduction: The 10q22 chromosomal region with genomic linkage to pre-eclampsia shows a parent-of-origin effect with maternal transmission1,2. By studying the methylation status of the CpG island within the STOX1 promoter region, we and others3,4 have been unable to identify a differentially methylated region (DMR) explaining this parent-of-origin effect. Recently, in intron 2 of stox1 in mice, a tissue-specic DMR has been identied5. Furthermore, in human lymphoblastoid cells a differential allelic expression ratio was detected for STOX1 SNPs6. Both of these studies indicate the existence of a DMR in humans leading to differential expression, and potentially explain the parent-of-origin effect observed. Methods: By computational analysis a region within intron 1 of the STOX1 gene was identied as a potential CpG island. This was conrmed using CTconverted DNA of buffycoat samples as a source of cells in which differential expression was observed6. Normal and androgenetic early placenta samples were tested in the same manner along with term placenta, SGHPL5 cells and adult tissue samples. Results: The methylation pattern identied in term placenta and adult tissue is consistent with an imprinted gene in which 50% of the alleles are methylated in tissues expressing STOX1, while 80% of the alleles are methylated in tissue with very low levels of STOX1 expression (liver). In early placenta only 20% of the alleles were methylated, while androgenetic placentas, consisting of paternal alleles only, showed 50% methylation. SGHPL5 cells, representing extravillous trophoblasts, were also 50% methylated. This indicates that in early placenta, in contrast to term and adult tissue, only some cell types (extravillous trophoblasts among others), are subject to imprinting. Conclusion: This study conrms that the parent-of-origin effect observed in the pre-eclampsia linkage analysis is caused by a DMR within STOX1, leading to cell type-specic imprinting in the early placenta. References 1) Oudejans et al., Hum Mol Reprod 2004. 2) van Dijk et al., Nat Genet 2005. 3) van Dijk et al., Nat Genet 2007. 4) Iglesias-Platas et al., Nat Genet 2007. 5) Suzuki et al., Genes Cells 2007. 6) Cheung et al., Am J Hum Genet 2008. Keywords: pre-eclampsia, STOX1, parent-of-origin, differentially methylated region
[P02.16]. FEATURES OF CONSTITUTIVE HETEROCHROMATIN REGIONS EXTRAEMBRYONIC AND EMBRYONIC TISSUE IN HUMAN EMBRYOS Tromova Irina*, Kuznetzova Tatyana. Saint-Petersburg State University, Russian Federation Epigenetics modications of chromatin are methylation DNA and histone modication. Centromeric chromatin is constitutive heterochromatin regions (CHR). DNA located in this site of chromosome, belongs to the class of satellite DNA. The implications of epigenetic silencing in normal developmental gene regulation, aging and cancer progression have made heterochromatin the focus of intense investigation. Special interest is represented large CHR autosome 1, 9, 15,16, 22 and Y-chromosomes of human in which presence of the structural genes is supposed and functional value is discussed. CHR in chromosomes of different tissues are characterized by tight condensation, late replication and methylation. Meanwhile decondensation, hypomethylation CHR in human trophoblast and tumor cells were registered. Stress-induced transcription of satellite III in some cell lines was shown. We studied denite peculiarities of CHR in direct and semi-direct slides from chorionic villi and embryonic tissue samples (8 and 3 accordingly) from articial abortions after pretreatment of slides with different enzymes (RNase A, DNase I, DNA- ligase T4) and dyeing by acridine orange. Separate and combined pre-treatment of slides with different nucleases (RNase A and DNase I) observed of red uorescence of constitutive heterochromatin (typical for single-stranded DNA and RNA) in 1qh, 9qh, 16qh, Yqh, 15cenh, 22cenh most metaphase plates. Chromosome arms were relatively uniform stained or banded similar to RFA in some metaphases. Subjection to DNA -ligase T4 resulted in total absence of uorescence ("black holes") in CHR and uniform green uorescence along chromosome arms. Staining pattern corresponded to untreated slides was observed in all control slides treated with corresponding buffer solutions. Our results advocate for unusual conformation packaging of CHR which could be attributed to the feasible transcriptional activity of pericentromeric satellite DNA in embryonic and cytotrophoblast cells during embryogenesis.
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[P02.17]. UNIQUE PROFILE OF DNA METHYLTRANSFERASE PROMOTER METHYLATION IN HUMAN PLACENTAL TISSUE AND PURIFIED FIRST TRIMESTER TROPHOBLASTS B Novakovic1,2, N Wong1,2, M Sibson1, J Craig1,2, R Saffery*1,2. 1Murdoch Childrens Research Institute, Australia, 2University of Melbourne, Australia One of the earliest lineage differentiation events in embryogenesis involves the formation of extraembryonic and embryonic lineages in the blastocyst. This is accompanied by a dramatic change in global DNA methylation levels, such that extraembryonic (and placental) tissue is globally hypomethylated in relation to all somatic tissues. Despite this, it is becoming increasingly clear that the placenta also shows a unique prole of gene specic hypermethylation. The family of DNA methyltransferases are responsible for both the maintenance (DNMT1) and de novo establishment (DNMT3a, -3b, -3L) of DNA methylation during cell division and development. In order to examine the potential role of these genes in regulating the placental epigenome, we investigated the promoter methylation status of each in human placental tissue and puried cell populations using state-of-the-art Sequenom MassARRAY EpiTyping. No promoter methylation of DNMT3A or -3B was seen in any tissue tested. Intriguingly however, both full term and rst trimester placenta show specic hypermethylation of the maintenance DNA methyltransferase-1 (DNMT1) gene and hypomethylation of the DNMT3L gene that modulates DNMT3A and -3B- mediated de novo methylation. This was conrmed by bisulphite sequencing methylation analysis which also revealed monoallelic methylation of DNMT1 in villous tissue and puried cytotrophoblasts, with no evidence of imprinting (parent of origin effect). DNMT1 was not methylated in any somatic tissue tested while DNMT3L is hypermethylated and silenced in most somatic tissues. In vitro methylation experiments conrmed that DNMT1 promoter methylation attenuates transcriptional activity in human trophoblasts. This pattern of concomitant epigenetic down-regulation of the maintenance DNMT1 gene with up-regulation of the DNMT3L regulator of de novo methyltransferases is anticipated to have a profound effect on the establishment of the placental epigenome. Keywords: DNA methylation, DNMT1, DNMT3L, Epigenome
[P03.01]. ALTERED PLACENTAL GENE EXPRESSION IN RESPONSE TO MATERNAL DEXAMETHASONE EXPOSURE IN THE MOUSE JSM Cuffe*1, H Dickinson2, WM Boon2, KM Moritz1,2. 1School of Biomedical Sciences, The University of Queensland, Australia, 2Department of Physiology, Monash University, Australia Introduction: Increased maternal glucocorticoid exposure has been shown to program adult onset disease in offspring. Previous studies have utilized a number of animal models to look at the effects of short term maternal glucocorticoid exposure, but rarely has this been done in the mouse. We tested the hypothesis that a short term maternal dexamethasone exposure during pregnancy in the mouse would affect the function of the developing placenta which may contribute to the programming of disease. Methods: C57/BL/6 mice were infused with dexamethasone (DEX) or saline (SAL) for 72 hours via osmotic minipump beginning at embryonic day (E) 12.5. Placentas were collected at E14.5 (during infusion) or E17.5 (after infusion) and embryo and placental weights recorded. Gene expression of GLUT1, GLUT3, Map2k1 and 11bHSD2 was examined by real time PCR. Results: Placental and foetal body weights tended to be reduced in response to DEX at E14.5 (P0.06), however at E17.5 there was no differences between groups in body or placental weight. DEX caused signicant alterations in gene expression as shown in the table below: Relative gene expression: (n9-10 samples from 4-6 litters, *P<0.05)
Gene GLUT1 GLUT3 Map2k1 11bHSD2 E14.5 E17.5 E14.5 E17.5 E14.5 E17.5 E14.5 E17.5 SAL 1.150.22 1.020.07 1.050.10 1.020.06 1.100.15 1.010.06 0.990.47 1.000.10 DEX 2.620.49* 1.540.49 1.150.08 0.720.07* 1.730.24* 0.710.08* 2.500.44 4.051.70
Conclusion: Using our novel mouse model of short term DEX exposure, we show a transitory reduction in both placental and foetal weight indicating DEX has directly inhibited growth and development. However, normal weight two days after completion of treatment suggests signicant catch up growth. Changes in glucose transporter gene expression suggest alterations in placental and foetal nutrient supply while changes in MAP2k1 expression suggests altered placental vasculogenesis. The temporal changes in expression demonstrate direct and compensatory effects of DEX exposure. Keywords: Glucocorticoids, mouse, placenta, genes
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[P03.02]. ALTERED GLUCOCORTICOID METABOLISM IN PLACENTAL TISSUE FROM FIRST TRIMESTER PREGNANCIES AT INCREASED RISK OF PRE-ECLAMPSIA S Mukherjee*, JE Cartwright, G Whitley StJ, AE Michael, B Thilaganathan. St Georges, University of London, United Kingdom Introduction: Glucocorticoids may exert important actions in the placenta in early pregnancy. The local actions of glucocorticoids can be modulated by 11b-hydroxysteroid dehydrogenase (11bHSD) enzymes which catalyse inter-conversion of cortisol with its inert metabolite, cortisone. Although, 11bHSD isoenzymes are known to be expressed in the placenta and decidua, their expression and activity have not been well characterised in the rst trimester placenta. Measurement of uterine artery resistance indices (RI) by Doppler Ultrasound in the rst trimester can be used to assess the risk of developing pre-eclampsia. The aim of this study was to compare 11bHSD expression and activity in rst trimester placental tissue from pregnancies screened as being at the highest and lowest risk of developing pre-eclampsia. Methods: Expression of 11bHSD enzymes was assessed in rst trimester placental tissue by western blot analysis and immunohistochemistry. Enzyme activities were assessed using radiometric conversion assays. Results: Western blot analysis conrmed expression of 11bHSD2 in rst trimester placental tissue and immunohistochemistry localised expression of the 11bHSD2 protein to the syncytiotrophoblast. 11bHSD1 expression was not detected in chorionic villous tissue. Enzyme activity assays conrmed NAD+-dependent inactivation of cortisol by 11bHSD2 in rst trimester placental tissue. Net cortisol oxidation was signicantly greater in placental tissue from pregnancies at higher risk of pre-eclampsia than in low risk pregnancies (50.915.9 versus 18.31.9 pmol cortisone/mg protein, n11 & 12, respectively; p<0.05). Discussion: 11bHSD2 expression is thought to protect the fetus from exposure to maternal cortisol. While other studies have suggested that 11bHSD2 is downregulated in term pre-eclamptic placentae, our studies suggest that there is increased activity in rst trimester placenta from pregnancies at higher risk of developing pre-eclampsia. It remains to be determined if it is related to the pathopysiology of pre-eclampsia or is a compensatory response to poor trophoblast development. Keywords: Trophoblast, Glucocorticoids, Pre-eclampsia, Uterine Dopplers
Results: There were 42 genes differentially expressed between the IUGR and healthy antenatal cohort. Corticosteroid administration to the IUGR group resulted in very signicant changes in mRNA transcripts, with 431 genes differentially expressed. The two samples from the IUGR group post corticosteroids showed a gene prole clustering distinctly different to the remaining six samples (two IUGR pre steroids and the four healthy antenates see heatmap/Figure 1). Gene set enrichment analysis showed that hypoxic, apoptosis, oxygen tissue binding and placental genes as notable gene pathways that increased signicantly when corticosteroids were given. Conclusion: Corticosteroid administration in severe onset IUGR is associated with acute elevation in mRNA transcripts associated with hypoxia, oxygen binding, apoptosis and placental regulation. These changes are consistent with animal and human observations that corticosteroid administration to IUGR pregnancies may have detrimental effects. These observations are being currently veried in a larger cohort.
[P03.03]. CORTICOSTEROIDS UP-REGULATE HYPOXIC, APOPTOTIC AND PLACENTAL GENES IN SEVERE GROWTH RESTRICTION: GENOME-WIDE TRANSCRIPTIONAL PROFILING OF mRNA TRANSCRIPTS IN MATERNAL BLOOD S Tong*1, C Whitehead1, D Wu2, K Palmer1, J Mockler1, EM Wallace1. 1Centre for Womens Health Research, Monash Institute of Medical Research, Australia, 2Bioinformatics Division, Walter and Eliza Hall Institute of Medical Research, Australia Introduction: RNA from the fetoplacental unit is released into the maternal circulation, and is remarkably stable. Therefore, it may be possible to transcriptionally prole the placenta non-invasively by measuring RNA in a maternal venepuncture sample. We set out to examine the prole of mRNA transcripts in maternal whole blood in association with severe early onset growth restriction (IUGR), and to determine mRNA changes with administration of corticosteroids. Methods: We collected maternal whole blood from 4 healthy antenates and two patients at 27 weeks gestation with severe early onset IUGR. We collected further samples from the two patients with IUGR 24 hours after administration of corticosteroids (and documented ultrasound doppler changes in response to the steroids). We used the Paxgene system [PreAnalytix] to collect and isolate the RNA, conrmed purity [Experion], and did a genome-wide microarray [Illumina platform]. We performed differential analyses, which included assessment of genes grouped according to biological pathways (Gene ontogeny analysis).
Figure 1. Heat map of 58 representative genes to show IUGR post steroids group (n2) cluster differently from IUGR pre-steroids (n2) and healthy antenates (n4).
Keywords: glucocorticoids, mRNA, hypoxia, microarray
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[P04.01]. DECIDUAL NEUTROPHILS A NOVEL FINDING: THEIR ROLE IN SECOND TRIMESTER PLACENTATION HA Amsalem*1,2, CD Dunk1, SL Lye1,2, A Hazan2. 1Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Canada, 2University of Toronto, Canada Objectives: The fetomaternal interface once thought to be an immune privilege site is now known to harbour a large population of maternal immune cells. These cells are crucial to the angiogenesis and vascular remodelling accompanying normal placentation. Accumulating data shows that neutrophils, a major part of the innate immune system, may play a crucial role in vascular remodelling within tumor tissue. Little is known regarding the presence and role of neutrophils in normal and aberrant placentation. We hypothesize that neutrophils inltrate second trimester decidua from the maternal peripheral circulation and contribute to vascular remodelling. Methods: Following informed consent, second trimester decidual tissues (n5) were collected from patients during social termination of normal pregnancy. Leukocytes were released from decidual tissues by mechanical mincing and immunostained for FACS analysis using Anti CD66b and CD15 (as neutrophil markers) and CD 45 (leukocytes marker). The same decidua was also used for immunohistochemistry staining in order to localize a specic cellular distribution of cells within the tissue. Decidua tissues were immunostained for CD66b, neutrophil elastase (neutrophil markers) and cytokeratine. Results: Our FACS analysis showed that 5-10% of the total CD45+ cells (leukocytes) in the second trimester decidua are neutrophils. NK subpopulations were used as purity control to rule out peripheral blood contamination. Immunostaining conrmed the presence of neutrophils within decidual tissue. Neutrophils were observed adhered to endothelium and inltrating the vascular wall. Large aggregates of neutrophils were seen within the decidual stroma. Our preliminary data suggest that neutrophil inltration is restricted to the decidua basalis. Conclusions: This observation is the rst demonstration of a resident neutrophil population in the second trimester decidua. Future studies will address their potential role at the fetomaternal interface. Keywords: decidua, neutrophils, inltration, vascular remodelling
[P04.02]. DECIDUAL MACROPHAGES FROM FIRST TRIMESTER PREGNANCIES AT INCREASED RISK OF PRE-ECLAMPSIA HAVE LOWER MHC CLASS II EXPRESSION AND ALTERED CYTOKINE PRODUCTION C Austen*, AP Johnstone, R Fraser, G StJ Whitley, B Thilaganathan, JE Cartwright. St Georges University of London, United Kingdom Introduction: Macrophages constitute 20-30% of the decidual immune cells at the site of implantation. The role these cells have in the regulation of placentation or the pathology of pre-eclampsia has not been determined. Measurement of uterine artery resistance indices (RI) by Doppler Ultrasound in the rst trimester can be used to identify pregnancies with a 30% risk of developing pre-eclampsia (bilateral uterine artery notching and mean RI>95th centile) or <1% risk (no notches and a mean RI<95th centile). The aim of this study was to compare rst trimester decidual macrophages isolated from pregnancies screened, prior to termination of pregnancy, as being at the highest and lowest risk of developing preeclampsia, had the pregnancy progressed. Methods: CD14+ macrophages were isolated from rst trimester decidua using antibody-coated magnetic beads. Expression of MHC Class I and II was determined by ow cytometry. The secretion of cytokines during 16h culture was assessed using a cytokine array (Proteome Proler Antibody array, R&D Systems). Results: Macrophages from high resistance pregnancies showed reduced expression of MHC Class II and lower levels of IL-1b, IL-6, MIP-1a and TNFa secretion compared with cells from normal resistance pregnancies. No differences were detected in MHC Class I expression. Discussion: First trimester decidual macrophages from pregnancies classied as at higher risk of developing pre-eclampsia differ in their MHC Class II expression and cytokine secretion compared to lower risk pregnancies. First trimester decidual macrophages in a lower risk pregnancy may have an increased activation state (indicated by the higher level of MHC Class II) which, in addition to the higher levels of pro-inammatory cytokine production, may be important in the promotion of trophoblast invasion. During a pregnancy with a high resistance index the altered decidual cytokine environment, contributed in part by the macrophages, may be a factor in the development of pre-eclampsia. Keywords: Macrophage, Pre-eclampsia, MHC, Cytokine
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[P04.03]. NECROTIC DEPORTED TROPHOBLASTS APPEAR TO ACTIVATE MACROPHAGES: A POTENTIAL MECHANISM CONTRIBUTING TO THE PATHOGENESIS OF PREECLAMPSIA Q Chen*1,2, JX Ding1, LM Chen1, HY Jin1, PR Stone2, LW Chamley2. 1The Hospital of Obstetrics & Gynaecology, Fudan University, China, 2Department of Obstetrics & Gynaecology, The University of Auckland, New Zealand Preeclampsia is characterised by an exaggerated maternal inammatory reaction. In normal pregnancy, trophoblasts die by an apoptosis-like process and are shed into the maternal blood then deported from the uterus. In preeclampsia it has been suggested that the shed trophoblasts are more necrotic. It is unclear how shed/deported trophoblasts are cleared from the maternal circulation but macrophages may be involved. Although trophoblasts do not express classical class I HLA, they are immunologically foreign to maternal immune system. We previously reported that phagocytosis of apoptotic trophoblasts by macrophages resulted in an antiinammatory/immune suppressing response. Here we compared the effects of necrotic and apoptotic shed trophoblasts on macrophages. Red uorescent-labelled shed trophoblasts were harvested from placental explants and either used directly (apoptotic) or induced to undergo secondary necrosis by freeze-thawing. The shed trophoblasts were fed to PMA-treated U937 macrophages activation examined by immunostaining for HLA-II expression. Peripheral blood mononuclear cells (PBMNCs) were cultured in the presence of supernatants from untreated U937 cells or U937 cells that had phagocytosed apoptotic or necrotic trophoblasts and the PBMNC proliferation determined by Alarmar Blue assay (Invitrogen). Confocal microscopy demonstrated that HLA-II expression was substantially increased on U937 cells that had phagocytosed necrotic shed trophoblasts compared to U937 cells that had phagocytosed apoptotic shed trophoblasts or untreated controls. The proliferation of PBMNCs treated with conditioned medium from U937 cells that had phagocytosed necrotic shed trophoblasts was signicantly greater (p<0.005) than the proliferation of PBMNCs treated with conditioned medium from U937 cells that had phagocytosed apoptotic trophoblasts or untreated U937 cells. These data suggest that changing the death process leading to trophoblast shedding towards a more necrotic process could result in an inappropriate maternal immune response initiated by maternal macrophages. Such an immune response might contribute to the exaggerated maternal inammatory reaction of preeclampsia. Keywords: trophoblast deportation, HLA-II, marcophages, phagocytosis
[P04.04]. CHARACTERIZATION OF ANTIGEN PRESENTING CELL SUBSETS IN PREGNANCY-ASSOCIATED MALARIA DURING A FOLLOW-UP STUDY IN BENIN N Fievet*2, S Ibitokou11, B Vianou11, C Agbowa1, M Oesterholt5,1, A Massougbodji1. 1University of Abomey Calavi, Benin, 2UR010 IRD, Benin, 3 Institute of International Health, University of Copenhagen, Denmark, 4 Wenner-Gren Institute, Stockholm University, Sweden, 5Radboud University Nijmegen, Netherlands, 6UR010, IRD, IFR 71 Universite Rene Descartes, France Introduction: The central phenomenon in the pathogenesis of Pregnancy Associated Malaria (PAM) is the accumulation of P. falciparum (Pf) infected erythrocytes in the placenta. The STOPPAM consortium conducts 2 cohort studies in pregnant women from Benin and Tanzania to evaluate the immunopathological consequences of Pf infections during pregnancy. Dendritic Cells (DCs) are of particular relevance in pregnancy-related infections given their role to induce pregnancy tolerance, and antigenspecic immunity, thus contributing in protecting the mother from infections without compromising fetal survival. Data on DCs in PAM are needed to understand the cellular mediated immunity (CMI) implication in a future PAM vaccine design. Methods: In Come, southwestern Benin, a longitudinal prospective study of 1000 pregnant women is ongoing. Pregnant women are enrolled before 24 weeks of pregnancy and followed with full clinical, haematological, and parasitological investigations at each ANV until delivery. CMI is performed 1) in a subgroup of 150 women at inclusion: 75 women with active Pf infection, matched for gravidity and gestational age with 75 women with neither Pf infection at inclusion nor history of such infection during earlier pregnancy; 2) in a subgroup of 120 women at delivery: 40 with an active placental Pf infection, 40 with no placental infection but having presented with Pf infection during pregnancy, and 40 with no Pf infection during whole pregnancy. Ex vivo DC and monocyte phenotyping, and activation of antigen presenting cells after LPS activation are evaluated using ow cytometry. Results and Discussion: We will compare data on women at inclusion and at delivery according to the timing and pathology of malaria infection. Hitherto, CMI was explored in 100 women at inclusion and in 25 at delivery. At time of the IFPA congress, all women will have been investigated. Keywords: Plasmodium falciparum, dendritic cells, monocytes, Placenta
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[P04.05]. DECIDUAL NATURAL KILLER CELLS FROM PREGNANCIES AT HIGHER RISK OF PRE-ECLAMPSIA FAIL TO INDUCE ENDOTHELIAL APOPTOSIS: A DEFECT IN SPIRAL ARTERY REMODELLING? R Fraser*, GStJ Whitley, AP Johnstone, B Thilaganathan, JE Cartwright. St. Georges, University of London, United Kingdom Introduction: During pregnancy uterine spiral arteries are remodelled into larger diameter, higher ow vessels. This change is essential for the developing fetus to obtain sufcient oxygen and nutrients. In preeclamptic pregnancies insufcient remodelling occurs. Trophoblasts play a role in spiral artery remodelling through induction of vascular cell apoptosis. However, the role of decidual natural killer cells (dNK), which are the major maternal immune component of the decidua, has not been determined in remodelling. The aim of this study was to establish the effect of dNK on vascular cells and to investigate whether there are functional differences in dNK isolated from pregnancies at higher risk of developing pre-eclampsia. Methods: Measurement of uterine artery resistance indices (RI) by Doppler Ultrasound in the rst trimester can be used to identify pregnancies with a 30% risk of developing pre-eclampsia (bilateral uterine artery notching and mean RI>95th centile) or <1% risk (no notches and a mean RI<95th centile). CD56+ dNK cells were isolated from rst trimester decidua by positive selection using antibody-coated magnetic beads. Endothelial cells were co-cultured with dNK cells, the NK92 cell line or NK cell conditioned medium. Apoptotic morphology was examined by time-lapse microscopy. Results: Endothelial cells underwent apoptosis in the presence of dNK cells isolated from normal RI pregnancies. This effect was seen in direct coculture but not in the presence of dNK conditioned medium or when NK92 cells were used. Apoptosis was inhibited in the presence of the caspase inhibitor zVAD-fmk. dNK from high RI pregnancies did not induce endothelial cell death above basal levels. Discussion: Decidual NK cells can cause endothelial cell death through direct interactions and may therefore have a role in spiral artery remodelling. The deciency in endothelial apoptosis induction shown by dNK from high RI pregnancies may contribute to the impaired remodelling seen in pre-eclamptic vessels. Keywords: natural killer cell, pre-eclampsia, endothelial cell, apoptosis
[P04.07]. AUTOIMMUNITY TO THE ENDOPLASMIC RETICULUM PROTEIN CALRETICULIN DURING HUMAN PREGNANCY
RESIDENT
N. M. Gude*1,2, J. L. Stevenson1, P. M. Sheehan1,2, S. P. Brennecke1,2. 1Royal Womens Hospital, Australia, 2University of Melbourne, Australia The calcium-binding endoplasmic reticulum resident protein calreticulin is signicantly increased in maternal blood throughout human pregnancy compared to the non pregnant state (Gu et al, Molec Hum Reprod, 14:309315, 2008). The role of circulating calreticulin in human pregnancy is not known. Calreticulin can generate an autoimmune response when present in the extracellular environment. This has been proposed to contribute to the progress of autoimmune diseases such as systemic lupus erythematosus. The aim of this study was to determine the prevalence of anticalreticulin IgG in a cohort of pregnant women, and to assess changes in antibody titre throughout gestation. 103 women without autoimmune disease or other pre-existing pathology were recruited at their rst antenatal visit and blood was taken at approximately fortnightly intervals. Autoantibodies were measured using a high stringency ELISA and titrated against a standard source of anti-calreticulin IgG. A positive reaction was taken as >mean plus 3 standard deviations of the nonspecic binding wells. Pregnancy outcomes were: 85 normal, 6 preeclampsia (2 with fetal growth restriction), 5 pregnancy-induced hypertension, 4 gestational diabetes, 2 preterm labour and 1 fetal growth restriction alone.
This is the rst time anti-calreticulin autoantibodies have been reported in human pregnancy. The prevalence in this cohort of pregnant women (5.8%) is similar to that observed by other studies for non pregnant, control populations (i.e. without autoimmune disease). Further work is required to determine if the presence of maternal autoantibodies to calreticulin during pregnancy is associated with altered risk of adverse pregnancy outcome. Keywords: calreticulin, autoimmunity, maternal blood, antibody
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[P04.08]. CD4+FOXP3+ T REGULATORY CELLS ABUNDANCE DURING PREGNANCY IS INFLUENCED BY INTERLEUKIN-10 IN CONCERT WITH FETAL ALLOANTIGENS LR Guerin*1, JR Prins2, JD Hayball3, SA Robertson1. 1Research Centre for Reproductive Health, Discipline of Obstetrics and Gynaecology, University of Adelaide, Australia, 2Department of Obstetrics and Gynaecology, University Medical Center Groningen, University of Groningen, Netherlands, 3Hanson Institute and Sansom Institute, Australia Maternal immune tolerance of conceptus alloantigens is critical in ensuring pregnancy success. T cells known as regulatory T cells (Treg cells) that express the hallmark markers CD4 and Foxp3 are essential for mediating fetal tolerance. However, the factors that control Treg cells during pregnancy are unknown. We endeavoured to analyse the role of interleukin-10 (IL-10) in regulating Treg cell function and abundance in pregnancy. Female IL-10 decient (IL-10-/-) or wild type (WT) C57Bl/6 were mated with either Balb/c (allogeneic) or C57Bl/6 (syngeneic) males. Treg cells were analysed using anti-Foxp3 antibodies and FACS analysis in the iliac and inguinal lymph nodes or in the uterus via immunohistochemistry throughout gestation. IL-10 deciency was shown to result in an elevation of the percentage of CD4+ cells expressing the Treg cell marker Foxp3 by approximately 30%, and to increase total cell numbers by w2-fold in all lymphoid tissues analysed. In females gestating allogeneic foetuses, there was an increase in Treg cells in the uterine draining (iliac) lymph nodes, but not other lymph nodes, with a peak of w10-fold more Treg cells at gestational day (gd) 10. This was accompanied by an w50% increase in the percentage of CD4+ cells expressing Foxp3 in both the iliac and inguinal lymph nodes. In mice gestating syngeneic fetuses there was no increase in the percentage of CD4+ cells expressing Foxp3 due to IL-10 deciency. When CD4+ Foxp3+ T cells were recovered from iliac lymph nodes of pregnant females and analysed in mixed lymphocyte reaction assays using paternal lymphocytes in vitro, there was no effect of IL-10 genotype on suppressive function. These ndings highlight an important role for IL-10 in Treg cell abundance and lineage commitment throughout gestation. Additionally they indicate a role for fetal alloantigens in promoting the conversion ofCD4+ cells to Foxp3-expressing Treg cells. Keywords: Treg, interleukin 10
[P04.09]. COLOCALIZATION OF HLA-G5, B7-H1 AND LAMP-1 PROTEINS IN THE HUMAN PLACENTA S.K. Kshirsagar, A.S. Trikhacheva, M.G. Petroff*. University of Kansas Medical Center, Kansas, United States The semi-allogeneic fetus enjoys immune privilege by promoting maternal-fetal immunological tolerance. The outermost trophoblastderived layers of the placenta maintain this status by forming a physical barrier with immunomodulatory properties between the mother and the fetus. Among the immunosuppressive proteins expressed by these cells are B7-H1 and HLA-G. In addition to being expressed on the cell surface, these proteins have been found to be associated with trophoblast-derived exosomes in the maternal circulation, suggesting a role for these factors in systemic modulation of the maternal immune system. The aim of this study was to determine whether placental B7-H1 and HLA-G proteins are associated in situ with the secretory lysosomal pathway, which can lead to exosome secretion. The expression of B7-H1, HLA-G and Lamp-1 in placental tissue from term and rst trimester placenta was examined by immunohistochemistry and immunouorescence microscopy. In the syncytiotrophoblast and extravillous trophoblast, B7-H1 and Lamp-1 colocalized at the maternal-fetal interface. Using the isoform specic antibody 12C3, HLA-G5 expression was observed in villous cytotrophoblast cells and extravillous trophoblast cells. Similar staining patterns were observed in rst trimester and term placentas. Cytospin preparations of the term trophoblast cells clearly showed colocalization of Lamp-1 and HLA-G5 in a punctate, intracellular pattern. Finally, HLA-G5 and Lamp-1, but not B7H1, also colocalized in the placental Hofbauer cells. Our results suggest that B7-H1 and HLA-G proteins are associated with secretory lysosomal pathway in the placenta, and support the hypothesis that they can be secreted via exosomes. This work was supported by NIH grants HD045611 and HD049480.
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[P04.10]. FETAL ANTIGEN INDUCES TOLERANCE IN MATERNAL CD4+ T CELLS A. Perchellet, M.G. Petroff*. University of Kansas Medical Center, Kansas, United States Tolerance of the maternal immune system is believed to be important for successful pregnancy as the fetus is semi-allogeneic and may be subject to anti-fetal responses. We examined maternal T cell tolerance to the fetus in mice using a system in which a model antigen, ovalbumin (OVA), is expressed exclusively in the fetus. This is achieved by breeding females that lack OVA to transgenic males that ubiquitously express membranebound OVA. By employing T cell receptor (TCR) transgenic mice specic for either a MHC class I or class II-restricted epitope of OVA (OT-I and OT-II, respectively) as mothers, we investigated the fate of fetus-specic CD8+ and CD4+ T cells during gestation. Both CD8+ and CD4+ T cells displayed an activated phenotype in the spleen and lymph nodes of OVA-bred OT-I and OT-II mice, indicating their encounter with fetal antigen in lymphoid tissues. A small percentage of CD4+ T cells were deleted in the periphery and thymus of OVA-bred OT-II mice, with evidence of TCR downregulation in the remaining T cells. However, deletion and TCR downregulation were not observed in OVA-bred OT-I mice. Both CD4+ and CD8+ T cells upregulated ICOS expression in the presence of specic fetal antigen, but only CD4+ T cells consistently upregulated the inhibitory receptors PD-1 and CTLA-4. More regulatory T cells were present in OVA-bred than in WT-bred OT-II mice, suggesting that fetal antigen specically stimulates expansion of these cells. These data indicate that fetal antigen-specic maternal CD4+ T cells are tolerized during gestation by several potential mechanisms, whereas tolerance of fetal antigen-specic CD8+ T cells is less effective. This notion is supported by the observation that fetal loss occurred in OVA-bred OT-I, but not OT-II, mice. This project is supported by NIH grants HD045611 and HD049480. A. Perchellet is supported by NIH training grant T32HD007455.
[P04.11]. MINOR HISTOCOMPATIBILITY ANTIGEN EXPRESSION IN TROPHOBLAST AND FETAL BLOOD CELLS: IMPLICATIONS FOR MATERNAL-FETAL IMMUNE TOLERANCE M.G. Petroff*, K.M. Adams-Waldorf, J. Zhao. University of Kansas Medical Center, Kansas, United States Pregnancy represents a unique physiological situation in which a mother establishes robust immunological tolerance to the semiallogeneic fetus. Increasingly, there is evidence that this tolerance includes accommodation by antigen-specic lymphocytes to specic paternally-inherited alloantigens. Murine transgenic systems have shown specic activation, proliferation, and deletion of fetal antigen-specic T cells, and in women, expanded cohorts of fetal antigen-specic T cells are frequently detected as a result of pregnancy. We have previously hypothesized that antigens derived from the syncytiotrophoblast and fetal blood cells access maternal antigen presenting cells and tolerize lymphocytes by way of trophoblast shedding and fetal microchimerism, respectively. Here, we address this question by asking whether the placenta and fetal blood leukocytes could be a source of antigens in human pregnancy, with a focus on two distinct minor histocompatibility antigens (mHAg): the autosomally-encoded mHAg HA-8, and the Y chromosome-encoded mHAg SMCY. RT-PCR analysis of whole placenta lysate as well as puried trophoblast cells revealed that both HA-8 and SMCY mRNA are expressed in placenta and trophoblast cells. SMCY was detected only in male placenta and trophoblast samples; placentas and trophoblast cells from female infants failed to yield an RNA product. Puried fetal cord blood leukocytes also expressed RNA for both mHAg. Finally, HA-8 protein was found by immunohistochemistry to be present within the cytoplasm of the syncytiotrophoblast of term placentas. In the basal plate placenta, HA-8 antibody also reacted lightly with extravillous trophoblast cells. These results provide new evidence that trophoblast cells and fetal blood leukocytes are a source of proteins that could be antigenic to maternal leukocytes, and support the hypothesis that maternal lymphocytes could recognize these fetal antigens if presented in the context of maternal antigen presenting cells. This work was supported by NIH grants HD045611 and HD049480.
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[P04.12]. GENE EXPRESSION OF TOLL-LIKE RECEPTORS (110) IN FIRST TRIMESTER TROPHOBLASTS, AS ASSESSED BY RT-qPCR ANALYSIS G.D. Olsen*, A.S. Gundersen, A-H. Leknes, T.D. Nguyen, R. Austgulen, A-C. Iversen. NTNU, Norway Introduction: The placenta constitutes a physical and immunological barrier to protect the fetus against invading infectious agents and inammation. So far, local maternal immune cells have been assigned the most active immunological role, but a number of observations, such as the documentation of Toll-like Receptor (TLR) gene expression, suggest that fetal trophoblasts contribute to placental immunity and inammatory responses. The aim of this study was to further explore the gene expression of TLRs in trophoblasts to extend knowledge of relevance for an immunological role. Gene expression of all TLRs in rst trimester trophoblasts was quantied and compared to corresponding ndings in broblasts and trophoblast cell lines. Methods: Primary trophoblasts and broblasts were isolated from rst trimester placentas (5-12 gestational weeks). The choriocarcinoma cell lines JEG3 and JAR and normal human dermal broblasts (NHDF) were included for comparison. Gene expression of TLR 1-10 was analyzed in all cell types by quantitative real time reverse transcription PCR (RT-qPCR). Results/Discussion: Interestingly, rst trimester trophoblasts demonstrated a relatively high expression of most TLRs, more pronounced than that observed in both the trophoblast cell lines and the different broblasts. A more restricted TLR expression level was observed in primary broblasts than in NHDF, whereas in trophoblast cell lines, JEG3 showed lower TLR expression levels than JAR. Furthermore, substantial variations in TLR gene expression levels were observed between individuals in rst trimester trophoblasts. Conclusion: The pronounced TLR gene expression in primary trophoblasts is rather suggestive of an active immunological role during placentation. Further studies on TLR protein expression and function in these trophoblasts are warranted. Keywords: Trophoblasts, Inammation, Toll-like receptors, RT-qPCR
[P04.13]. HUMAN DECIDUAL TISSUE CONTAINS DISARMED CD8+ EFFECTORMEMORY T CELLS T Tilburgs*1,2, CMC Schonkeren1, M Eijkmans1, DL Roelen1, SA Scherjon1, FHJ Claas1. 1Leiden University Medical Center, Netherlands, 2Harvard University, United States During pregnancy maternal lymphocytes at the fetal-maternal interface play a key role in the immune acceptance of the allogeneic fetus. Decidual NK cells contain immune modulatory properties and facilitate trophoblast invasion into maternal tissue. More recently, CD4+CD25bright regulatory T cells have shown to be concentrated in decidual tissue where they are able to suppress fetus-specic and non-specic responses. However, decidual CD8+ T cells form the largest fraction of T cells at the fetal-maternal interface but limited data is present on the characteristics of these cells. Therefore we performed phenotypic analysis of the decidual and peripheral CD8+ T cell pool with CD45RA, CCR7, CD28 and CD27 expression using nine-colour owcytometry. In addition, we examined expression of the cytolytic molecules perforin, granzyme B and granzyme K to determine the cytotoxic potential of the decidual CD8+ T cell subsets. Our data demonstrate that decidual CD8+ T cells mainly consist of differentiated Effector Memory cells while unprimed nave cells are almost absent. Unlike peripheral blood Effector-Memory CD8+ T cells, the decidual EffectorMemory CD8+ T cells do not express perforin and have a reduced expression of granzyme B. Apparently, the functional features of decidual CD8+ T cells do not correspond their matching phenotype in peripheral blood. These data show that decidual CD8+ T cells may pursue alternative means of effector cell differentiation and indicate that decidual CD8+ T cell differentiation and regulation may play a crucial role in maternal immune tolerance to the fetus. Keywords: Decidua, CD8+ T cells, Effector-Memory differentiation, Human
[P04.14]. CD11c IDENTIFIES MACROPHAGES
TWO
DISTINCT
SUBSETS
OF
DECIDUAL
BL Houser, ML Nicotra, T Tilburgs*, JL Strominger. Harvard University, United States Placentation is a dening characteristic of Eutherian mammals. However, how the maternal immune system tolerates the fetal allograft during human pregnancy remains unclear. After NK cells, macrophages comprise the second largest leukocyte population in the decidua, at 20-25% of all immune cells. Here we demonstrate that there are two distinct subsets of decidual macrophages based on CD11c expression (CD11cHI and CD11cLO). Approximately, 35% of decidual macrophages are CD11cHI and 65% are CD11cLO. These two populations have unique cell morphologies and specic surface markers. Gene expression analysis by RNA microarray revealed over 200 genes that were differentially expressed between these two populations. Functional analysis revealed that the two subsets do not functionally differ in their phagocytic capacity. However, compared to in vitro derived macrophages, both decidual macrophage populations are less phagocytic. These data show that two previously undescribed subsets of macrophages are found in the decidua. Keywords: Decidua, Macrophages, Human, CD11c
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[P05.03]. EPIDERMAL GROWTH FACTOR (EGF) STIMULATES UPREGULATION OF MMP-9 AND TIMP-1 IN BOVINE PLACENTAL CELLS VIA MAPK SIGNALLING PATHWAY Marc Dilly*, Nina Hambruch, Jan Dirk Haeger, Christiane Pfarrer. Department of Anatomy, University of Veterinary Medicine, Hannover, Germany The bovine synepitheliochorial placenta is characterized by restricted trophoblast invasion, a unique feature of which the regulatory mechanisms are not yet fully understood. Among other factors, matrix metalloproteinases (MMPs) and counteracting tissue inhibitors of metalloproteinases (TIMPs) serve to control cell migration and tissue remodelling. MMP-9 is present in the bovine placenta throughout gestation; its proteolysis is predominantly regulated by the action of endogenous TIMP-1. Epidermal growth factor (EGF), as regulator of fundamental cell properties, is capable to up-regulate MMP-9 activity in a variety of cells types. However, there is no direct evidence for the stimulation of MMP-9 and TIMP-1 expression by EGF in the bovine placenta. In addition, the signalling pathways involved in this regulation are not clear. In this in vitro study, cultured maternal caruncular epithelial cells (BCEC-1) and trophoblast cells (F3) isolated from bovine placenta were used to examine the possible involvement of the mitogen-activated protein kinase (MAPK) pathway in the regulation of the MMP-9/TIMP-1 system by EGF and MAPK inhibitor application. The activity of MAPK was determined by Western blot while mRNA levels of MMP-9/TIMP-1 were compared by densitometric analysis of specic RT-PCR products. MMP activity was analyzed by zymography. We demonstrated that EGF increases both MMP-9 and TIMP-1 mRNA expression in BCEC-1 and F3 cells. This effect could be abolished by inhibiting MAPK activation. In the same time, Western analysis showed a tremendous activation of MAPK exclusively in F3 cells, whereas zymography revealed that EGF elevates MMP-9 activity predominantly in BCEC-1 cells. The results presented suggest that EGF activates the MAPK pathway in bovine placenta cells, and this activation is necessary for the up-regulation of MMP-9 and TIMP-1 expression. Thus EGF may be involved in the regulation of restricted trophoblast invasion and dened tissue remodelling during bovine gestation. Funded by the German Research Foundation (DFG). Keywords: Bovine, EGF, MMPs, MAPK
[P05.04]. ACTIVIN A REGULATES HUMAN EXTRAVILLOUS TROPHOBLAST CELL INTEGRINS AND ADHESION TO EXTRACELLULAR MATRIX E Dimitriadis*, C Stoikos, LA Salamonsen. Prince Henrys Institute of Medical research, Australia Introduction: Successful pregnancy depends on adequate invasion of extravillous trophoblast (EVT) into the uterine decidua. Locally produced activin A has been proposed to have a role in EVT invasion however the mechanisms by which this occurs are poorly understood. We hypothesized that activin A regulates trophoblast invasion by modulating EVT adhesive properties. This study investigated whether activin A inuences human EVT integrin molecule production and EVT adhesion to various ECM. Methods: The human EVT cell line, HTR-8/SVneo (HTR8) was used as a model for human EVT. The expression of activin receptors (R) ActivinR1a, ALK4 and ActivinRIIA/B on HTR8 cells was examined by RT-PCR. The effect of activin A on HTR8 integrin molecule expression was assessed by integrin antibody arrays while activin As role on trophoblast cell adhesion to bronectin (FN), collagen (COL) 1, COLIV, vitronectin (VN) and laminin (LN) was measured by cell-matrix adhesion assays. The effect of activin A on phosphorylated (p) and total SMAD abundance in HTR8 cells was assessed by Western blot. Results: HTR8 cells expressed ActivinR1a, ALK4 and ActivinRIIA/B mRNA. Activin A (1, 10, 50, 100 ng/ml) dose dependently increased pSMAD2 abundance while SMAD2 was unaffected in HTR8 cells. The activin inhibitor, SB431542 (SB: 10mM) abolished pSMAD2 protein abundance but pSMAD2 increased when SB was added with activin A (50 ng/ml) compared to SB alone. HTR8 cells adhered maximally to FN, COL1 and COLIV. Activin A (50ng/ml for 24h) decreased cell binding to bronectin, COLI and COLIV (p<0.05), and cell surface integrin subunits a1 a2 a3 a5, b1, b2 and b4 (p<0.05) in HTR8 cells. Conclusion: This is the rst study to demonstrate that activin A regulates human trophoblast cell surface adhesion molecule production and adhesion to various ECM. This suggests a mechanism by which activin A regulates trophoblast cell invasion during early pregnancy. Keywords: trophoblast adhesion, activin a, integrins, trophoblast invasion
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[P05.05]. ASPARTYL-ASPARAGINYL b-HYDROXYLASE: A GENE CRITICAL FOR PLACENTATION AND FETAL GROWTH F Gundogan*1,2, AD Bedoya2, ES Lau2, P Mark3, JR Wands2,3, SM de la Monte2,3. 1Women and Infants Hospital, United States, 2Brown Alpert Medical School, United States, 3Rhode Island Hospital, United States Introduction: Aspartyl-asparaginyl b-hydroxylase (AAH) is a type 2 transmembrane protein that hydroxylates epidermal growth factor-like domains of Notch and Jagged, which have known roles in cell migration and invasion. Placenta is the only non-transformed organ that expresses high levels of AAH, and extravillous cytotrophoblast, which are motile and invasive, express higher levels of AAH than the non-motile villous counterpart. In humans, impaired placentation accompanied by intrauterine growth restriction (IUGR) was found to be associated with reduced levels of AAH in extravillous trophoblast. In the present study, we directly addressed the roles of AAH in placentation and trophoblast motility using in vitro and in vivo gene silencing approaches. Methods: HTR-8 SVneo human trophoblastic cells were transfected with siRNA targeting AAH (siAAH) or no specic sequences (siScr) using the Amaxa electroporation system. Directional motility was measured using the ATP luminescence based motility and invasion assay. To study the effects of siAAH in vivo, pregnant Long Evans dams were anesthetized and subjected to laparotomy to microinject siAAH or siScr directly into the mesometrial triangle on gestational day 17. The siRNA molecules were co-transfected with GFP expressing plasmids to monitor delivery and transfection efciency. Placentas harvested 24 h later, were used to measure AAH, Notch, Jagged and HES (downstream target of Notch) by qRT-PCR. Results: SiRNA silencing of AAH signicantly reduced the mean directional motility in HTR-8 SVneo trophoblastic cells relative to siScr transfected control cells. In vivo intra-placental delivery of siAAH was successfully accomplished based on the robust GFP uorescence and demonstration of reduced AAH mRNA levels by qRT-PCR analysis. Correspondingly, inhibition of AAH expression was associated with signicant reductions in Notch, Jagged, and HES-1 mRNA levels, and signicant IUGR of the pups (1.460.02 g. versus 1.770.04 g. in control, P<0.0001). Conclusions: AAH has a critical role in mediating trophoblast motility, which is required for placentation. Inhibition of AAH expression, such as that caused by maternal consumption of alcohol, leads to impaired placentation and intrauterine growth restriction. Therapeutic measures to support or bolster AAH expression may help reduce the risk of IUGR. Keywords: AAH, motility, placentation, IUGR
[P05.06]. BOVINE TROPHOBLAST EMBEDDED SPHEROIDS
CELLS
INVADE
COLLAGEN
GELS
FROM
Jan Dirk Haeger*, Nina Hambruch, Marc Dilly, Christiane Pfarrer. Department of Anatomy, University of Veterinary Medicine, Hannover, Germany Introduction: To overcome the limitations of two-dimensional cell culture systems, three-dimensional spheroids which are considered to be more like in vivo have been developed in the past. Spheroids have been formed with equine chorionic girdle cells and human cytotrophoblasts. Objective: We aimed to generate spheroids with bovine placental trophoblast cells and to show that these spheroids are suitable to test the inuence of growth factors on the invasion of bovine trophoblast cells in vitro. Methods: Trophoblast cells were seeded in drops on dishes which were turned upside-down and incubated (hanging drop). Each drop consisted of a dened cell number, 25% methocoel, matrigel (0.8-1%) and regular culture media. Spheroids were harvested and characterized morphologically by light-, transmission and scanning electron microscopy (LM/TEM/ SEM). To determine the viability of spheroidal trophoblast cells spheroids were incubated with calcein-AM and ethidium-homodimer-1. Additionally, spheroids were harvested during formation (1, 2 and 3 days after seeding) and the nuclei were stained using Bisbenzimid to detect fragmentation of cell nuclei (IF). For in vitro invasion assays trophoblast spheroids were embedded in collagen gels which were overlayed with serum free media containing EGF (50 ng/ml). Results: Bovine trophoblast spheroids were clearly delimited and covered by extracellular matrix (LM/SEM). Cells contributing to spheroids were indiscriminable from each other (LM). The outer spheroidal layer consisted of differentiated cells possessing an apical pole directed to the outside (LM/ TEM). The inner core of the spheroids contained degenerating cells while the cells of the outer rim were viable (TEM/IF). Spheroidal trophoblast cells invaded the collagen gels. EGF strongly enhanced this process. Conclusion: Using bovine trophoblast cells we have generated spheroids that are useful to further examine the inuence of growth factors on the invasion of bovine trophoblast cells in vitro and the underlying signalling pathways. Funded by the German Research Foundation (DFG). Keywords: bovine, trophoblast cells, spheroids, EGF
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[P05.07]. MIGRATION AND DIFFERENTIATION OF A BOVINE TROPHOBLAST CELL LINE IS STIMULATED BY EPIDERMAL GROWTH FACTOR (EGF) IN A CONCENTRATION-DEPENDENT WAY N. Hambruch*, M. Dilly, J.-D. Haeger, C. Pfarrer. Department of Anatomy, University of Veterinary Medicine, Hannover, Germany In bovine placentomes, trophoblast giant cells (TGC), differentiating from uninucleate trophoblast cells (UTC), are crucial for feto-maternal interactions as they have the unique ability to migrate into and fuse with the maternal epithelium. Since this differentiation and migration is essential for successful gestation, we established a bovine trophoblast cell line (F3) to investigate the inuence of potentially regulatory factors on the migratory activity in vitro. One likely candidate is epidermal growth factor (EGF) which is known to regulate fundamental cell properties, such as growth, differentiation and invasion. Consequently, we aimed to study EGF mediated effects on the regulation of F3 cellular growth, differentiation and motility with special respect to different EGF concentrations. F3 cells were incubated with EGF in concentrations ranging from 10 to 100ng/mL in serum free media for 24h. Subsequently, cell proliferation was measured by colorimetric MTT assay. Additionally, a wound-healing assay (motility assay) was carried out to analyse the inuence of EGF on the migratory activity. Furthermore, the ratio of binucleate TGC to UTC was determined after stimulation with 10 or 100ng/ml EGF. Incubation of F3 with EGF led to a signicant enhancement (p<0,001) in proliferation compared to serum deprived cells, with highest mitogenic responses at 10 and 50ng/ml, whereas 100ng/ml showed the weakest effect. Consistent with these results the stimulation with 10ng/ml EGF resulted in a higher motility than with 100ng/ml as shown by woundhealing assay. When comparing the effect of EGF on the ratio of binucleate TGC to UTC rst experiments indicated that high EGF concentrations favour the formation of TGC. In conclusion, our data demonstrate that EGF is able to stimulate growth and motility of bovine trophoblast cells in a concentration-dependent manner. Furthermore, rst results indicate, that EGF may play a role in the regulation of TGC differentiation. Funded by German Research Foundation (DFG). Keywords: Bovine, Trophoblast, EGF-signalling, Cell culture
[P05.08]. EXPRESSION OF N-ACETYLGLUCOSAMINYLTRANSFERASE V IS DOWNREGULATED BY TRANSFORMING GROWTH FACTOR b1 AND HYPOXIA IN EXTRAVILLOUS TROPHOBLAST (EVT) CELLS K Hayashi*, E Yamamoto, K Ino, K Niimi, S Kondo, F Kikkawa. Nagoya University, Japan Objectives: N-Acetylglucosaminyltransferase V (GnT-V) is one of the most relevant glycosyltransferases to tumor invasion and metastasis, and catalyzes b1-6 GlcNAc branching on N-glycans. We have shown that GnT-V regulated EVT invasion through glycosylation of a5b1 integrin and GnT-V expression was downregulated in EVTs invading the decidua. In the present study, we investigate the molecular mechanisms involved in regulation of GnT-V expression. Methods: 1. We cultured HTR-8/SVneo (EVT cell line) and under normoxia (20%) and hypoxia (1%) for 24hr and 48hr. The effect of hypoxia on GnT-V expression was investigated by Western blot analysis and RT-PCR. 2. GnT-V expression level was examined in the cell lines with supernatant of decidual tissue culture for 48hr, RT-PCR and Western blot were performed. 3. To clarify cytokines in decidua involved in regulation of GnT-V expression, we cultured the two cell lines with TGF-b1, TNFa, INF-g, IL-6 for 48hr and RT-PCR and Western blot were performed. Results: 1. The expression of GnT-V in HTR8/SVneo was lower under hypoxia than normoxia by western blot analysis and RT-PCR. 2. The level of GnT-V expression was decreased by addition of supernatant uid of decidual culture. 3. TGF-b1 signicantly decreased the level of mRNA and protein of GnT-V in a dose-dependent response in HTR-8/SVneo. Conclusion: These results suggest that hypoxia and TGF-b1 regulate the level of GnT-V expression and this mechanism may be involved in regulation of EVT invasion. Keywords: EVT, GnT-V, TGFbeta1, hypoxia [P05.09]. ESTRADIOL MODULATES THE EXPRESSION OF CHEMOKINE RECEPTORS ON A HUMAN MAST CELL LINE AND PROMOTES THEIR MIGRATION TO THE UTERUS F Jensen*, A Teles, M Woudwyk, AC Zenclussen. Experimental Obstetrics & Gynecology, Medical Faculty, Otto-von-Guericke University, Magdebug., Germany Human embryo implantation is a complex process involving blastocyst attachment to the endometrial epithelium and trophoblast invasion. Uterine histamine is a key regulator of implantation due to its capacity of altering vascular permeability. As histamine is produced by mast cells (MCs) which are present in the uterus as well as at the fetal-maternal interface during pregnancy, we aimed to analyze MC migration to trophoblast cells by using a two chamber in vitro system. Since it is known that CCL11, CCL14, CCL16 and CCL22 are expressed in the uterus throughout the menstrual cycle, and their expression level uctuate under hormonal inuence, we further analyzed the effect of estradiol on the expression of their receptors in MCs as a possible mechanism for MC migration. We employed the well-characterized human MC line HMC-1. We rst analyzed the migratory capacity of HMC-1 towards primary human trophoblasts of rst trimester and towards choriocharcinoma cells (JEG-3 cell line). HCM-1 cells were further incubated with physiological concentrations of estradiol and MC degranulation as well as the expression of CCR4, CCR5 and CXCR4 protein and mRNA were analyzed. We conrmed that MCs strongly migrate to both human primary trophoblasts and JEG-3 cells. Physiological concentrations of estradiol lead to MC degranulation while signicantly up-regulating the expression of CCR4, CCR5 and CXCR4 in HMC-1 cells. The modulatory effects of estradiol on the expression of chemokine receptors clearly brings to ligth a novel mechanism as to how MCs may migrate to the uterus/fetal-maternal interface. Because MCs degranulation products as e.g. histamine, are key regulators in implantation, we speculate that MCs are recruited to the uterus by estrogen inuence via upregulation of chemokines receptor expression, while their degranulation may prepare the uterus for a possible implantation. Keywords: Mast cells, Chemokines, Trophoblast
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[P05.10]. VASCULAR-DERIVED CHEMOKINES PROMOTE TROPHOBLAST INVASION RJ Keogh*1,2, S Chau1,2, M Grgurinovic2, P Murthi2, S Rogerson2, S Brennecke1,2. 1Royal Womens Hospital, Australia, 2University of Melbourne, Australia Introduction: There is a dynamic interaction between trophoblast and vascular smooth muscle cells (VSMC) during the remodelling of uterine spiral arteries in the rst trimester of human pregnancy. Characterization of this interaction shows that movement of trophoblast is directional with greater persistence and speed in the presence of VSMC (1). This directed movement of trophoblast is likely to be regulated by chemokines. Chemokines are a subgroup of cytokines that have an ability to cause chemotaxis (directed movement) of nearby responsive cells. In addition to acting as chemo-attractants, chemokines also have other important functions including facilitating cell adhesion, proliferation and survival. VSMC produce chemokines which have a direct role in vascular remodelling and trophoblast express receptors for a number of different chemokines, including those made by VSMC. Methods: We have screened arterial SMC for chemokine production using a protein prole array. Candidate chemokines, for which trophoblast express receptors, were identied and their effects on trophoblast function were tested using HTR8/SVneo cells as a model of invasive extravillous trophoblast. Wound healing assays were used to assess effects on migration and gelatin zymography was performed to assess matrix metalloproteinase activity as an indicator of invasive potential. Results: Four candidate chemokines were identied all of which have the potential to control trophoblast cell functions. Three of these chemokines have been tested thus far (CXCL1, CCL2, CCL5). All three chemokines were found to stimulate trophoblast cell migration in a concentration-dependent manner. In addition, all three chemokines were found to up-regulate matrix metalloproteinase 2 and 9 activity. Ongoing work will examine the effects of these chemokines on trophoblast proliferation. Discussion: We conclude that CXCL1, CCL2, and CCL5 are important factors contributing to the control of directed trophoblast movement into uterine spiral arteries. Reference (1) Hamzic, E. et al. Experimental Cell Research (2008) 314, 1455-64. Keywords: Chemokine, Trophoblast invasion, Vessel
[P05.11]. RCHO-1 TROPHOBLAST MODEL SYSTEM TO STUDY THE EXPRESSION AND FUNCTION OF HIGH TEMPERATURE REQUIREMENT FACTOR A1 F Ajayi, P Samuels, DA Kniss*. Ohio State University, United States Preeclampsia continues to be a worldwide complication of human pregnancy and in the US the incidence is 5-8%. Theories as to the pathogenesis of the disease suggest that inappropriately shallow invasion of extravillus trophoblasts into the uterine spiral arterioles contributes to the failure to establish a low resistance circulatory environment, and heralds the pathobiology that ultimately gives rise to the clinical symptoms of preeclampsia. Moreover, abberant vascular remodeling within the uteroplacental circulation creates a state of relative local hypoxia that induces further cellular pathophysiological sequelae. Recent work by our group and by others has demonstrated that High temperature requirement factor A1 (Htra1, a 55-kDa serine protease) may play a role in placentation, in particular the migratory and invasive phases by the trophoblast. Patients who developed early-onset, severe preeclampsia had elevated levels of circulating Htra1 (Ajayi et al., work in progress). In addition, ectopic expression of Htra1 using an in vitro model of extravillus trophoblast (HTR-8 SV/neo) inhibited cell migration and invasion in conventional Matrigel assays (Ajayi et al., AJOG, 2008). Using a rat choriocarcinoma model of trophoblast (RCHO-1, kind gift from Michael Soares) we conducted experimental studies to examine the ontogeny of Htra1 expression as cells differentiated from trophoblast stem cells into trophoblast giant cells. The expression of Htra1 mRNA (measured by realtime RT-PCR, qRTPCR) and its cognate protein (measured by immunoblotting and immunouorescence) was low to absent in cells grown in maintenance medium, but was substantially up-regulated within 24 h following the transition to differentiation medium. Importantly, there was a concomitant diminution in Id2 (stem cell marker) and increase in CSH1 (differentiation marker) during differentiation of trophoblast stem cells into multinucleated giant cells. Preliminary studies indicated also that hypoxia in vitro was associated with increased Htra1 expression. These early data provide a readily testable model system in which to probe the biology of Htra1 expression and function in the early stages of extravillus trophoblast development. (This work was supported in part by Perinatal Resources, Inc., and The Ohio State University Perinatal Research and Development Fund.) *Maternal-Fetal Medicine Fellow-in-Training. Keywords: Preeclampsia, Trophoblast stem cells, Htra1, Trophoblast invasion
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[P05.12]. THE ROLE OF AKT ISOFORMS IN HUMAN TROPHOBLAST MIGRATION P Haslinger*, S Sonderegger, J Otten, K Biadasiewicz, M Knoer. Department of Obstetrics and Fetal-Maternal Medicine, Austria Objective: The protein kinase AKT is a well-known regulator of diverse cellular processes including proliferation, migration, differentiation and survival. With respect to the placenta AKT was shown to be a critically involved in basal and growth factor-dependent trophoblast migration. However, three different isoforms of AKT exist which, depending on the biological process, may have agonistic or antagonistic functions. Hence, the aim of this study was to analyse expression of AKT1, AKT2 and AKT3 in different trophoblast model systems and to investigate their individual roles in trophoblast proliferation and migration. Methods: Expression of AKT1, AKT2 and AKT3 was analysed by real-time PCR and Western blotting (isoform-specic antibodies) using whole tissues of rst and third trimester placentae, puried rst and third trimester trophoblasts/stromal broblasts, rst trimester villous explant cultures, puried EVT as well as trophoblastic cell lines (JEG-3, HTR-8/ SVneo, SGHPL-5). Constitutive, stable (puromycin-selected) knock-down cell pools of AKT1, AKT2 and AKT3 were generated in SGHPL-5 cells by transduction with retroviral vectors (LMP) expressing shRNAmir (microRNA-adapted short hairpin RNA) against human AKT1, AKT2 and AKT3, respectively (OpenBiosystems, Huntsville, AL, USA). Proliferation and migration were analysed by counting cumulative cell numbers and by performing transwell assays in the absence or presence of EGF. Results: All AKT isoforms were detected in the different trophoblast cell models using real-time PCR and Western blotting. Transcript and protein levels of AKT did not largely vary between the different trophoblast cultures. Western blot analyses revealed that the extent of knock-down was between 80% and 90% for the different AKT isoforms. Compared to non-silencing controls, knock-down of AKT1, AKT2, and AKT3 did not affect trophoblast proliferation. Basal migration was only diminished in AKT3 knock-down cells, whereas EGF-dependent migration was strongly reduced in both AKT1 and AKT3 gene-silenced pools. AKT2 knock-down had only subtle effects of EGF-dependent migration. Conclusion: None of the different AKT isoforms is involved in trophoblast cell proliferation. Basal migration may largely depend on AKT3. AKT1 and AKT3 seem to play redundant roles in EGF-dependent trophoblast migration. S.S. and P.H are supported by grant P-17894-B14 of the Austrian Science Funds, K. Biadasiewicz is supported by grant Nr. 12487 of the Austrian National Bank, Austria. Keywords: trophoblast, invasion, AKT, signaling
[P05.13]. IDENTIFICATION OF VEGFR-2 AS A NOVEL DECORIN BINDING RECEPTOR ON THE HUMAN EXTRAVILLOUS TROPHOBLAST CONTROLLING ACQUISITION OF AN ENDOVASCULAR PHENOTYPE G Khan*1,2, N Lala1, G Gannareddy1, RN Bhattacharjee1, PK Lala1. 1University of Western Ontario, Canada, 2Defense Institute of Physiology and Allied Sciences, New Delhi, India Introduction: The human placenta is an invasive structure in which a cell population known as the extravillous trophoblast (EVT) migrates out of chorionic villi and invades the uterine endometrium and its arteries, adopting an endovascular phenotype. Poor uterine arterial invasion and remodelling by EVT cells is associated with IUGR in the fetus and preeclampsia in the mother. We have identied two decidua-derived negative regulators of EVT cell proliferation, migration and invasiveness: TGF-beta, and a TGF-beta binding small leucine rich proteoglycan decorin (DCN) co-localised with TGF-beta in the decidual ECM (Xu, et al. Biol Reprod 67, 681-89,2002). DCN actions on EVT cells were differentially mediated by multiple tyrosine kinase receptors EGF-R, IGFR-1 and VEGFR-2 (Iacob, et al. Endocrinol 149,6187-97,2008). Objectives: Since DCN binding to VEGFR-2 has never been reported before in any cell type, we directly tested this binding and the identity of VEGFR-2 binding sites of DCN protein in our human rst trimester EVT cell line HTR8/SVneo, and further examined whether this binding could retard VEGFinduced acquisition of an endovascular phenotype. Methods and Results: EVT cell lysate proteins were subjected to farwestern blots by using puried DCN as bait and VEGFR-2 as prey proteins. DCN binding to EVT cell proteins was detected with DCN antibody and re-probed with VEGFR-2 antibody, showing DCN-bound VEGF-R2. Pure VEGFR-2 served as positive control. Similar proof was obtained by coimmunoprecipitation of DCN and VEGFR-2 in EVT cell lysate proteins, probed with DCN antibody. Certain DCN peptides that preferentially blocked DCN-VEGR-2 binding in EVT cell proteins also blocked EVT proliferation, indicative of possible VEGFR-2 binding sites of DCN. In a cell free system, using surface plasma resonance spectroscopy, we approximated the afnity of binding between pure DCN and pure VEGFR-2, providing a dissociation constant (Kd) of 73 mM. Finally, EVT cells when plated on growth factor reduced matrigel formed sparse endothelial-like tubes (acquisition of endovascular phenotype), which was stimulated in the presence of VEGF121. This VEGF-induced stimulation was inhibited by DCN pre-treatment in a dose-dependant manner. Discussion: That DCN binds to VEGFR-2 is a novel nding for any cell type. DCN-VEGFR-2 interactions in controlling acquisition of an endovascular phenotype by EVT cells may suggest that DCN over expression or activity may contribute to the development of preeclampsia. We are currently testing this possibility. (Supported by a CIHR grant to PKL) Keywords: Extravillous trophoblast, Decorin, VEGFR-2, Preeclampsia
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[P05.14]. ENDOGLIN IN HUMAN EXTRAVILLOUS TROPHOBLAST INDUCED BY TGF-BETA Y Mano*, T Kotani, S Sumigama, H Hayakawa, K Shibata, F Kikkawa. Nagoya University Graduate School of Medicine, Japan Endoglin (CD105) is a co-receptor for transforming growth factor-beta (TGF-beta). Endoglin is known to be expressed in syncytiotrophoblasts and its soluble form increases in preeclamptic serum. On the other side, several studies showed that endoglin was involved in cancer invasion. However, the role of endoglin in human trophoblast function remains unknown. The aim of this study was to investigate the expression of endoglin in evtravillous trophoblast (EVT), which poses invasive phenotype. We showed that endoglin was expressed on interstitial and endovascular EVT in human rst-trimester implantation site by immunohisctochemistry. Then, we investigated what factors would control the expression of endoglin. Semi-quantitative RT-PCR and Western blotting revealed that the expression of endoglin was increased under hypoxia (1%O2) condition. Both TGF-beta1 and TGF-beta3 (10ng/ml) also signicantly induced endoglin in human EVT cell line HTR-8/SVneo cells. We gained primary cultured EVT from rst-trimester human villous explants culture. In primary cultured EVT, the similar results were observed. These data suggested that endoglin was expressed in EVT, and the expression is regulated by hypoxia, TGF-beta1 and TGF-beta3. Now we are going to investigate the role of endoglin in EVT to introduce siRNA of endoglin into HTR-8/SVneo. Keywords: endoglin, extravillous trophoblast, transforming growth factor-beta, hypoxia
[P05.16]. EFFECTS OF ADIPONECTIN ON DIFFERENTIATION, INVASION AND MIGRATION OF HUMAN TROPHOBLASTIC CELLS A Onogi*, K Naruse, H Shigetomi, Y Yoshizawa, T Noguchi, T Sado. Dept. of Obstetrics & Gynecology, Nara Medical University, Japan Introduction: Adiponectin (Adn) is an adipocyte-derived cytokine leads insulin sensitivity and anti-inammatory action through TNF-a-suppression. We reported a paradoxical increase of Adn in preeclampsia (PE), but the manner of Adn effects on trophoblasts remains unclear. In this study, we cultured trophoblasts separated from human placenta and trophoblastic cell lines with different concentrations of Adn and evaluated the effects of Adn in several methods. Methods: Human term placentas were taken at selective cesarean section with informed consent. Separated trophoblasts by Percoll gradient were harvested on bronectin-coated plate overnight with or without Adn addition. Cytotrophoblasts(CTBs) were separated from early terminated pregnancy sample with similar method and cultured on MatrigelR overnight. Cytokines / proteinases / inhibitors in supernatants were measured with Multiple Cytokine Assay after conrmation of cell viability using MTT assay. Immunocytochemistry for cytokeratin and HLA-G were performed on early CTBs to assess the migration/differentiation to invasive phenotype. Adn were also added in several concentrations at culture of trophoblast cancer cell line JEG-3, JAR and BeWo, and invasion assay and wound healing assay were performed. Results: Effects of high concentration Adn culture of term trophoblasts were not signicant on cytokines / proteases / inhibitors secretion and cell viability. Addition of Adn in primary CTBs culture from early pregnancy increased connection and differentiation of the cells in lower concentrations, but inhibited them in higher concentrations. Ability of invasion after Adn addition were varied between cell lines, but signicant increase was shown in JAR after 10mg/ml Adn addition (p<0.05). Migration was significantly decreased in BeWo in 100ng/ml Adn (p<0.05), but not signicant in other higher or lower concentrations. Conclusion: Differentiation, migration and invasion of cytotrophoblast from early pregnancy and cancer cell line were strongly effected by Adn. Rather than the possible effects of Adn on placenta in PE, these might be basic evidences to explain the high rate of recurrent miscarriage or fetal growth restriction in hypoadiponectinaemia, like diabetes mellitus complicated pregnancy. Keywords: Adiponectin, trophoblast invasion, trophoblast migration, diabetes mellitus
[P05.15]. THE ROLE OF AUTOPHAGY ON THE INVASION OF EXTRAVILLOUS TROPHOBLAST Akitoshi Nakashima*1, Mikiko Tatematsu1, Tamotsu Yoshimori2, Shigeru Saito1. 1University of Toyama, Japan, 2University of Osaka, Japan Objectives: Extravillous trophoblast (EVTs) suffer from severe environment, such as hypoxia and low nutrition, during early pregnancy. Generally, hypoxia reduces cell viability, but EVTs maintain the invasiveness for successful pregnancy. Autophagy (AtP) is a bulk degradation system for promoting cell survival under nutrient depletion. In this study, we showed the specic role of autophagy on EVTs-invasion under hypoxia by using autophagy-defect cell line. Methods: EVT cell line, HTR-8/SVneo cells (HTR8) were incubated in DMEM with CoCl2 250mM (2%O2). We constructed AtP-defect cell line, HTR-4B, which is stably transfected with Atg4B dominant negative mutant by retrovirus vector. Using this cell line, intracellular ATP quantication was examined by luciferase driven bioluminescence. MMPs-mRNA levels were analyzed by real time RT-PCR. Results: We provided the three major ndings in IFPA meeting 2008. 1) Autophagy occurred in EVT primary cells and HTR-8 under hypoxia. 2) 3-MA, AtP-specic inhibitor, signicantly reduced the numbers of invading cells under hypoxia but not normoxia. 3) AtP was observed in EVT cells in human early pregnant specimen. In this study, we estimated the number of invaded cells between HTR8-4B, and HTR8-cont. Hypoxia signicantly decreased the numbers of invaded cells by 81% in HTR8-4B, compared with normoxia, whereas hypoxia increased that of cells by 153% in HTR8-cont. No signicant differences were detected between HTR8-4B and HTR8cont in the growth rate and the cell death rate under hypoxia. Intracellular ATP levels were signicantly decreased in HTR8-4B, but not in HTR8-cont under hypoxia. Furthermore, MMP2, MMP9 and uPA levels were also signicantly decreased in HTR8-4B compared with HTR8-cont. Conclusions: These ndings suggested that autophagy produced energy to EVT cells, which were invading to decidua under hypoxia. On this process, autophagy machinery may interact with producing MMPs on invaded EVT cells. Keywords: Autophagy, Hypoxia, ATP, MMPs
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[P05.17]. INTERLEUKIN-11 INHIBITS HUMAN TROPHOBLAST INVASION VIA STAT-3 INDICATING AN IMPORTANT ROLE DURING PLACENTAL DEVELOPMENT P Paiva*1,2, L Salamonsen1,2, U Manuelpillai2, E Dimitriadis1,2. 1Prince Henrys Institute of Medical Research, Australia, 2Department of Obstetrics and Gynaecology, Monash University, Australia Successful pregnancy depends on the precise regulation of extravillous trophoblast (EVT) invasion into the uterine decidua, primarily by deciduaderived factors. In humans, during early pregnancy, interleukin (IL)-11 is maximally expressed in the decidua1, with its receptor, IL-11-receptor alpha (Ra) also identied on invasive EVT in vivo2. While a role for IL-11 in EVT migration has been established2, whether it also plays a role in regulating EVT invasion is unknown. We investigated whether IL-11 inuences human EVT invasion and the signalling pathways and underlying mechanisms involved using the HTR-8/SVneo immortalized EVT cellline and primary EVT as models for EVT. The effect of IL-11 on tyrosine phosphorylation (p) of signal transducer and activator of transcription (STAT)-3 was determined by Western Blot. EVT invasion was assessed using in vitro Matrigel invasion assays. To elucidate the mechanisms by which IL-11 may inuence EVT invasion, matrix metalloproteinase (MMP) and urokinase plasminogen activator (uPA) activity were assessed by gelatin and plasminogen zymography / uPA activity assay respectively. Tissue inhibitor of MMPs (TIMPs)-1 and -2, plasminogen activator inhibitor (PAI)-1 and -2 and uPA receptor (uPAR) were assessed by ELISA whereas TIMP-3 was assessed by Western Blot. EVT adhesive properties and integrin expression were assessed by in vitro adhesion assays. IL-11 (100 ng/ml) signicantly inhibited invasion of EVT cells by 40-60% (p<0.001). This effect was abolished by inhibitors of STAT-3 but not of mitogen-activated protein kinase pathways. IL-11 (100 ng/ml) had no effect on MMP-2 and -9, TIMP 1-3, uPA, uPAR, PAI-1 and -2 in EVT conditioned media and / or cell lysates. IL-11 (100 ng/ml) also did not regulate EVT cell adhesion or integrin expression. These data demonstrate that IL11 inhibits human EVT invasion via STAT-3 indicating an important role for IL-11 in the decidual restraint of EVT invasion during normal pregnancy. References (1) Dimitriadis et al. (2003) Reprod Biol Endocrinol. 1, 34-38. (2) Paiva et al. (2007) Endocrinol. 148, 5566-72. Keywords: Interleukin 11, STAT3, Trophoblast
[P05.18]. EXPRESSION OF PREGNANCY RELATED SERINE PROTEASE HtrA3 IS TIGHTLY UPREGULATED DURING HUMAN STROMAL CELL DECIDUALIZATION H Singh*, L Salamonsen, G Nie. Prince Henrys Institute of Medical Research, Melbourne, Australia Background: Adequate invasion of the trophoblast cells is necessary for implantation and placentation. Expression of serine protease HtrA3 is highly upregulated in the decidualizing stromal cells in late secretory phase of the menstrual cycle and throughout pregnancy. It is highly expressed in the 1st trimester in most trophoblast cell types, but not in the invading interstitial trophoblast. Unlike cancer cells in which HtrA3 is down-regulated, trophoblast generally exhibit controlled invasion. The current study investigated expression of HtrA3 during decidualization of human endometrial stromal cells (HESC) in vitro and its function. Methods: Stromal cells were isolated from normal human endometrial biopsies (n3) and decidualized in vitro with estrogen, progesterone and cyclic-AMP. HtrA3 expression at various time points (0 - 96h) in decidualized and non-decidualized HESC was assessed by quantitative RT-PCR. Indirect immunouorescence and western was performed to determine the cellular localisation and protein expression of HtrA3. Results: Both HtrA3 mRNA and protein expression was signicantly increased in decidualized stromal cells compared to controls. HtrA3 mRNA expression for both isoforms (long and short) was signicantly increased within the rst 48h of decidualization. Approximately 25-40 fold increase was observed within initial 24h of decidualization for the long and short form respectively reaching maximum levels at 48h. The level of decidual HtrA3 mRNA gradually decreased with further decidualization, but expression level remained signicantly higher than controls. Homogeneous pattern and increase in intensity of HtrA3 staining was observed in decidualized stromal cells at 96h in comparison to non-decidualized cells. Conclusions: HtrA3 is tightly regulated during decidualization of HESC in vitro. It might be possible that the signicant initial increase of HtrA3 expression has a role in promoting decidualization. Function of HtrA3 in trophoblast invasion and its regulation in normal and complicated pregnancies will be explored. Keywords: HtrA3, Decidualization, Trophoblast, Invasion
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[P05.21]. EXPRESSION OF N-ACETYLGLUCOSAMINYLTRANSFERASE IVa IN EXTRAVILLOUS TROPHOBLAST AND GESTATIONAL TROPHOBLASTIC DISEASE E Yamamoto*, K Niimi, K Hayashi, T Kotani, K Ino, F Kikkawa. Nagoya University, Japan Objectives: Hyperglycosylated hCG (hCG-H) is a glycoprotein variant of the hormone hCG and reported to be detected in serum and urine of early pregnant women, invasive mole and choriocarcinoma, but not of molar patients. N-acetylglucosaminyltransferase IVa (GnT-IVa) is one of glycosyltransferases to attach abnormal biantennary N-linked sugar chains to hCG. Our aim is to clarify the expression of GnT-IVa in gestational trophoblastic disease and extravillous trophoblasts (EVTs) in human placenta. Methods: Western blot and RT-PCR were performed for expression levels of GnT-IVa protein and mRNA in choriocarcinoma cell lines (Jar, JEG-3, Bewo, CC1, CC3, CC4 and CC6), EVT cell line (HTR-8/SVneo), various gestational placenta and hydatidiform mole tissues. Expression and localization of GnT-IVa were analyzed by immunohistochemistry in choriocarcinoma, invasive mole, hydatidiform mole and human placenta. Results: GnT-IVa was expressed in all choriocarcinoma cell lines, especially strongly in Jar, JEG-3, CC4, and CC6, and weakly in HTR-8/SVneo. However, GnT-IVa expression was not detected in various gestational placenta and molar tissues by RT-PCR and Western blot. Immunohistochemical study revealed that GnT-IVa was strongly expressed in choriocarcinoma and invasive mole, but not in hydatidiform mole. In human placenta, GnT-IVa staining was detected in EVT cells in the rst trimester, and it was negative all trophoblasts of the second and third trimester placentas. Conclusion: GnT-IVa was expressed specically in choriocarcinoma, invasive mole and EVT in early gestation. These results suggested that GnT-IVa may be involved in EVT invasion as well as malignant potential of gestational trophoblastic disease. Keywords: GnT-IVa, EVT, Hyperglycosylated hCG, hCG
[P06.01]. MYOFERLIN AND DYSFERLIN ARE DISPENSABLE FOR CELL-CELL FUSION IN BeWo J. M. Robinson*, W. E. Ackerman, D. D. Vandre. The Ohio State University, United States Introduction: Cell-cell fusion is fundamental for the formation of multinucleated syncytia, which arise normally during the genesis of skeletal muscle and placental syncytiotrophoblast (STB). It has recently been shown that targeted disruption of the myoferlin (MYOF) gene in mice impairs myoblast fusion during skeletal muscle syncytialization. Given that MYOF and the closely related protein, dysferlin (DYSF), are expressed in trophoblast, we speculated that MYOF might serve an analogous function during cytrophoblast cell fusion. Methods: Lentivirus-based shRNA constructs were used to knock down the expression of MYOF and DYSF in BeWo cells. Both native and knockdown cells lines were assessed for the ability to undergo forskolin-induced fusion. Results: MYOF, but not DYSF, was expressed in mononuclear BeWo cells. Following treatment with 20 mM forskolin, DYSF expression increased progressively from 24 to 72 h, while MYOF expression remained constant. Three separate shRNA constructs were used to generate stable BeWo cell lines exhibiting at least 80% knock down of MYOF at the protein level. In parallel, four shRNA constructs were used to knock down DYSF expression to a similar degree. All three of the MYOF knock-down lines (including one in which MYOF was undetectable by immunoblotting) were found to undergo forskolin-induced cell fusion to a similar degree as native BeWo. In addition, the MYOF knock-down lines retained the ability to upregulate DYSF in response to fusion. The DYSF knock-down lines exhibited MYOF expression typical of native cells and, in the absence of DYSF upregulation, underwent normal fusion in response to forskolin. Discusson: These results suggest that neither MYOF nor DYSF are required for forskolin-mediated intercellular fusion in BeWo. Importantly, this establishes that these cell lines can be used as a novel system in which to address the function of MYOF and DYSF in plasma membrane repair using fused structures resembling STB. Keywords: Dysferlin, Myoferlin, BeWo
[P06.02]. PLACENTATION MODEL IN PURANE (THRICHOMYS APEREOIDES LUND, 1839). C Amrosio*. University of Sao Paolo, Brazil Thrichomys apereoides, a hystricognath rodent species belonging to the family Echimyidae, is characterized by high agility, a mainly vegetarian lifestyle, activities concentrated at dawn and a habitat in rocky areas with dense vegetation. We rstly characterize its type of placentation and the evolution of placental features. The investigated material includes four placentas at mid gestation, processed and analyzed by standard macroscopy and light microscopy. The placenta possesses a disc shape with few lobules. Lobules are clearly delimited by interlobules, with few maternal lacunae. In both regions we found predominantly cytotrophoblast, but also syncytiotrophoblast. Complementary studies on the ultrastructure must be done, however the placenta can be characterized as hemochorial by means of light microscopy. Centrally at the junction zone there is an area with a relatively large volume, classied as the subplacenta. The subplacenta is not lobulated and is characterized by a large number of trophoblast cells and syncytiotrophoblast surrounded by mesenchyme. An inverted vitelline placenta is presented, possessing groups of giant cells and layers of spongiotrophoblast. The visceral portion of the yolk sac has long villous projections and is high vascularized. The parietal portion of the yolk sac shows just one cell layer disposed on the placenta. In summary, placentation in Thrichomys is very similar to other members of South American and African hystricognaths, indicating a remarkable stable pattern of evolutionary transformations in the placenta. Thus, including the guinea pig a wide range of model species is principally useable in comparison to human placentation.
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[P06.03]. StarD7 A FUSOGENIC PROTEIN IN TROPHOBLASTS S.C. Angeletti1, Q. Chen2, S.M. Pearson2, S. Genti-Raimondi1, L.W. Chamley*2. 1Department of Clinical Biochemistry CIBICI, National University of Cordoba, Argentina, 2Department of Obstetrics and Gynaecology, University of Auckland, New Zealand StarD7 is a novel protein of unknown function that belongs to the START lipid domain family and is expressed in the cytoplasm of human trophoblast cells. It was previously found that StarD7 is able to interact with phosphatidylserine. We have previously shown that StarD7 is partially relocated from the cytoplasm to the plasma membrane during in vitro cytotrophoblast differentiation into syncytiotrophoblast. This study further investigates the function of StarD7 in trophoblasts. MTT-based cell proliferation assays were preformed in triplicate using BeWo cells that were incubated with increasing concentrations of recombinant StarD7 protein (0, 5, 10 or 20 mg/ml). Cell viability was determined by propidium iodide staining and detection of LDH in the culture supernatants. Statistical analysis was by t-test and a P value < or 0.05 was considered to be signicant. Syncytialization of BeWo cells was assessed by immunostaining for desmoplakin. The proliferation of BeWos was signicantly inhibited by incubation with StarD7 at 5, 10 and 20 mg/ml. Incubation of BeWos with StarD7 did not cause a signicant increase in cell death as measured by either propidium iodide or LDH release at any of the concentrations we tested. Fluorescent microscopy suggested that there were few intracellular desmosomes between adjacent BeWo cells after treatment with StarD7 at 20 mg/ml. BeWo cultures treated with StarD7 at these concentrations had similar levels of intracellular desmosomes to those found in control cultures treated with 5 mM forskolin. These results together demonstrate that exogenous Star D7 causes BeWo cells to cease proliferating but does not cause their death. The reduction in desmosomes indicates loss of intracellular boundaries leading us to conclude that StarD7 can initiate/facilitate the syncytialization of BeWo cells. Our results suggest that the phospholipid-binding protein StarD7 may play a physiological role during the differentiation of cytotrophoblasts into syncytiotrophoblast. Keywords: START lipid domain, Trophoblast proliferation arrest, Syncytialization, Cell fusion
[P06.04]. CROSSTALK BETWEEN EXTRINSIC AND INTRINSIC CELL DEATH PATHWAYS THROUGH CASPASE-8 IN MATERNAL FOOD RESTRICTED RAT PLACENTAS AT E20 L Belkacemi*, MG Ross, M Desai. Department of Obstetrics and Gynecology, David Geffen School of Medicine at UCLA and LABIOMED Research Institute at Harbor-UCLA Medical Center, CA, USA, United States Introduction: Maternal food restriction (MFR) during pregnancy leads to intrauterine growth restricted (IUGR) fetuses. Placental dysfunction is among the leading causes of IUGR. Increased placental apoptosis has been associated with IUGR. Apoptosis occurs via extrinsic death receptor pathway (Fas) and/or the intrinsic pathway (mitochondria). Activation of caspase-8 occurs directly via Fas pathway or indirectly via mitochondrial pathway. Caspase-8 activates intrinsic BID protein to form truncated BID (tBID). Using a rat model, we have shown that MFR results in reduced placental growth and increased apoptosis at E20. Since the Fas pathway is responsible for placental trophoblasts turnover, we sought to determine the role of caspase-8 in the Fas-induced apoptosis. We focused our study on the two placental positions (proximal and mid-horn) with the extremes of nutrient/oxygen supply, and two distinct placental zones (basal, site of hormone production; and labyrinth, site of feto-maternal exchange). Methods: Pregnant rat dams were fed an ad libitum diet (AdLib) or were 50% MFR beginning at E10 of gestation. At E20 rats were euthanized, and gestational sacs dissected. The placentas were separated and weighed. Six placentas from left mid- and proximal horns were xed in paraformaldehyde for TUNEL and activated caspase-3 staining. The corresponding right mid- and proximal horn placentas were separated into basal and labyrinth zones and analyzed for the expression of caspase-8 and tBID proteins (Western blot). Results: As compared to AdLib, MFR mid- and proximal horn placentas showed signicant reduction in weight and increased apoptosis in both zones (basal and labyrinth). Consistent with this, MFR placentas had increased caspase-3 expression. Furthermore, caspase-8 and tBID protein expression were signicantly increased in both zones in mid- and proximal placentas. Lastly, both MFR and AdLib mid-horn placentas had signicantly upregulated tBID as compared to respective proximal placentas. Conclusion: Thus, the mechanism for MFR-induced placental apoptosis via caspase-8 appears to be mediated primarily through the extrinsic Fas pathway with potentially secondary signaling from intrinsic pathway. Keywords: Apoptosis, placenta, IUGR, caspase-8
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[P06.05]. MATERNAL FOOD RESTRICTION IMPACTS ON MATERNAL-FETAL DEVELOPMENT AND WATER HOMEOSTASIS IN RAT PREGNANCIES L Belkacemi*, MH Beall, Q Liu, M Desai, MG Ross. Dept of Obstetrics and Gynecology, David Geffen School of Medicine at UCLA and LABIOMED Research Institute at Harbor-UCLA Medical Center, CA, United States Introduction: Mammalian gestations contain signicant amounts of water, both as a major constituent of the fetus, and as amniotic uid. This water is derived from the mother across the placenta, presumably via an osmotic mechanism. We have previously shown that maternal plasma hypertonicity reduces AF volume (AFV). Maternal food restriction (MFR) during pregnancy results in intrauterine growth restricted (IUGR) fetuses and newborns. IUGR is often associated with oligohydramnios. To assess the etiology of oligohydramnios, we examined the impact of 50% MFR on maternal water intake, plasma osmolality and body weight, and the subsequent effects on fetal plasma and AF osmolality and AFV at E20 (near term). Methods: From E10 to E20, rats were provided either an ad libitum (AdLib) diet (N6) or were restricted to 50% of the food of AdLib-fed rats (MFR; N6). From E10 to E20, MFR and AdLib dam body weights and water intake were recorded daily and maternal plasma osmolality quantied at E20. At E20, rat dams were euthanized and mid- and proximal horn gestational sacs dissected. Fetal weights were recorded. AFV was quantied as the difference in sac weight before and after drainage. Fetal plasma and AF osmolalities were quantied by freezing point depression. Results: At E10, prior to the initiation of MFR, there was no difference in body weight between MFR and AdLib dams. At E20, MFR dams had signicantly lower body weights (P<0.05), decreased water intake (P<0.05) but increased plasma osmolality as compared to AdLib dams. However, the ratio of body weight to water intake was not signicantly different between the two groups. Fetal plasma osmolality was signicantly increased in the MFR group while MFR fetal body weight was signicantly decreased. Furthermore, AFV was signicantly decreased in both mid- and proximal horn sacs whereas the AF osmolality was increased in those sacs (P<0.05). Conclusion: Although MFR leads to decreased maternal body weight and water intake at E20, the water intake adjusted for body weight were comparable between MFR and AdLib dams, suggesting that MFR-induced changes are not dependent upon maternal uid intake. However, the increased maternal osmolality in the MFR dams may signicantly alter water ux to the fetus, leading to fetal hyperosmolality and oligohydramniosis. Keywords: Amniotic uid, maternal food restriction, osmolality, water homeostasis
[P06.07]. DYNAMIC STUDIES ON CHANGES IN PLACENTAL STRUCTURE AND BLOOD FLOW IN AN ANIMAL MODEL OF PREECLAMPSIA USING HIGH RESOLUTION MRI G Bobek*1, T Stait-Gardner1, B Bahman1, J Preis1, W Price1, A Hennessy1,2. 1 University of Western Sydney, Australia, 2Heart Research Institute, Australia Introduction: The placenta appears to be central in the aetiology of preeclampsia. It has been postulated that reduced placental perfusion as a result of aberrant cytotrophoblast invasion and remodelling of the maternal spiral arteries is the initiating event that leads to the widespread dysfunction of the maternal vascular endothelium. This study presents our initial results into the use of magnetic resonance imaging (MRI) to directly examine murine placental structure and placental blood ow in normal pregnancy and in preeclampsia. Methods: Embryo placenta units from 14.5 day pregnant C57BL/6JArc mice were imaged using a Bruker Avance 11.74 Tesla wide-bore spectrometer with micro-imaging probe capable of generating gradients of 1.5 T/m. The measurements were made using the FLASH (Fast Low Angle SHot) method with echo time 6.000 ms, repetition time 397.28 ms and 20 averages (scan time w34 minutes). The eld-of-view was 23.000 mm 23.000 mm with slice thickness 0.500 mm. A 256256 matrix size resulted in voxel dimensions of w90 mm 90 mm 500 mm. Results: Our results demonstrate clear and detailed structural features of individual embryo placental units; resolving the amniochorionic membrane, detailed embryonic features, umbilical cord and placental vasculature. The high resolution images enable the selection of precise regions of interest to facilitate blood ow measurements in the placenta.
Discussion: MRI offers a non-invasive technique to conduct dynamic studies on changes in placental structure and blood ow in animal models of preeclampsia. We have demonstrated a substantial enhancement in placental image resolution above those previously reported. Using MRI techniques including BOLD (Blood Oxygen Level Dependent) MRI and DWI (Diffusion Weighted Imaging) for quantifying the ow dispersion within the placenta, we will be able to dynamically follow changes in placental perfusion and structure to investigate links between cytokine imbalance, shallow placental invasion and subsequent hypertensive response. Keywords: magnetic resonance imaging, placenta, perfusion, preeclampsia
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[P06.08]. NITRIC OXIDE IS INVOLVED IN THE SHIGA TOXIN MECHANISMS RESPONSIBLE FOR PREMATURE DELIVERY OF DEAD FETUSES J Burdet*1, E Zotta1, A M Franchi2, C Ibarra1. 1Laboratorio de Fisiopatogenia, Departamento de Fisiologa, Facultad Medicina, Universidad de Buenos Aires, Argentina, 2CEFYBO-CONICET, Universidad de Buenos Aires, Argentina Shiga toxin-producing Escherichia coli (STEC) infections could be one of the causes of fetal morbimortality in pregnant women. The main virulence factor of STEC is Shiga toxin type 1 or 2 (Stx1, Stx2). We have previously reported that intraperitoneal (i.p.) injection of culture supernatant from E. coli recombinant expressing Stx2 and containing lipopolysaccharide (LPS) in rats in the late stage of pregnancy induced premature delivery of dead fetuses. It has been reported that LPS may combine with Stx2 to facilitate vascular injury that may lead to a pathological cascade that involves the production of nitric oxide (NO). Objective: Our aim was to evaluate if NO is involved the effects of Stx2 on pregnancy. Materials and Methods: Pregnant rats on days 14-16 of gestation were i.p. injected with culture supernatant from recombinant E. coli containing 0.4 mg/ml Stx2 and 30 ng/ml LPS. A group of rats was previously injected with aminoguanidine (AG), an inducible NO synthase (iNOS) inhibitor and the development of preterm labor was evaluated. Western blot analyses were performed to determine the iNOS expression in placentas from Stx2-treated and untreated rats. Results: Stx2 and LPS induced fetal resorption, placental abruption, intrauterine hemorrhage and fetal death at 1-2 days post-injection. Pretreatment of 24 h with AG caused signicant reduction of Stx2 effects on the feto maternal unit but did not prevent the premature delivery of dead fetuses. Histological studies show no signicant differences in placenta tissues from rats treated with AG and Stx2 compared with those treated only with AG or with controls. Western blot assays showed a higher expression of iNOS in placentas from Stx2-treated rats than in those previously treated with AG. Conclusions: Our results suggest that NO is partially involved in the mechanisms of the premature delivery of dead fetuses caused by Stx2 and LPS. Keywords: Shiga toxin, preterm labor, pregnancy, nitric oxide
[P06.10]. A CASE OF A RETAINED PLACENTA PERCRETA FOLLOWED BY A VIABLE BIRTH FIVE YEARS LATER H.C. Chihara*, Y.N. Nagai, T.W. Watanabe, T.A. Adachi, A.N. Nagai, K.T. Tsutsumi. Department of Obstetrics and Gynecology, Nagai Clinic, Japan Placenta accreta is considered to be due to the absence of the decidua basalis and the associated invasion of the myometrium by the placental villi that may reach the peritoneal covering. We report on our experience with a patient in whom placenta increta recurred at the site of a previous placenta percreta, but who delivered a live baby and the uterus was saved. The patient was a 33-year-old multipara with two previous deliveries by Caesarean section. At the age of 28 years, she conceived naturally and delivered her second child by caesarean section. However, in this pregnancy she was found to have a placenta percreta that was attached over an area from the uterine body to the fundus. Since it was impossible to detach the placenta, the operation was terminated without removing it. The retained placenta gradually shrank and apparently mostly disappeared during the following year, until only remnants of the placenta remained on the serous membrane. On magnetic resonance imaging (MRI), the myometrium at the uterine fundus was thinning or missing, while the serous membrane was preserved. After 5 years, the patient conceived naturally. The placenta was attached in the region of the previous placenta percreta. According to the observations made during the Caesarean section performed at 37 weeks of gestation, the diagnosis was again placenta increta, but this time it could be separated, and the uterus could be saved. This case study illustrates the reparative capacity of the endometrium and shows that it is not always necessary to perform a hysterectomy in uncomplicated abnormal implantations of the placenta. It also demonstrates the time course of resorption of retained placenta percreta tissues. We will also report on the peri-operative ndings from the Caesarean section in the third pregnancy, including ultrasound images, MRI results and hysteroscopic appearance. Keywords: placenta accreta, placenta percreta, magnetic resonance imaging, hysteroscope
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[P06.13]. ALTERED INSULIN RECEPTOR SPLICING RESULTS IN DIFFERENTIAL INSULIN SIGNALLING IN GDM U. Hiden1, E. Glitzner1, L. Lassance1, I. Cetin2, U. Lang1, G. Desoye*1. 1Medical University Graz, Austria, 2University of Milano, Italy Objectives: The human insulin receptor (IR) exists in two isoforms that differ by inclusion/exclusion of exon 11, hence IR11+ and IR11-. Exon skipping results in different signalling efciency and ligand afnity. Diabetes alters IR splicing in muscle, liver and adipose tissue. Here, we hypothesized altered insulin receptor splicing in the placenta resulting from the diabetic environment in GDM, which may further alter insulin signalling. Methods: IR splicing was determined in normal (n28) and GDM (n17) placentas using RT-PCR. The mRNA expression of IR splicing factors was measured to identify factors involved in splicing alterations. Cytokeratin-7 and vWF were measured to exclude that changes in cellular composition account for altered IR splicing patterns. Insulin signalling differences in IR11+ vs. IR11- was determined by immunoblotting of Erk1/2 and PKB activation in mouse NIH3T3 cells over-expressing human IR11+ or IR11-. Isolated placental endothelial cells were treated in vitro by hyperinsulinemia and hyperglycaemia to identify possible factors altering IR splicing in vivo. Results: IR11+ over-expressing cells activated the Erk1/2 pathway 5-fold stronger than the PKB pathway. In contrast, IR11- over-expressing cells induced PBK signalling 3-fold stronger than Erk1/2 signalling. In GDM, the amount of IR11- was reduced by 29% (p0.03) whereas IR11+ remained unchanged. Changes in prevalence of IR isoforms did not result from altered placental cellular composition as no change was found in the proportion of CK7 to vWF. In isolated placental endothelial cells, IR11proportion was reduced by 15% after treatment with glucose (12mM) plus insulin (1nM). Conclusions: Reduced placental expression of IR11- vs. IR11+ in GDM gives rise to altered insulin signalling and effects. In vitro experiments indicate that altered insulin receptor isoform expression may result from hyperglycaemia and hyperinsulinemia in the fetal circulation that affect IR splicing in endothelial cells. As a consequence these may show reduced PKB mediated insulin effects. (Jubilee Fund grant no: 10896, 12601, 13307) Keywords: insulin, signalling, gestational diabetes
[P06.14]. AUTOMATED CELL-DETECTION TECHNOLOGIES FOR SCIENCE AND DIAGNOSTICS A. Heindl1, R. Ecker2, G. Bises1, T. Thalhammer1, I. Ellinger*1, A. Seewald3. Medical University Vienna, Austria, 2TissueGnostics, Austria, 3Seewald Solutions, Austria Introduction: As molecular cell phenotypes in cell-cohorts and tissues, also referred to as cytomes, result from genotype and environmental exposure, there exists phenotype-heterogeneity in healthy tissues that is pronounced in case of disease. These different phenotypes contain information about the present disease status (diagnosis) as well as its therapy dependent future development (prediction). Consequently, there is growing interest in basic medical research and diagnostics for multimolecular analysis of cytome heterogeneity in combination with exhaustive bioinformatic knowledge extraction (cytomics). Cytomicstechnologies are often microscope-based (slide based cytometry) using automated in-situ identication of cells and quantication of associated marker molecules. However, automated in-situ identication of special cellular shapes (e.g. multi-nucleated cells, cells without nucleus, and recognition of sub-cellular compartments) by means of high-content image analysis has not been established properly so far. Our project aims to nd a new approach for holistic pattern-recognition based on humanlike interpretations. Methods: Classical digital image-processing and pattern recognition approaches combined with machine-learning techniques are used to access also implicit expert knowledge. Cell recognition will be improved by including multiple cellular parameters like nuclei, generic markers, celltype specic markers in the analysis in comparison to current state-of-theart systems. Results: This versatile system is developed and primarily applied on sections of tissues that are not able to be analyzed with the current stateof-the-art technology. The unique functionality of this approach is exemplied by demonstrating placental tissue with its multinuclear syncytiotrophoblast and endothelial cells lacking nuclei due to the preparation procedure. A rst application of the novel automated cell-detection analysis is done in a biomedical pilot project on the characterization of the IgG transport pathway in placental chorionic tissue. Discussion: This study aims on improvement of automated in-situ identication of various placental cell-types in order to allow application of cytomics state-of-the-art technologies in placental basic research but also diagnosis. Keywords: Slide-based cytometry, cytomics, automated microscopy, IgG transport
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[P06.17]. PLACENTAL BARRIER IN NECROMYS LASIURUS AND ORYZOMYS SP. (CRICETIDAE: SIGMODONTINAE) PO Favaron1, AM Carter2, CE Ambrosio1, AC Morini1, ALR Franciolli1, MA Miglino*1. 1Sao Paulo University, Brazil, 2University of Southern Denmark, Denmark, 3Universidade Federal Rural do Semi-Arido, Brazil, 4 Humboldt-University Berlin, Germany The family Cricetidae has 681 recognized species. It includes Sigmodontinae, an important subfamily of Neotropical rodents, for which there is little information on placenta and placentation. This study focuses on the characteristics of the placental barrier in Necromys lasiurus and Oryzomys sp. Four placentae from each species were obtained from Collection of Zoology Museum of Sao Paulo University, Brazil. Labyrinth tissues were xed in 2.5% glutaraldehyde and prepared for TEM standard procedures. The placental barrier in these species consists of three trophoblast layers (TI, TII and TIII) and the fetal endothelial cell. TI is closest to maternal blood spaces; it consisted of a thin membrane, often discontinuous and had relationship with TII. Due to the strong contact between TI and maternal blood spaces, maternal blood cells were often observed to be surrounded by TI. The middle layer TII or middle layer showed a villous appearance with projections that on account of the discontinuous structure of TI in some places were in close contact with both TII and maternal blood. Cells of TII were dispersed in the extracellular matrix, had a single nucleus and little organized nucleolus with areas of condensed chromatin dispersed through the nucleus. TIII had a very strong relationship with the fetal capillary endothelium basement membrane as junctions were observed junctions in the area of apposition of these membranes. TIII was connected to TII by close and intermediate junctions, and desmosomes. The labyrinth is the region of most contact between maternal and fetal circulations. Thus N. lasiurus and Oryzomys sp. showed three trophoblast layers separating the two blood streams, so the placenta is classied as haemotrichorial subtype as in other Cricetid species described by Carpenter (1972 and 1975), King and Hastings (1977), Takata (1997), and Limongi and Ferro (2003). Keywords: placentation, rodents, haemotrichorial
[P06.18]. HIGH D-GLUCOSE UP-REGULATES hCAT-1 EXPRESSION AND ACTIVITY BY INCREASING SLC7A1 EXPRESSION IN HUMAN UMBILICAL VEIN ENDOTHELIUM Marcelo Gonzalez*1,2, Paola Casanello1,2, Luis Sobrevia1,2. 1Ponticia Universidad Catolica de Chile, Chile, 2Cellular and Molecular Physiology Laboratory, Chile L-Arginine is taken up by human umbilical vein endothelial cells (HUVEC) via the cationic amino acid transporter 1 (hCAT-1) isoform encoded by SLC7A1 gene. High D-glucose increases L-arginine transport, but mechanisms regulating hCAT-1 expression are unknown. Methods: Primary cultures of HUVEC (passage 2) were cultured under standard conditions. hCAT1-mediated L-arginine transport (0-1000 mM Larginine, 2 mCi/ml L-[3H]arginine, 37 C), hCAT1 protein abundance and mRNA copies were determined. Results: HUVEC exposed to D-glucose (5-25 mM, 0-24 hours) exhibit increased L-arginine (100 mM) uptake at 8 and 24 hours of incubation, associated with higher maximal velocity (Vmax) and capacity (Vmax/Km) of L-arginine transport (EC50 15 0.2 mM). High D-glucose increases hCAT1 mRNA expression (4 and 24 hours) and protein abundance (8 and 24 hours), an effect blocked by actinomicyn D. SLC7A1 promoter transcriptional activity was higher in high D-glucose (24 hours) as well as the Sp1 nuclear abundance and binding to SLC7A1 promoter. Conclusion: D-Glucose increases L-arginine transport by increasing hCAT1 expression, likely due to increased Sp1 binding to SLC7A1 promoter in HUVEC. Supported by FONDECYT 1070865/1080534, CONICYT 23070213. M. Gonzalez holds a CONICYT-PhD fellowship. Keywords: L-arginine transport, High glucose, hCAT-1, endothelium
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[P06.19]. IGF-2 RECEPTOR SIGNALLING AND TROPHOBLAST CELL SURVIVAL IN TERM PLACENTAL EXPLANTS LK Harris*, JD Aplin, PN Baker, IP Crocker, M Westwood. University of Manchester, United Kingdom Introduction: Insulin-like growth factor-II (IGF-II) enhances proliferation and survival of cytotrophoblast by signalling through the IGF-1 receptor. Classically, the IGF-2 receptor (IGF-2R) is thought to serve as a clearance rather than a signalling receptor, by trafcking excess IGF-II for lysosomal degradation. We hypothesized that knockdown of IGF-2R in the syncytiotrophoblast of term placental explants would enhance cytotrophoblast responsiveness to IGF-II. Methods: Term placental explants were transfected with siRNA specic for IGF-2R or a control sequence; knockdown was validated by RT-qPCR and immunohistochemistry. 48h post-transfection, explants were transferred to serum-free culture medium and stimulated with IGF-I, IGF-II or Leu27IGF-II, which only binds IGF-2R. After a further 24h, proliferation and apoptosis were assessed by immunostaining for Ki67 and M30, respectively. Results: siRNA decreased IGF-2R mRNA by 96.5% after 48h, and reduced protein expression in syncytiotrophoblast. In control explants, all IGF treatments increased the number of Ki67-positive cytotrophoblasts (P<0.001; 2-way ANOVA); however, Leu27IGF-II did not enhance proliferation after IGF-2R knockdown. Similarly, all IGF treatments decreased the number of M30-positive cells in control explants (P<0.001), but Leu27IGF-II did not decrease apoptosis after IGF-2R knockdown. As predicted, IGF-II-mediated rescue of cytotrophoblast apoptosis was increased after IGF-2R knockdown, relative to IGF-II-treated control explants. However, IGF-2 had no additional effect on cytotrophoblast proliferation in explants with reduced IGF-2R. Discussion: The absence of IGF-2R may increase the availability of IGF-2 for signalling through IGF-1R, resulting in enhanced cell survival. Mitogenic and pro-survival effects of Leu27IGF-II were only observed in the presence of IGF-2R, suggesting that this ligand may displace IGF-2 from IGF-R2 and/or signal through IGF-2R; further work is required to delineate the mechanisms involved. As increased cytotrophoblast apoptosis is observed in pregnancies complicated by fetal growth restriction, development of strategies to decrease IGF-2R expression in the syncytiotrophoblast may provide a therapeutic avenue for treating this condition. Keywords: cytotrophoblast proliferation, cytotrophoblast apoptosis, IGF-2, FGR
[P06.20]. CASPASE DEPENDENT REMODELLING OF CYTOSKELETAL ALPHAFODRIN IN FUSING TROPHOBLASTS AND BeWo CELLS Martin Gauster*, Monika Siwetz, Kristina Orendi, Gerit Moser, Gernot Desoye, Berthold Huppertz. Medical University Graz, Austria Introduction: Fusion of cytotrophoblasts with the syncytiotrophoblast is an essential step in differentiation of the human villous trophoblast. While knowledge about potential fusogenic factors deepened in the recent past, details of membrane cytoskeleton remodelling during trophoblast fusion are not yet identied. This study focussed on remodelling of submembranous spectrin-like a-fodrin during intertrophoblastic fusion. Methods: Primary term trophoblasts and forskolin challenged BeWo cells were subjected to quantitative real-time RT-PCR, immunouorescence and immunoblotting analyses to elucidate expression, localization and fragmentation of a-fodrin. Calpain inhibitors (calpeptin and calpain inhibitor III), inhibitors of caspases 3, 8 and 9 (DEVD, IETD and LEHD) and a general caspase inhibitor were applied to identify involved proteases in a-fodrin fragmentation. Results and Discussion: Experiments with primary trophoblasts and forskolin challenged BeWo cells revealed a biphasic strategy of the cells to achieve reorganization of a-fodrin during fusion. One strategy was down-regulation of a-fodrin mRNA, which was observed early in fusing trophoblasts and BeWo cells. The second strategy was proteolytic fragmentation of already existing full-length a-fodrin, which was cleaved into 120 and 150kDa fragments. Application of functional calpain inhibitors did not block a-fodrin fragmentation, suggesting that afodrin remodelling is a calpain independent process during trophoblast fusion. Inhibitors of caspases 3, 8 and 9, however, attenuated generation of the 120kDa fragment, indicating that all three caspases participated in the cleavage process. In fusing BeWo cells activation of caspases 3, 8 and 9 was detected. The fact that a general caspase inhibitor completely blocked fragmentation (120 and 150kDa fragments), argues for an exclusive role of caspases in a-fodrin remodelling in trophoblasts. Finally, immunouorescence double staining of human rst trimester placenta revealed colocalization of active caspase 8 with a-fodrin positive vesicles in fusing villous cytotrophoblasts. These results suggest that a bundle of caspases mediate remodelling of submembranous a-fodrin during trophoblast fusion. Keywords: villous trophoblast, syncytial fusion, caspase, calpain
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[P06.22]. MMP-2 EXPRESSION IN PLACENTAE AT HIGH ALTITUDE PREGNANCY F Lyall*1, L Marks1, S Zamudio2. 1University of Glasgow, United Kingdom, 2 New Jersey Medical School, United Kingdom Introduction: Metalloproteinases (MMPs) have been implicated in trophoblast invasion and placental vascular remodelling. The expression of some MMPs may be regulated, at least in part, by changes in oxygen tension. Pregnancies at high altitude are exposed to chronic hypoxia and we have previously shown that villous endothelial MMP-9 expression is reduced in high altitude pregnancies compared with moderate altitude or sea level pregnancies1. Methods and Hypothesis: We hypothesised that MMP-2 expression would also be altered at high altitude. Placentae were studied from uncomplicated pregnancies at sea level (n5), moderate altitude, 1600m (n6) and high altitude 3100m (n9). Immunohistochemistry was used to determine MMP-2 expression on villi. Immunostaining was quantied by an investigator blinded to the tissue identity and an arbritary scale of 0-4. The data was tested using the Shapiro-Wilk analysis and was not normally distributed hence non-parametric analysis was used. Results: MMP-2 was expressed in trophoblast, endothelium, muscle and stroma at all altitudes. In contrast to MMP-9 no difference in immunostaining was found between the two groups in any of the cell types. There was a trend for reduced endothelial staining but this did not reach statistical signicance. Conclusions: In contrast to MMP-9 we have found no evidence to support the hypothesis that MMP-2 is abnormally expressed in the placenta, at term, in uncomplicated pregnancies at high altitude. Whether enzyme activity is altered requires further investigation. 1 Marks L, Zamudio S & Lyall F (2002) MMP-9 expression is abnormal in placentae at high altitude: A link to chronic hypoxia? Hypertens Preg 21 (suppl 1) 111 Keywords: oxygen, high altitude, MMP, immunohistochemistry
[P06.23]. ENDOMETRIAL BEHAVIOUR OF COATI (NASUA NASUA) JC Morini Junior*1, PO Favaron1, AC Morini1, ALR Franciolli1, MA Miglino1, CE Ambrosio2. 1School of Veterinary Research and Animal Science, Brazil, 2 Faculty of Animal Science and Food Engineering, Brazil Two species of coatis, Nasua narica and Nasua nasua lives from Arizona to Argentina. Coati (Nasua narica) and racoon placentation have been observed, but less information is available about Nasua nasua. These aspects are important to be studied for comparative placentation especially with another carnivore species. This study focuses on the characteristics of the uterus that was supposedly pregnant obtained from UNIFEOB trial center, Sao Paulo, Brazil. The possible pregnancy was done because of a less increase of one of the horns and also the presence of corpus luteum at the ovarium of the same side. Tissues were xed in 10% formaldehyde and prepared for light microscopy and immunohistochemistry by standard procedures. The bicornual uterus was composed by perimetrium, myometrium, and endometrium. At the myometrium were found a large number of vessels strongly stained by vimentin. Two distinct arrangement of muscle cells were observed at this layer, one circular and other longitudinal separated by vessels. The endometrium consists in a simple cuboidal epithelium with numerous glands with high activity of secretion. Those glands and the endometrium layer of the lumen present cytoqueratin positive stain at its cytoplasm. As we know, cytoqueratin was expressed at the cytoskeleton of trofoblast cells, but it was found at the lumen of endometrial gland cells, however, a disorganized area was found related to embryo site of implantation. The embryo cannot be found, and because of it we cannot conrm the gestation, but the high activity of the uterine glands its rearrangement more deep and the positive stain of cytokeratin gave us evidence that the maternal environment was prepared for the embryo attachment as know in domestic carnivores. Keywords: Coati, Placenta, Uterus, Immunohistochemistry
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[P06.24]. ENDOTHELIAL PROGENITOR CELLS ARE NOT DECREASED IN MATERNAL SERUM FROM PATIENTS WITH DIABETES IN PREGNANCY DW Morrish*, J Dakour, C Chik, A Shuaib. University of Alberta, Canada Introduction: Endothelial progenitor cells (EPC) are critical in response to vascular injury and repair. EPC levels are reduced in patients with vascular disease. Pregnancy and diabetes are considered states of endothelial stress and share similar characteristics with vascular disease states. However, the lack of an unique antigen to identify EPCs has lead to different methods of identifying EPCs. The widely used Hall method (culture on brinogen) identies non-endothelial hematopoietic cells that nonetheless show correlations of decreased numbers with vascular disease. Alternate methods identify late clonogenic EPCs, eg Yoder et al, are of true endothelial origin. Using these methods, fetal cord blood levels of EPCs are reduced in diabetes in pregnancy (Ingram et al, Diabetes 57:724, 2008). Objective: To determine if diabetes in pregnancy results in reduced maternal EPC levels using clonogenic EPC methods. Methods: Human mononuclear cells from whole blood were isolated on Ficoll gradients, resuspended in complete EGM-2 medium supplemented with growth factors and seeded on type I collagen plates. After 24 h, cells were washed and adherent cells cultured in complete EGM-2-10% FBS with daily medium changes for 7 d, then every 2 d, until colonies formed. Results: Colony counts at 2 weeks of culture were: normal pregnant women: 2.10.6 (SEM; n23); pregnant diabetic: 1.40.3 (n24; preeclampsia: 0.80.5 (n4); normal nonpregnant women: 1.00.6 (n3). ANOVA showed no difference between normal pregnant and diabetic pregnant women. Preeclampsia and nonpregnant women showed lower colony counts but numbers were insufcient for analysis. Conclusion: Unlike the fetus, true endothelial-derived EPCs are not reduced in the mother with diabetes in pregnancy, suggesting other etiologies for maternal vascular abnormalities in diabetic pregnancies. Keywords: endothelial progenitor cells, diabetes, preeclampsia
[P06.25]. HOMEOBOX GENES ARE DIFFERENTIALLY EXPRESSED IN FETOPLACENTAL ARTERY AND VENOUS ENDOTHELIAL CELLS OF THE HUMAN PLACENTA P Murthi*1,2, NA Pathirage1,2, U Hiden3, M Dieber-Rotheneder3, G Desoye3, B Kalionis1,2. 1Department of Obstetrics and Gynaecology, The University of Melbourne, Australia, 2Department of Perinatal Medicine, Pregnancy Research Centre, The Royal Womens Hospital, Australia, 3Department of Obstetrics and Gynaecology, Medical University of Graz, Austria Introduction: Angiogenesis is fundamental to normal placental development and aberrant angiogenesis contributes substantially to placental pathologies. The complex process of angiogenesis is regulated by transcription factors leading to the formation of endothelial cells that line the vasculature. Homeobox genes are important transcription factors that regulate vascular development in embryonic and adult tissues. Previous studies in our laboratory have shown that a number of homeobox genes are expressed in placental endothelial cells (1,2). Recent studies by Lang et al (3) have shown that human feto-placental endothelial cells have a mature arterial and juvenile venous phenotype, which have distinct differentiation potentials. In this study, we hypothesised that homeobox genes are differentially expressed in the feto-placental artery (HPAEC) and in venous endothelial cells (HPVEC). Methods: HPAEC and HPVEC were obtained from uncomplicated term placentae as previously described (3). Briey, HPAEC and HPVEC were isolated by separate perfusion of chorionic arteries and veins with HBSS containing 0.1 U/ml collagenase, 0.8 U/ml dispase (Roche), and antibiotics (Gibco). The homeobox gene expression prole of HPAEC and HPVEC was determined using the Human Homeobox (HOX) Genes RT2 Proler PCR Array (SAbiosciences) which determined the expression of 84 HOX genes involved in multicellular organismal development. Results: Both known and novel homeobox genes showed greater than two fold increased relative expression in HPAEC compared with HPVEC. Known placental HOX genes included HLX, HEX, HOXB7, MEIS1, TLX1 and HOXC6. Novel homeobox genes, which showed increased relative expression levels were CUX1 and HOPX. Discussion: This is the rst study to report on differential expression for homeobox genes in HPAEC and HPVEC. Further analyses on the functional role of these homeobox genes in HPAEC and HPVEC maturation and differentiation will provide a better understanding of the molecular regulation of placental angiogenesis. References 1. Murthi P, et al. Placenta. 2007;28(2-3):219-23. 2. Murthi P, et al. Placenta. 2008;29(7):624-30. 3. Lang I, et al. Differentiation. 2008;76(10):1031-43. Keywords: Placental endothelial cells, homeobox transcription factors, angiogenesis, gene expression
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[P06.26]. ANGIOPOIETIN-LIKE PROTEIN 2 (AngPTL2) IN HUMAN MATERNAL SERUM, AMNIOTIC FLUID AND TERM PLACENTAL TROPHOBLAST IN PREGNANCY K Naruse*1, M Endo2, A Onogi1, H Shigetomi1, Y Yoshizawa1, T Sado1. 1Dept. of Obstetrics & Gynecology, Nara Medical University, Japan, 2Dept. of Molecular Genetics, Graduate School of Medical Sciences, Kumamoto University, Japan Introduction: Angiopoietin-like protein 2 (AngPTL2) is a family member of angiopoietins playing key roles in angiogenesis, vascular maintenance, cell migration and apoptosis. Although roles of AngPTL2 in tumorgenesis and pathogenesis in tumors and metabolic diseases were dened from former researches, little is known about its alteration and expression in pregnancy and placenta. In this study, we measured peripheral AngPTL2 through pregnancy and searched its expression on cultured trophoblasts separated from human placenta, to assess its possible role in human pregnancy. Methods: Paired serum samples from 1st, 2nd and 3rd trimester of pregnancy, or prior to and at the next day of selective cesarean section at term from respective 7 women were taken at the routine medical examination with informed consent. Amniotic uid samples were taken from 13 women at the time of selective cesarean section and centrifuged / ltered before the measurement. Serum and amniotic uid concentrations of AngPTL2 was measured with ELISA. Human term placentas were taken at selective cesarean section or term vaginal birth. Trophoblast was separated with trypsin digestion and Percoll gradient method, harvested on bronectin-coated plate overnight and xed for staining. Immunocytochemistry for AngPTL2 and cytokeratin (5+6+8+18) were performed using respective antibodies and compared together. Results: AngPTL2 concentration in the serum were signicantly increased in the 3rd trimester of pregnancy (1st, 2.400.20; 2nd, 2.930.40; 3rd, 3.940.37 ng/ml, p0.0019). Additionally, AngPTL2 in the maternal serum was signicantly decreased at the next day of selective cesarean section (3.450.37 vs. 2.410.16 ng/ml p0.029). Concentration in amniotic uid was relatively low (0.060.01 ng/ml). Immunostaining of the AngPTL2 were positive on the term placental trophoblasts and were matched with cytokeratin-positive cells. Conclusion: Maternal peripheral AngPTL2 increase in pregnant women was shown rst time, and placenta was suggested as a source of AngPTL2 in pregnancy. AngPTL2 may contribute to the placental vascular maintenance or other protective roles in the cells at the feto-maternal border. Keywords: AngPTL2, Angiogenesis, Placental vascular maintenance, fetomaternal border
[P06.27]. METHOTREXATE AND EPIDERMAL GROWTH FACTOR INHIBITION REGRESS PLACENTAL-DERIVED TISSUES IN VITRO AND IN VIVO: TOWARDS A NOVEL COMBINATION MEDICAL THERAPY TO TREAT ECTOPIC PREGNANCY UW Nilsson*1,2, YE Gao1,2, TG Johns1, BR Williams1, ED Williams1, S Tong1,2. 1 Centre for Cancer Research, Monash Institute of Medical Research, Australia, 2Centre for Womens Health Research, Monash Institute of Medical Research, Australia Introduction: Ectopic pregnancies are serious gynaecological emergencies that can cause fatal haemorrhage. Most (75%) require surgery. Systemic methotrexate (MTX) is sometimes used. Since efcacy is patchy, it only works for small ectopics. We hypothesise that Epidermal Growth Factor Receptor (EGFR) inhibition MTX could efcaciously resolve stable ectopic pregnancies of any size. The placenta relies on the EGFR pathway for survival. EGFR inhibitors, currently on the market to treat cancers, have few side effects. Furthermore, EGFR inhibitors have supra-additive anti-tumour activity when combined with chemotherapeutics. We set out to develop a novel approach to medically treat ectopic pregnancy using the EGFR inhibitor getinib (Iressa) MTX. Methods/Results: In vitro: We rst conrmed by immunostaining that 1st trimester trophoblast, BeWo and JEG-3 cells express EGFR. Next, we added various treatments to placental-derived cells and assayed viability 72 hours later (CellTiter-Blue [Promega]). Getinib alone did not cause trophoblast cell death of JEG-3 (see gure) or BeWo cells (not shown) compared to control. MTX alone (1-1000uM) decreased cell viability in a dose-responsive manner (not shown). Interestingly, when getinib was combined with MTX, a dose-dependent, supra-additive decrease in cell viability occurred in both JEG-3 (gure A) and BeWo cells (not shown). In vivo: SCID mice (n3-5 per group) were inoculated with JEG-3 cells (106), and treated with different doses of getinib and MTX, all as single agents (see gure). Compared to vehicle-treated mice, there was a signicant inhibition of xenograft growth with both doses of getinib where only w30% had palpable tumours by 19 days. There was also a dose dependent inhibition of xenograft growth with MTX. Conclusion: Getinib MTX causes signicant regression of placental derived tissues. It may be a novel therapeutic approach to treat stable ectopic pregnancies medically where conceivably, tablets (Getinib + MTX) could replace surgery.
Keywords: Ectopic pregnancy, Epidermal growth factor, Methotrexate, Therapy
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[P06.28]. RNA INTERFERENCE-INCUCED REDUCTION IN ASCT2 EXPRESSION, A SYNCYTIN RECEPTOR, SUPPRESSES CELL FUSION OF HUMAN PLACENTAL BeWo CELLS T Nobuzane*, S Matsuyama, Y Kudo. Department of Obstetrics and Gynecology, Hiroshima University, Japan Introduction: Syncytin, the human endogenous retrovirus envelope proteins, is highly expressed in the placental syncytiotrophoblast layer, and contributes to placental fusion events. ASCT2 is a syncytin receptor and known to be identied as the amino acid transporter B0. The decrease of fusion activity in hypoxia was observed. Additionally the alteration of these gene expressions in pre-eclampsia has been reported. To investigate the relationships between syncytin, ASCT2 and fusion activity, we use siRNA technique in human placental BeWo cells. Methods: BeWo cells were treated with siRNA for either syncytin or ASCT2. The efcacy of these specic siRNA treatments was analyzed and cell fusion was assessed by ow-cytometry. Results: The mRNA expression of syncytin and ASCT2 was reduced after siRNA treatment. The fusion activity was reduced only after treatment with siRNA for ASCT2. Discussion: These results suggest that ASCT2 might be involved in the process of cell fusion in BeWo cells. Keywords: cell fusion, ASCT2, BeWo cells
[P06.30]. DETERMINATION EMBRYOGENESIS
OF
THE
FIRST
LINEAGES
DURING
CATTLE
D. Berg, C. Smith*, D. Wells, R. Broadhurst, M. Berg, P.L. Pfeffer. Agresearch, New Zealand The rst lineage decision during mammalian embryogenesis is between inner cell mass (ICM) and trophectoderm (TE). By blastocyst stages these lineages are clearly discernable morphologically. In mice, ICM and TE require the function, and are characterised by the mutually exclusive expression, of Oct4 (ICM) and Cdx2 (TE). This has led to models implicating these factors in early lineage determination. We were thus surprised to nd only marginal lineage-specic enrichment of these genes in cattle blastocysts, using quantitative PCR. Lineage tracing conrmed that blastocyst TE cells were fated to remain trophectodermal. However, sandwiching Day7 blastocyst or Day14 gastrulation-stage TE cells within morula cells revealed that blastocyst TE was not yet committed. We speculated that lack of TE commitment in cattle was linked to the observed retarded downregulation of Oct4 in these cells. We could demonstrate that mouse and cattle Oct4 regulation is fundamentally different at blastocyst stages. Unlike mouse embryos, cattle embryos transgenically modied (via nuclear transfer) with mouse Oct4 regulatory sequences driving a GFP reporter were unable to shut off the mouse Oct4 reporter at blastocyst stages. Conversely, both transgenic mouse and cattle blastocysts were unable to restrict a cattle Oct4 reporter to the ICM. These experiments indicate that not only does the cattle embryo lack the necessary factors present in the mouse for shutting off Oct4 transcription in the blastocyst TE compartment, but that the cattle Oct4 gene is missing the mouse-TEspecic negative regulatory elements. Was Cdx2 responsible for Oct4 downregulation? pCAG-GFP-AntiCdx2-miR mediated knock-down of Cdx2 expression had no effect on Oct4 transcription but led to severe TE defects at gastrulation stages. The rodent-ruminant differences in timing of both commitment and Oct4/Cdx2 lineage restriction may be adaptations to different requirements: whereas mouse embryos implant as blastocysts, cattle embryos are less hasty, attaching only post-gastrulation. Keywords: lineage determination, bovine, trophectoderm, preimplantation
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[P06.31]. INSULIN INCREASE D-GLUCOSE TRANSPORT IN HUMAN UMBILICAL VEIN ENDOTHELIAL CELLS C Puebla*, CA Sepulveda, JL Vega, P Casanello, L Sobrevia. Cellular and Molecular Physiology Laboratory (CMPL) and Perinatology Research Laboratory (PRL), Department of Obstetrics & Gynaecology, School of Medicine, Ponticia Universidad Catolica de Chile, Chile Introduction: Umbilical cordon represent the medium to communication between mother and fetus. Nutrients are in contact directly with the endothelium, which is affected by the different concentrations of these molecules. In endothelial cells, facilitative and insulin-insensitive transporters are the principal protagonist in the transport of D-glucose, but it is controversially discussed wheter the endothelium represents an insulinresponsive tissue, inclusive the insulin-sensitive GLUT4 isoform seems to be expressed in human umbilical vein endothelial cells (HUVEC) from normal pregnancies. We studied if D-glucose transport in HUVEC from normal pregnancies exposed to high D-glucose is modulated by insulin. Methods: HUVEC were isolated by collagenase (0,2 mg/ml) from human umbilical cord vein from normal pregnancies and exposed (24 hours) to 5 mM (normal) or 25 mM (high) D-glucose in absence or presence of insulin (10 nM, 8 hours). 2-deoxi-D-[3H]glucose (2DG) transport (1,6 mM, 1 mCi/ ml, 5 sec to 40 sec, 22 C) was measured. Results: 2DG transport is lower in HUVEC exposed (24 h) to high Dglucose. Then in presence of insulin the 2DG transport increase in HUVEC exposed to high D-glucose, but in normal D-glucose the effect was absent. Discussion: D-glucose transport is decreased in HUVEC exposed to high Dglucose, but in presence of insulin this transport is increased to normal condition, which could be by the presence of insulin-sensible transporter GLUT-4; eventually this phenomenon is a compensatory effect: cells try to decreased extracellular glucose levels to protect the fetus. The fact that insulin did increase D-glucose transport only in cells exposed to high Dglucose, could suggest that this pathological environmental condition could increase insulin-sensitivity for GLUT-4. FONDECYT 1070865/1080534/7070249 (Chile), C Puebla and JL Vega hold CONICYT PhD fellowships. Keywords: Endothelium, Transport, Glucose
[P06.32]. ENDOCRINE GLAND-DERIVED VASCULAR ENDOTHELIAL GROWTH FACTOR (EG-VEGF) EXPRESSION IN TWO TRANSIENT ORGANS: PLACENTA AND THYMUS OF DOGS A COMPARATIVE STUDY Fernanda R. Agreste*1, Patrcia R. Moriconi1, Bruno Cogliati1, Pedro P. Bombonato1, Carlos Eduardo Ambrosio1, Christiane Pfarrer2. 1Faculty of Veterinary Medicine and Animal Sciences, University of Sao Paulo, Brazil, 2 University of Veterinary Medicine Hannover, Germany Recently, EG-VEGF (endocrine gland-derived vascular endothelial growth factor) was described in human and mouse placenta and is likely to play an important role in placentation as it stimulates proliferation, migration, and fenestration selectively in capillary endothelial cells from endocrine glands. However, the EG-VEGF mRNA level determined in others organs like liver and kidney, suggests that EG-VEGF may have direct effects in these tissues and thus is not restricted to endocrine tissues. Thymus and placenta are transient organs showing common features of elevated metabolic activity and modications in cellular proliferation and differentiation when undergoing continuous stress. In contrast to most domestic species the dog placenta does not produce steroid hormones. Our aim was therefore to localize and quantify EG-VEGF throughout development and initial involution of two transient organs, canine thymus and placenta. Thymuses from 30, 40, 50 and 60 days-old fetuses, 6 and 12 months-old dogs (n6 per group) and dog placentae from 20, 40 and 60 days of gestation (n4 per group) were examined by immunohistochemistry and Western blot. EG-VEGF was detected in all stages of development and involution in thymus and placenta. In thymus, protein was localized in medullar endothelial and epithelial cells while no staining occurred in the cortical region. In placenta, EG-VEGF was detected mainly in endothelial cells and syncytiotrophoblast of labyrinth. Protein quantication revealed higher (p<0.05) values during development of thymus (days 30-60) and placenta (days 20-40), decreasing at the end of gestation for the placenta. During thymus involution (6-12 months) EG-VEGF levels were constant but lower (p<0.05) than during gestation. Our results suggest that EG-VEGF may play important roles during thymus and placenta development and involution, which have to be specied in further studies. Possible functions include modulatory effects on thymic and placental microenvironment, inuencing cellular proliferation, differentiation and secretion. Funded by CAPES, FAPESP and DAAD. Keywords: placenta, EG-VEGF, canine, thymus
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[P06.33]. UNALTERED 5-HYDROXYTYPTAMINE (5-HT) RECEPTOR EXPRESSION IN PLACENTA, DESPITE INCREASED CIRCULATING 5HT IN PRE-ECLAMPSIA S. D. Sivasubramaniam*, P. Muralitharan, L. De Girolamo. Nottingham Trent University, United Kingdom This study aimed to investigate the status of circulating 5HT and the mRNA expressions of three main vascular 5-hydroxytryptamine (5-HT or serotonin) 1B, 2A and 7 receptors in normotensive (NT) and pre-eclamptic (PE) placentae. 5HT mainly produces vasoconstriction in the peripheral vasculature and in placental vessels. Previous studies have demonstrated that 5-HT can play roles in placental development and pregnancy maintenance. It may also take part in regulating fetal development. Since the effects of 5-HT are brought about by 7 different types (and their subtypes) of 5-HT receptors, it would be interesting to study the status/expression of these receptors in placental tissues. Circulating levels of 5-HT were indirectly determined by the analysis of urinary 5-HT and its metabolite 5-hydroxyindoleacetate (5-HIAA) by ELISA. The status and the expression of 5-HT receptors between NT and PE placentae were compared by RT-PCR and quantitative real time (qRT-PCR) respectively in relation to two house-keeping genes GAPDH and b-actin. Pre-eclamptic women had signicantly higher levels of 5-HT (124.823.6mg/24h urine) compared to NT counterparts (59.59.8mg/24h urine) (p<0.05).Whilst the levels of the metabolite 5-HIAA were similar in the two groups. Thus the ratio of 5-HIAA:5-HT in PE women was 50% lower than NT controls (p<0.05). RT-PCR results have shown the presence of 5HT1B, 2A and 7 receptors in NT and pre-eclamptic samples. However we have found there are no signicant differences in the mRNA expressions of these receptors between NT and PE placentae. For example the 5HT2A expression in relation to b-actin in NT and PE placentae were 4.40 (SEM1.24) and 4.02 (SEM1.27) respectively. Similar patterns were observed for 5HT1B and 5HT7 receptor expressions. These results suggest that despite a denite increase in the circulating 5HT levels in PE, there is no change in the mRNA expressions of 5HT1B, 2A and 7 receptors in PE placentae. Keywords: 5-hydroxytryptamine, pre-eclampsia, qRT-PCR
[P06.34]. CLINICAL ANALYSES FOR TRANSITIONAL CASES OF INFERTILITY AND RECURRENT PREGNANCY LOSS E.T. Takahashi*, R.K. Kawaguchi, T.T. Tadao. Department of Obstetrics and Gynecology, The Jikei University, School of Medicine., Japan Objectives: In the clinical practice, it is not a little case that the patient who experience recurrent pregnancy loss (RPL) becomes to be infertility thereafter, or tends to be aborted repeatedly after infertile therapy. Those transitional conditions have not been noticed so far. We investigated the difference of possible causes and clinical status among those conditions from perspectives of reproductive failure. Patients and Methods: Out of patients of infertility (694) and RPL (691) treated at The Jikei University Hospital from 2003 2008, 4 groups were selected as follows. A : transitional cases from RPL to infertility (24). B : transitional cases from infertility to RPL (43). C : cases of infertility (40). D : cases of RPL (80). Results: 1. Although the ratio of pregnancy did not differ signicantly among 4 Groups, that of miscarriage showed higher in A and B than C and D, and moreover that of live-born showed the fewest in A compared with D, also with signicant difference. 2. The incidence of positive for APAs approximately amounted to about 50 percent of these factors in all Groups. Especially, in B, APAs was recognized signicantly higher in elderly-age than young-age (under 35 years). 3. The reproductive outcome after therapies was examined in all Groups. Concerning infertility, C showed signicantly fewer rate of miscarriage than A and B regardless of the therapies, ART or not. On the other hand, although anti-coagulant therapy was treated for RPL, D showed signicantly fewer rate of miscarriage than A and B. Conclusion: 1. The state of reproductive failure in transitional cases have a tendency to be unfavorable reproductive outcome. 2. In transitional cases of reproductive failure, active treatment such as ART is recommended from the early period. Keywords: anti-phospholipid antibody, infertility, recurrent pregnancy loss
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[P06.35]. THERAPEUTIC OUTCOME IN RECURRENT SPONTANEOUS ABORTIONS WITH ANTIPHOSPHOLIPID ANTIBODIES(aPLs). w THE INFLUENCE OF TITERS, VARIETIES, ISOTYPES, POSITIVE NUMBERS OF ANTIPHOSPHOLIPID ANTIBODIES N Umehara*, R Kawaguchi, S Wada, K Sugiura, K Ooura, T Tanaka. Department of Obstetrics and Gynecology, The Jikei University School of Medicine, Japan Objective: Low dose heparin in combination with low dose aspirin is the standard therapy for patients with histories of recurrent pregnancy losses and positive for antiphospholipid antibodies, but the inuence of antibody class, titer, isotype of IgG and IgM, positive number of the aPLs are not yet clear. Our objective is to investigate the reproductive outcome of anticoagulant therapy to patients of recurrent spontenous abortion with antiphospholipid antibody, with special attention to the impact of species, titer, and isotype of the antibody. Method: I had a checkup 282 examples with histories of two or more spontenous abortions, visited in this hospital from April, 2000 to July, 2005, and detected serum levels in 10 varieties of aPLs. Of 156 patients of recurrent abortion with antiphospholipid antibody, 101 cases got pregnant and anticoagulant therapy was performed according to our anticoagulation protocol, so these 101 cases were evaluated. Furthermore, we classied them into low and high titer groups, IgG or IgM isotypes groups, single positive or plural positive groups with aPLs. In each groups, we analyzed successful rate of pregnancy prospectively. Results: Species, titer or isotypes of antiphospholipid antibodies did not affect the rate of maintained pregnancy in recurrent abortion when anticoagulant therapy was actively introduced. Regarding on isotypes and the number of positive antibodies, aspirin treatment alone was sufcient for successful reproductive outcome in only the IgM-isotype and single antibody positive cases (95.2%), while in the IgG-isotype and plural antibodies positive cases, the rate of maintained pregnancy was increased by combination treatment with aspirin and heparin (95.2%) than aspirin alone (78.6%). Conclusions: The anticoagulant therapy was effective to patients of recurrent abortion with antiphospholipid. However, combination therapy with aspirin and heparin may be overtreatment for only the IgM-isotype positive case. Keywords: recurrent spontaneous abortion, antiphopholipid antibodies, reproductive outcomes, anticoagulant therapy
[P06.36]. MONOCHORIONIC TWIN FETUSES SHOWING A REVERSAL OF DONORRECIPIENT PHENOTYPES IN SEVERE TWINTWIN TRANSFUSION SYNDROME WITHOUT OLIGO-POLYHYDRAMNIOS SEQUENCE A Mitsumori*, O Akutagawa, M Kazuka, H Funayama, K Isaka. Tokyo Medical University, Japan Antenatal sonographic diagnosis of twintwin transfusion syndrome (TTTS) is based on ndings of a twin oligo-polyhydramnios sequence (TOPS) observed in monochorionic twin fetuses. However, TTTS can develop without a signicant characteristic intertwin discordance in the amniotic uid volumes. We present an uncommon form of TTTS without TOPS showing severe anemia in one twin and polycythemia in the other. A 34-year-old Japanese woman was referred to our hospital at 30 weeks of gestation for perinatal management of a monochorionic-diamniotic twin pregnancy. Although no abnormalities were noted by ultrasound examination before her rst visit to our hospital. Both fetuses had a normally lled bladder and a normal amniotic uid volume. The patient was admitted for close perinatal management at 30 weeks. Because of increased uterine activity, the intravenous administration of ritodrine hydrochloride was initiated. At 34 weeks, the fetal heart rate monitoring twin 1 showed loss of variability. Because of non-reassuring fetal status of twin 1, weighed 2341 g and was pale, whereas infant 2 weighed 1920 g and was plethoric. Apgar score at 1 and 5 minute after birth were 2/4 and 6/6 for infant 1 and 2, respectively. And hemoglobin contents were 4.7g/dl and 20.3g/dl. The placenta was monochorionic diamniotic, with a velamentous insertion of the edematous umbilical cord of twin 2. The placental territory of twin 1 was pale, three times larger than that of twin 2, which was plethoric. In conclusion, perinatologists involved in the care of monochorionic twins should keep the differential diagnosis of acute or chronic TTTS in mind, even in the absence of TOPS. TAPS can occur in the absence of the characteristic interwin discordance of the amniotic uid volume.
[P07.01]. PLACENTAL AND FOETAL ORGAN GROWTH ARE REGULATED BY DIFFERENT COMBINATIONS OF GROWTH FACTORS R. S. F. Lee*, N. Li, D. N. Wells. AgResearch, New Zealand The cloning of cattle by somatic cell nuclear transfer (SCNT) is characterized by increased incidence of abnormal development and excessive placental and foetal growth. In SCNT foetuses from a Friesian dairy cow cell line, we found no apparent correlation between placental and foetal weight, suggesting that placental and foetal growth may be independently regulated. We examined the expression of several growth-regulating genes, such as IGF2, H19, IGF2R, IGFBP-2, GPC3 and CDKN1C (p57kip2), in mid-gestation SCNT placenta and foetal tissues that normally show overgrowth (liver, kidney and heart) using northern blots. SCNT samples were compared with samples from gestation-matched half-siblings generated by articial insemination (AI) or from the transfer of in vitro produced (IVP) embryos. The mean expression levels for IGF2, H19, IGF2R and GPC3 in either foetal or placental samples were not different among the SCNT, AI or IVP groups. Mean IGFBP-2 expression was signicantly lower (P<0.05) in the SCNT and IVP placenta compared with the AI controls, consistent with IGFBP-2 being a negative regulator of IGF-II action and the observed placental overgrowth in SCNT, and to a lesser extent, IVP. No difference was detected in kidney and heart samples but mean expression in SCNT liver tended to be higher. In contrast, there was no difference in the expression of CDKN1C in placental, liver and heart tissues. Mean expression in the kidney was signicantly lower (P<0.001) in SCNT, where there was a signicant inverse correlation (P<0.005) between the level of CDKN1C expression and kidney weight. Thus, the growth of each foetal organ and the placenta is regulated by different combinations of factors that act in an autocrine/paracrine manner rather than systemically. In SCNT foetuses, this may not be as well coordinated between the different organs and placenta resulting in loss of allometric growth regulation. Keywords: IGFBP-2, CDKN1C, SCNT, overgrowth
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[P07.02]. ENDOCRINE GLAND-DERIVED VASCULAR ENDOTHELIAL GROWTH FACTOR (EG-VEGF) EXPRESSION IN CLONED AND NON-CLONED BOVINE PLACENTA FR Agreste1, LA Fatima1, C Pfarrer2, PC Papa*1. 1Sector of Anatomy, Department of Surgery, Faculty of Veterinary Medicine and Animal Sciences, University of Sao Paulo, Sao Paulo, Brazil, 2Department of Anatomy, University of Veterinary Medicine Hannover, Germany Endocrine gland vascular endothelial growth factor (EG-VEGF), also known as prokineticin 1 (PK1), has recently been described in the human and mouse placenta. It promotes proliferation, survival, and chemotaxis of endothelial cells in steroidogenic tissues, such as adrenal cortex capillary endothelial cells. The bovine placenta is multiplex, villous and synepitheliochorial due to migratory trophoblast giant cells (TGC). To determine the spatio-temporal distribution of EG-VEGF in non-cloned and cloned bovine placenta, placentomes from non-cloned pregnancies from early gestation until near term (6 groups: days 30, 60, 90, 150, 210 and 270 of gestation, n4 per group) and cloned placenta near term (n4) were evaluated by immunohistochemistry and quantied by western blot. EG-VEGF immunoreactivity was observed in uterine glands during implantation. In normal and cloned bovine placenta the immunoreaction was conned to maternal epithelium, binucleate TGC, fetal and maternal blood vessels. The quantication of EG-VEGF in normal bovine placenta revealed that high levels occurred in early placentation; these decreased from mid gestation (p<0.05) until near term. In cloned bovine placentae the expression was higher (p<0.05) than in non-cloned placentae. In summary, EG-VEGF is present in the synepitheliochorial bovine placenta in all studied gestational phases but the levels are related to gestational age. Thus, we may conclude that EG-VEGF expression in the bovine placenta seems to be important throughout gestation, probably inuencing endothelial and/or epithelial cells function. Moreover, placenta derived from cloned gestations, which presented documented differences regarding vascularization pattern and expression of vascular related growth factors, showed an up-regulation of EG-VEGF expression, regardless of fetus gender. This up-regulation could reect the attempt to compensate hypoxia in these placentae. Funded by CAPES, FAPESP and DAAD. Keywords: EG-VEGF, bovine, SCNT
[P07.03]. EXPRESSION OF ALPHA6BETA1 AND ALPHAVBETA3 INTEGRINS AND THEIR EXTRACELLULAR MATRIX LIGANDS IN PLACENTOMES FROM CLONED AND NON-CLONED CALVES LP Artoni1, N Hambruch2, PC Papa1, C Pfarrer*2. 1Institute of Anatomy, Department of Surgery, University of Sao Paulo, Sao Paulo, Brazil, 2 Department of Anatomy, University of Veterinary Medicine Hannover, Hannover, Germany Integrins are transmembrane glycoproteins which are involved in cell-cell and cell-extracellular matrix (ECM) adhesion and signal transduction. Integrins may be involved in the migration and fusion of trophoblast giant cells (TGC) with uterine epithelial cells in bovine placentomes. Somatic cell nuclear transfer is frequently associated with aberrant placentation as larger placenta with lower number of placentomes, hydroallantois and large offspring syndrome. Since integrins are considered to be constitutive proteins being responsible for the maintenance of the architecture within tissues, we aimed to verify a potential relation of integrins with common placental alterations observed in cloned animals. Bovine placentomes were collected and divided into 3 groups: 1) mid and 2) late gestation derived from natural mating (n9) and 3) late gestational bovine clones (n7). ECM proteins Collagen IV, Fibronectin and Laminin were localized by indirect immunohistochemistry and integrin subunits alpha6, beta1, alphaV and beta3 were shown by immunouorescence. The antibody specicity was conrmed by Western blot. In cloned and non-cloned animals the integrin subunits alpha6 and beta1 were observed near the basal membrane of maternal and fetal epithelial cells and stromal endothelium, and co-localized with laminin in some TGC. The integrin subunits alphaV and beta3 were co-localized with the collagen IV and bronectin in maternal and fetal stroma. Western blot conrmed the integrin and laminin antibodies specicity. No difference in the integrin or extracellular matrix proteins expression could be found between mid and late gestational placentomes from natural mating pregnancies and cloned gestation. The absence of differences found between cloned and non-cloned gestation suggests that the interaction of integrins with their ECM ligands cannot be directly related with the observed placental alterations. Nevertheless, further studies are necessary to elucidate the mechanisms that might be responsible for the alterations observed in the gestation of bovine clones. Funded by CAPES, FAPESP and DAAD. Keywords: Integrins, bovine, SCNT
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[P08.01]. ORGANOGENESIS OF THE CARDIO-RESPIRATORY SYSTEM OF BOVINE EMBRYOS MANIPULATED IN LABORATORY M. L. V. Alberto*, F. V. Meirelles, A. C. R. Galdos, R. Ricci, A. G. T. Pessolato, M. A. Miglino. Medical College of Zootechny and Veterinary Medicine, Section of Domestic and Wild Animals Anatomy USP/SP, Brazil Morphogenic changes of cardio-respiratory system of cattle from in vitro fertilization and nuclear transfer are the main factors responsible for high incidence of embryo, fetal and postnatal mortality. The study of development of heart and lung was made using techniques of light microscopy. In the embryos derived from in vitro fertilization at 28 days of gestation was found the laryngotracheal tube and its septation through the fold tracheoesophageal. In this same period, the embryos showed pericardial cavity, atrium divided into right and left portions, cone heart, venous sinus, myocardium and epicardium layer. Was observed sprouting in the right lateral portion of the trachea, cranial to its bifurcation in embryos with 36 days of gestational age. At 44 days of gestation the lung buds of the embryos showed the main bronchi originated of segmental bronchi. The mesenchyma of support in differentiation contained blood vessels dispersed, unlike embryos from nuclear transfer. These embryos at 68 days of gestation showed lung in pseudoglandular phase containing bronchioles buds and absence of blood vessels. At 70 days of gestation, the heart of the fetus showed signicantly large ventricle, small lobby and undeveloped lung. The placenta is considered a vital organ of pregnancy. Therefore a aw in the formation and development process can cause serious changes in organogenesis of the embryo and growth of the fetus until birth. Keywords: cardio-respiratory system, bovine embryos, lung buds, heart
[P08.03]. INITIAL DEVELOPMENT OF BOVINE PLACENTATION (BOS INDICUS) FROM THE POINT OF VIEW OF THE ALLANTOIS AND AMNION A. C. Assis Neto*1, C. E. Ambrosio2, M. F. Oliveira3, F. T. V. Pereira1, M. A. Miglino2. 1Sao Paulo State University, Brazil, 2University of Sao Paulo, Brazil, 3Universidade Federal Rural do Semiarido, Brazil, 4Sao Paulo State University, Brazil, 5University of Sao Paulo, Brazil The aim of this study was to perform a morphological characterization of the initial bovine placental development, from 20 and 70 days postinsemination (p.i), with emphasis on the differentiation of the allantois and amnion. After collection, the conceptuses were dissected, macroscopically measured and photographically documented. The extraembryonic membranes were cut into fragments measuring 5 cm2, and afterwards xed in 4% paraformoldehyde for analysis by light microscopy, and 2.5% glutaraldehyde for use in scanning and transmission electron microscopy. The extraembryonic and fetal membranes presented variable degrees of development throughout the periods analyzed. The macroscopic appearance of vascularization of the allantois, its attempt to merge with the chorium, and the effective appearance of the rst cotyledons in development were events observed from 30 to 40 days of pregnancy. The measurements of the amnion increased gradually as gestation developed. The allantoic epithelia presented cellular dimorphism from 20 to 25 days of pregnancy, but was shown to be immature from 60 to 70 days of pregnancy. Financial support: FAPESP, CAPES, CNPq, FUNDUNESP Keywords: Placenta, Allantois, Amnio, Bovine
[P08.02]. LOOKING FOR THE VITELLINE PLACENTATION IN EARLY BOVINES EMBRYOS Celina Almeida Furlanetto Mananares, Ana Flavia de Carvalho, Rudolf Leiser, Carlos Eduardo Ambrosio, Maria Angelica Miglino*. 1UNIfeob, Sao Joao da Boa Vista, Sao Paulo, Brazil, 2Department of Veterinary Anatomy, Histology and Embryology, Justus Liebig-University Giessen, Germany, 3 University of Sao Paulo, FMVZ, Surgery Department, Brazil Yolk sac is an important organ to embryonic circulation and metabolic process mainly during the transition of chorionvitteline placentation process to chorionallantois one. About 25% to 40% of embryo lost occurs during fertilization process until implantation. The yolk sac is the mainly source of nutrition before true placenta will completely formed and your importance are related to hematopoiesis. The aim of this work is describes the primitive gut transitional area with yolk sac connection and maintain importance to the embryo until placental formation. Were collected 59 embryos during 10 to 50 days of pregnancy and were grouped by Crownrump measure and external characteristics, xed, and processed by procedures for embedding in parafn. Sections were stained by HE. The embryo yolk sac was observed with active cells between it connection with primitive gut at anterior portion. Gut cells presented at begin of differentiation of epithelium columnar cells and endoderm follow down by undifferentiated layer of mesenchymal cells from the mesoderm. Gross aspect of this fetal membrane is centrally compact with two free elongated tips at 10 to 20 days of pregnancy and became decreases it size up to 40 days after fecundation. The yolk sac was formed by blood vessels island surrounding to endodermic cells and mesenchyme. Also was found nucleated blood cells (hemangioblast) from fetal origin. Mesenchymal layer was thin with elongated cell characterized by proteoglycan synthesis presented into cell matrix. At transitional strait channel between yolk sac and primitive gut with delicate roundest cells and into the lumen was found hemangioblast follow in fetus way direction and also coming into the mesenchyme. Although this last tissue type present hemangioblast into small capillary net. So, how is the real function of yolk sac and the vitelline nutrition marked the rst environment adversity of the embryo life? Keywords: development embryonary, yolk sac, vitelline placentation
[P08.04]. PLACENTAL GROWTH IN MINK: ELUCIDATED BY MITOSIS, APOPTOSIS AND TURNOVER RATE THROUGH OUT GESTATION Vibeke Dantzer*, Henrik Winther. 1IBHV, LIFE, Copenhagen University, Denmark, 2BImmunoHistology, Dako A/S, Produktionsvej 42, DK-2600 Glostrup, Denmark Objective: The development of placenta, growth and angiogenesis is a prerequest for successful gestation. We here demonstrate the spatio- and temporal localization of the mitotic activity, caspase activity and endothelial turnover rate in a small carnivore, the mink, having an incomplete zonary, villous, endothelio-chorial placenta and delayed implantation. Methods: Gestational age was estimated by uterine dilations (ampullae) and the fetal crown/rump (C/R) length (term 7cm). By immunohistochemical the activity of Ki-67 and Caspase-3 activity (18 mink) and 11 mink, injected with bromodeoxyuridin (Br-dUrd) 24-168 hrs before eutansia, was studied. Results: Ki-67 activity at non pregnant was only seen in the stroma. At invasion the endometrium was very active also in glandular epithelium being lower in the luminal epithelium. Trophoblast cells were very active and especially during primary invasion, where the apposing maternal tissue except, vascular endothelium become non-reactive, this invasion pattern was seen up to 50-60 mm here close to the glandular part. The activity in glandular epithelium remained almost to late stage. The maternal endothelium was active to about late mid gestation and then declined rapidly showing only few mitotic cells at 60 mm, however the mitotic activity was dominated by trophoblast and fetal vasculature declining later than the activity in the maternal endothelium Caspase activity was high in tissues apposed to invasion. Conclusion: 1) early mink placentation is characterized by high mitotic activity in the invasive trophoblast and uterine glands; 2) growth is high to late mid gestation mainly at the fetal placental side 3) maternal endothelial cell turnover rate at mid gestation is 5-7 days. Keywords: Mink, mustelidae, placental growth pattern, Mitosis, Apoptosis
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[P08.05]. MATERNAL DIETARY ARGININE SUPPLEMENTATION GESTATION IMPROVES REPRODUCTIVE EFFICIENCY IN PIGS
DURING
[P08.07]. STUDY OF THE EQUINE PLACENTA UP TO 120 DAYS OF GESTATION IN LIGHT MICROSCOPY (LM) AND TRANSMITION ELECTRON MICROSCOPY (TEM) Ana Flavia de Carvalho1, Ana Claudia Cristiane Ferraz1, Priscila Leal do Nascimento1, Celina Almeida Furlanetto Mananares1, Ana Carolina Furlanetto Mananares1, Andre Luis Rezende Franciolli*2. 1Dept. of Morphology, UNIfeob, Sao Joao da Boa Vista, Sao Paulo, Brazil, 2Dept. of Surgery, Faculty of Veterinary Medicine, University of Sao Paulo, Sao Paulo, Brazil The epitheliochorial placentation in horses is a result of placenta development. The aim of this study was to morphologic analyze equine embryonic membranes under LM and TEM, to provide key parameters related to the equine reproduction. It was observed 43 placentas (0-120 days of gestation) xed in 10.0% formaldehyde plus 2.5% glutaraldehyde in 0.1M phosphate buffer (pH 7.4), and processed by parafn embedding and HE, tricromic Masson and PAS reaction. Under LM, trophoblast in early gestation (36 days) presented few villous with columnar low epithelium layer and brush border, uni or binuclear cells, that multiply rapidly and acquire an invasive phenotype, basal membrane and a highly vascularizated conjunctive tissue. As gestation proceeded, the villous became more complex revealing primary villous on days 53-88. The alantoic membrane presented squamous and mesenchymal cells, full of vessels, fusioned to chorion forming the chorioalantoic membrane. The amnions presented squamous cells, supported by mesenchymal cells, fusioned with amnion resulted in amniochorion membrane. Yolk sac had large round cells, supported by mesenchymal cells with hemangioblasts islands and mesothelial boundries. In TEM, chorion presented epithelium columnar cells (uni, bi or multinuclear cells), mesenquimal tissue rich in collagen bers, forming trophoblast. A large amount of mitochondria in cytoplasm of cellular apex and eletrondense vesicles was observed. Allantoic cells presented round nuclei and subdivided cytoplasm, a high concentration of mitochondrias and endoplasmic granular reticulum in the cellular apex or base, respectively. The amnion presented an only layer of attened cells supported by conjunctive tissue. Also, it was observed a large amount of eletrondense granules in the cellular apex surrounded by a round shape shadow. In conclusion, we suggested that equine embryonic and fetal membrane is similar to other artiodactyls membranes as in pigs, ruminants (cattle and sheep) and camelids (llamas). Keywords: placenta, Early palcentation in mare, morphologic aspects
MJ De Blasio*1, JA Owens1, CT Roberts1, R Smits2, M Nottle1. 1The Robinson Institute & School of Paediatrics and Reproductive Health, University of Adelaide, Australia, 2QAF Meat Industries Pty Ltd, Corowa NSW, Australia Introduction: Arginine (a non-essential amino acid) and its conversion to nitric oxide (NO) by nitric oxide synthase (NOS) can regulate the formation of new blood vessels and vasodilation. This may reduce resistance and increase blood ow to the uterus and placenta, and hence delivery of nutrients for fetal growth and survival. In rats, arginine deciency causes IUGR and increases fetal death and perinatal mortality, whereas dietary arginine supplementation reverses this. Human IUGR is associated with impaired NO synthesis, arginine transport, and eNOS activity in umbilical vein endothelial cells, but supplementing with arginine has produced inconclusive results. We hypothesised that maternal arginine supplementation (MAS) in the pig (a species with naturally occurring IUGR due to a large litter size) during early gestation, when placental angiogenesis and vascularity increase, would increase birth weight and placental weight. Methods: Gilts (parity 0) and sows (parity 3) (n150/parity) were fed a control or arginine supplemented (+25g/d arginine) diet (2.5kg/d), in early gestation (17-33d). Birth and placental weights of progeny were measured at term (115d). Results: MAS reduced pregnancy loss in both parities and increased number of progeny born alive (Control vs. Arginine: 10.7 vs. 11.9, +1.2 p0.048) in primiparous pigs. MAS increased litter birth weight (12.3 vs. 14 kg, +1.7kg p<0.05) and litter placental weight (2.63 vs. 3.93kg, +1.3kg p<0.05) in parity 0, but not parity 3 pigs. Discussion: Maternal arginine supplementation during critical periods of placental development may enhance placental-fetal blood ow and nutrient transfer, thereby improving fetal growth and survival and ameliorate the effects of IUGR. Keywords: Arginine supplementation, early gestation, pigs [P08.06]. EQUINE PLACENTA BY SCANNING ELECTRON MICROSCOPY (SEM) Ana Flavia de Carvalho, Priscila Leal do Nascimento, Andre Luis Rezende Franciolli*. 1Dept. of Morphology, UNIfeob, Sao Joao da Boa Vista, Brazil, 2 Dept. of Surgery, Faculty of Veterinary Medicine, University of Sao Paulo, Brazil, 3Presbiteriana Mackenzie University, Brazil The epitheliochorial placentation in horses is result of placenta development. The aim of this research was to analyze the morphology using SEM of the equine embryonic membranes, to provide key parameters related to the equine reproduction. We used 13 placentas (0-120 days of gestation) xed in 2.5% glutaraldehyde in 0.1M phosphate buffer pH 7.4. The chorion with less than 36 days of gestation showed chorionic projections with the cellular apical surface rounded and heptagon shape and covered by microvilli, with 3.1 cm of CR (36 days of gestation) round cellular surfaces without microvilli (smooth chorion). The chorion with 6.7 cm to 12.3 cm of CR (57 to 79 days of gestation), showed numerous round projections, with goffer shape to endometrial direction. The chorion with 10.3 cm to 20.2 cm of CR (71 to 107 days of gestation) showed ultrastructural features similar to animals with 6.7 cm to 12.3 cm of CR. This showed a hexagonal cell, apex with a few trophoblast cells without microvilli and others with microvilli of different sizes. The allantoic membrane in all gestational periods observed showed characteristic slightly rough, with microvilli in the cells surface forming images pentagon, hexagonal and heptagon. Round structures were observed with "button" shape, suggesting adhesion between both chorion and allantoic. The amnion had a similar to the allantoic, whose cells varied in tetragonal to heptagonal shape it is delimited by microvilli strings. In the amniotic surface were observed a number of structures with severe shapes, similar to buttons, as observed in allantoic membrane. The yolk sac in all groups showed droplets secretion in the cell apex showing exocytose, and its surface was irregular due to the secretion granules. In conclusion, we believe that equine embryonic membrane is similar to other artiodactyls membranes as pigs, ruminants and camelids. Keywords: placenta, equine, SEM, fetal membranes
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[P08.08]. CATHEPSIN B, CATHEPSIN L AND CYSTATIN C IN PORCINE UTERI AND PLACENTAE: A MODEL FOR PROTEIN MODIFICATION DURING UTILIZATION AND FLUID-PHASE TRANSPORT ACROSS UTERINE EPITHELIA, AREOLAE AND NEONATAL GUT G.A. Johnson*1, G. Song2, D.W. Bailey1, K.A. Dunlap1, R.C. Burghardt1, F.W. Bazer2. 1Department of Veterinary Integrative Biosciences, Texas A&M University, United States, 2Department of Animal Science, Texas A&M University, United States Proteases, Cathepsins (CTSB) and (CTSL1), and their inhibitors, Cystatin C (CST3), remodel endometrium and placenta for transplacental transport of gases, micronutrients and macromolecules. We examined CTSB, CTSL1 and CST3 expression and hormonal regulation in endometria and placentae of pigs. 1) gilts were hysterectomized on Day 9, 12, or 15 of the estrous cycle, or Day 9, 12, 15, 20, 25, 30, 35, 40, 50, 60, or 85 of pregnancy. 2) cyclic gilts were injected (Days 11-14) with estrogen (E2) and hysterectomized on Day 15 or 90 of pseudopregnancy. 3) gilts were ovariectomized on Day 12, injected with progesterone (P4) for 28 days, and hysterectomized on Day 40. CTSB increased in luminal epithelium (LE) after Day 30 of gestation. CTSB in LE was not affected by E2, but was increased by P4. CTSB was abundant in chorionic epithelium by Day 20 and remained through Day 85. CTSL1 increased in LE, GE and trophectoderm by Day 20 of gestation. Endometrial CTSL1 was not affected by E2, but was increased by P4. CTSL1 was highly expressed in the chorionic epithelia that form areolae to absorb secretions of uterine glands. CST3 was expressed in presumptive immune cells within endometria, but increased in LE by Day 25 of gestation. CST3 was not affected by E2 at Day 15, but increased in LE on Day 90 of pseudopregnancy. P4 decreased CST3 in LE, but increased expression in immune cells. We next examined CTSL1 in the jejunum of neonatal pigs which absorbs IgG for passive immunity. CTSL1 was expressed in enterocytes of villi. These results show that CTSL1 is expressed by areolae, GE, and neonatal intestine, each of which have roles in uid-phase transport of proteins, and that interactions between CTSL1, CST3 and CTSB may modify proteins during utilization and transport across uterine, placental and gut epithelia. Keywords: Pig, proteases, areolae, uterus
[P08.10]. EXPRESSION AND MODULATION OF Wnt, Bmp, AND PUTATIVE ANTAGONIST GENES IN THE BOVINE CARUNCLE ENDOMETRIUM DURING THE IMPLANTATION STAGE M B Aires1, K Y Degaki1, V Dantzer2, T E Spencer3, A T Yamada*1. 1University of Campinas, Brazil, 2Faculty of Life Sciences, Denmark, 3Texas A&M University, United States Development of villous-like projection on cow caruncle (CAR) surface as local endometrium response to establish the embryo-chorion anchorage site for synepithelialchorial placentation, closely resembles epithelialmesenchyme interaction during embryo organogenesis of gut, urinay and respiratory mucosa that are strictly controlled by morphogens like WNT and BMP proteins families. The present work comparatively evaluated the evolution of villous-like projection in the pregnant and non-pregnat (NP) cow (Bos spp) caruncle based on morphological analysis and, Wnt and Bmp morphogen gene families expression by PCR and in situ hybridization. By histological analysis of CAR mucosa during implantation was established four developmental stages: S1trophoblast cells uterine epithelium adhesion, S2 initial villous-like projections on the CAR surface, S3 expansion and anastomoses of villous-like projections, and S4 placentome consolidation. Anti-PCNA immunostaining demonstrated intense proliferative activity from S1toS4 on CAR epithelial and stromal cells, resulting in a 5-fold expansion of endometrial tissue. By RT-PCR and in situ hybridization, the expression of the Bmp2 and Wnt7a genes was seen upregulated in the CAR, with the highest expression level detected at S1 compared to NP. Otherwise, Wnt2 and Wnt5a expression levels were lower in the CAR. Dkk1 and Sfrp2 were widely expressed in the LP and S1-S4 CAR and IC 45 endometrium. Noggin expression was consistently lower in the CAR, whereas Sostdc1 expression was higher in the CAR than IC at S1 and then declined in both regions until S4. Therefore, simultaneous expression of Wnt and Bmp together with their antagonists in the CAR and IC regions seems to be responsible for the capability of cow endometrium quick remodeling and ability to develop synepithelialchorial placentation. Furthermore, they also attest to the complex synergistic/antagonistic signaling mechanisms of several still unknown factors modulating the cow uterine mucosa response during embryo implantation and synepithelialchorial placentation. Grants: CNPq and CAPES. Keywords: placentome, caruncle endometrium, morphogen, WNT and BMP
[P08.09]. CAVEOLAE AND CAVEOLINS IN THE CLONED TRANSGENIC CATTLE PLACENTA FTV Pereira*1, FV Meirelles2, F Bressan2, M Miranda2, AC Assis Neto1, MA Miglino2. 1Paulista State University, Brazil, 2University of Sao Paulo, Brazil Caveolae and caveolin-mediated endocytosis are internalization pathways in the placental transport. However, their roles in a cattle placenta have not been addressed. In this study, we compared the involvement of caveolae- and caveolins -1,-2 in cloned transgenic cattle. Fetal broblasts expressing the GFP gene were used as nuclei donors to cloning by nuclear transfer (NT), to produce the gestations by embryo transfer. Transmission electron microscopy (TEM) and immunhistochemistry (IHC anti-caveolins -1, -2) were performed on placentomes and chorioallantois from 5 cloned (60 and 90 days of gestation) and 10 controls in the same gestation period. The tissues were glutaraldehyde or formalin xed. At the TEM we could observe and characterize the structures called caveolae in blood capillaries of the chorioallantoic membrane and placentomes by natural (control) and cloned transgenic cattle gestation. The caveolae appears as a plasmaleme vesicles and invaginations in both of the plasmic membrane in luminal and abluminal portion. However, we have observed many microvilli in the trophoblast of the placentome, such as in the chorioallantoic membrane. The fetal and maternal villi were immunoreactive to caveolin-1, but the strongest reaction was observed in the endometrial stroma. The chorioallantois trophoblast and placentome were immunoreactive to caveolin -2, mainly in a trophoblast giant binucleate cell. The results obtained by the TEM and IHC indicated that the caveolae plus microvilli are very important sites on placental transport and signaling in the natural and cloned transgenic cattle gestation. Funded: FAPESP. Keywords: Caveolae, Caveolins, Cloned cattle, Transgenic
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[P09.01]. PLACENTAL IGFBP-1, IGF-I, IGF-II AND THE MICRONUTRIENTS ZINC AND FERRITIN IN THE PAKISTANI SGA POPULATION Shahzad Akram*1, Christine Carlsson-Skwirut1, Zulqar Bhutta2, Olle Soder1. 1Karolinska Institutet, Sweden, 2The Aga khan University, Pakistan Background: The insulin-like growth factor (IGF) axis plays an important role in normal gestational development. Alterations in this axis can result in serious consequences, such as small for gestational age (SGA) babies. Micronutrient supplementation, namely zinc, has been shown to decrease the risks of preterm pregnancy and may thus regulate this axis. OBJECTIVE: The principal objective of this study was to correlate placental levels of the IGF-axis, namely, IGFBP-1 with IGF-I and IGF-II, and the micronutrients zinc and ferritin, in SGA and large for gestational age (LGA) babies in the Pakistani population. Methods: Human placental samples were obtained from 89 women in rural eld sites in Pakistan. Samples were divided into SGA and LGA groups based on population percentiles. Results: Higher levels of IGFBP-1 were seen in the SGA group as compared to the LGA group (p<0.01). Multivariate regression for IGFBP-1 levels signicantly correlated with mRNA expression levels of IGF-II in the SGA group (p<0.05). No signicant correlations were seen with IGF-I expression, zinc levels, or ferritin levels. Conclusion: We have shown the important differences in IGFBP-1 levels in placenta, comparing SGA and LGA groups. This suggests the key role this protein plays in growth regulation, particularly towards the end of gestation. Though no signicance was seen when analysing the binding protein with respect to the micronutrients, the correlations suggest their role in regulation of IGFBP-1. The importance of a well-balanced system cannot be stressed enough, as both deciencies and excessive amounts of IGFBP-1 can tip the balance of growth in either direction. Keywords: SGA, IGFBP-1, IGF-I, micronutrients
[P09.02]. IGF-I, OESTROGEN RECEPTOR, AND PROGESTERONE RECEPTOR EXPRESSION, AND MATERNAL ANTHROPOMETRY IN GROWTH RESTRICTED PREGNANCIES: A LOOK AT THE SWEDISH POPULATION Shahzad Akram*, Lena Sahlin, Ewa Ostlund, Gabriel Fried (Late), Olle Soder. Karolinska Institutet, Sweden Background: Foetal growth restriction is a complex problem of pregnancy, arising from multiple aetiologies, including environmental, nutritional and genetic problems. A key regulatory element of cellular and tissue growth, at the endocrine level, is the insulin-like growth factor (IGF) axis. Both excesses and deciencies in this axis contribute to foetal growth problems, the most common outcome being babies being born small for their gestational age (SGA). Aim: To determine the relations of placental levels of IGF-I, oestrogen receptor (OR), and progesterone receptor (PR) expression with maternal anthropometry, looking at birth weight outcomes. Methods: Placental samples were obtained from 33 patients, following delivery, from the Karolinska Hospital, Stockholm, Sweden. Experimental methods included hybridisation probe analysis for nucleic acids and PCR techniques. Preliminary Results: Placental expression of IGF-I, OR, and PR were all decreased in the SGA group, compared to the NC group. However, only those of IGF-I were statistically signicant (p<0.05). Furthermore, no signicant correlation was seen between maternal anthropometry and the above mentioned factors. Conclusion: The differences in placental expression of emphasises the key role the growth factor and receptors play in birth weight outcomes, particularly in babies born SGA. The expression of these receptors may thus potentially be used as markers for SGA. Though maternal anthropometry has been seen to contribute to birth weight outcomes, through maternal constraint, it did not appear to be a signicant contributing factor in the Swedish population. Keywords: IGF-I, IUGR, PR, OR
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[P09.04]. PEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR g (PPARg) MEDIATED MECHANISM FOR PLACENTAL APOPTOSIS IN MATERNAL FOOD RESTRICTED GESTATIONS L Belkacemi*, MG Ross, Q Liu, M Desai. Department of Obstetrics and Gynecology, David Geffen School of Medicine at UCLA and LABIOMED Research Institute at Harbor-UCLA Medical Center, CA, USA, United States Introduction: Maternal food restriction (MFR) during pregnancy leads to intrauterine growth restricted (IUGR) fetuses that have a higher risk of adult obesity, hypertension and diabetes. IUGR may occur secondary to insufcient maternal nutrient supply and/or reduced placental nutrient/ oxygen transfer. Notably, increased placental apoptosis has been associated with IUGR. The mechanism may involve peroxisome proliferatoractivated receptor g (PPARg) which is an anti-apoptotic transcription factor and mediates cellular proliferation and differentiation. We have shown that MFR placentas have reduced growth at gestational ages, E16 and E20. Therefore, we investigated the impact of MFR on placental apoptosis and PPARg expression at ages, E16 and E20. We focused our study on the two placental positions (proximal and mid-horn) with the extremes of nutrient/oxygen supply, and two distinct placental zones (basal, site of hormone production; and labyrinth, site of feto-maternal exchange). Methods: Pregnant rat dams were fed an ad libitum diet (AdLib) or were 50% MFR beginning at E10 of gestation. At E16 and E20, fetal and placentas were recorded. Six placentas from left proximal and mid-horns were used for TUNEL staining. The corresponding right mid- and proximal horn placentas were separated into basal and labyrinth zones and analyzed for zone and region specic expression of PPARg (Western blot). Results: MFR-induced fetal growth restriction was evident only at E20. In contrast at E16, as compared to AdLib, the MFR mid-horn placentas had reduced basal zone and similar labyrinth zone weights. No weight changes were seen in proximal horn placentas. By At E20, MFR mid- and proximal horn placentas showed signicant reduction in the basal and labyrinth zones. Interestingly, the level of apoptosis was signicantly increased in the MFR from mid- and proximal horn placentas at E16 and E20. Consistent with this, PPARg protein expression was signicantly downregulated at both E16 and E20 in the MFR placentas. Conclusion: The placentas show early impact (E16) of MFR as evident by increased apoptosis and reduced growth. This in turn may contribute to fetal growth restriction in MFR pregnancies. Further, MFR induced placental apoptosis may, in part, be mediated by PPARg. Keywords: apoptosis, IUGR, placenta, PPARg
[P09.05]. DIFFERENTIAL REGULATION OF GROWTH AND GENE EXPRESSION OF CULTURED HTR-8/SVneo HUMAN TROPHOBLASTS BY NUTRIENT RESTRICTION AND HYPOXIA P.M. Brannon*, S. Jones, J.Y. Lee. Cornell University, United States Maternal undernutrition (characterized by restricted energy, protein and micronutrient intake) decreases placental and fetal growth through unknown mechanisms. Hypoxia (Hx) and reduced nutrient availability subsequent to reduced placental blood ow are proposed mediators. We investigated the effects of NR and Hx on growth, gene expression and hCG secretion in the HTR-8/SVneo human trophoblast cell line (Tb). Cells (1- 6 x 105) were plated in 75 cm2 asks in RPMI media with 1.25%-5% FBS and cultured with 1-20% oxygen. We determined cell number by trypan blue exclusion or spectrouorometric DNA analysis, protein by Lowry, mRNA by RT-PCR and hCG by ELISA. Data from 3 replicate experiments with 3-4 samples/treatment were analyzed by ANOVA or 2-way ANOVA. Growth was log-linear with 1-4 x 105 plated cells for 96 h; 2 x 105 cells were plated for subsequent experiments. A 75% reduction (NR) of media glucose, essential amino acids and Gln, and vitamins reduced growth 52% at 72 h. Reduced oxygen (1, 2 or 4%) decreased growth at 72 h by 10-53% compared to 8 or 20% oxygen. Glut 3 mRNA increased even with 8% oxygen (1 fold) and maximally increased 7 fold with 1% oxygen; HIF1a and Ki67 mRNA decreased dose responsively to a minimum in cells grown at 1% oxygen (0.6 and 0.5 fold, respectively); VEGF mRNA increased biphasically with a maximum (1.4 fold) at 2% oxygen. Because mRNA responses occurred with 8% oxygen, 20% oxygen was selected as the normoxic level; 1% oxygen was selected as Hx because it resulted in maximal change in several mRNAs. In a 2x2 factorial experiment at 72 h, Hx, independent of NR, decreased growth 43.5%; NR independent of Hx decreased growth 34%; and Hx X NR interacted such that Hx only increased VEGF mRNA in complete media (1 fold). Hx, but not NR, independently increased glut3 mRNA (1 fold), Igf2 mRNA (0.62 fold), cell size (protein/DNA ratio, 141%) and secreted hCG (242%). Hx and NR both independently, but not interactively decreased the growth of Tb. The differential effect of Hx on glut3 and Igf2 mRNA and secreted hCG also suggests Hx acts independently to regulate some gene expression. However, the absence of an effect of Hx on VEGF mRNA in NR suggests that Hx and NR may also interact through one or more regulatory pathways. Keywords: maternal malnutriton, nutrient restriction, hypoxia, growth
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[P09.07]. TRANSPLACENTAL GLUCOSE TRANSPORT IN PLACENTAL MALARIA C.L.L. Chua*1, A.J. Umbers1, E. Chaluluka2, S.J. Rogerson1, P. Boeuf1. 1 University of Melbourne, Australia, 2University of Malawi, Malawi Each year, 50 million pregnancies are at risk of malaria. Placental malaria infection is associated with fetal growth restriction, resulting in low birth weight babies being born to infected mothers. The pathogenetic mechanisms in placental malaria which lead to compromised fetal growth are unknown. However, the sequestration of malaria parasites in the placenta often results in an inammatory response being initiated in the intervillous spaces (intervillositis). These events cause placental structural damages and may impair placental functions, including nutrient transport. Given that transplacental glucose transport is an essential factor controlling fetal growth, we hypothesised that impairment in glucose transport may be a potential mechanism underlying fetal growth restriction in placental malaria. Using placental samples from Malawi, which consisted of malaria-infected placentas with or without intervillositis, we quantied the expression of the glucose transporter GLUT-1 in the syncytiotrophoblast microvillous and basal membranes. GLUT-1 expression proles were then compared to that obtained on uninfected placentas from the same region. In this pilot study, GLUT-1 expression in the basal membrane was lower in the malariainfected placentas with intervillositis than in those without intervillositis (P0.064) or control placentas (P0.021). Given that GLUT-1 expression in the basal membrane is the limiting factor for transplacental glucose transport, we propose that the inammatory response associated with placental malaria has an adverse effect on the placental ability to transport glucose to the fetus. Our results suggest that malaria-induced placental intervillositis can cause fetal growth restriction through decreased nutrient transfer and shed new lights on the pathogenesis of fetal growth restriction during malaria in pregnancy. Keywords: Placental malaria, fetal growth restriction, intervillositis, GLUT-1 expression
[P09.08]. ROLE OF MICRORNAS IN PLACENTAL PROGRAMMING OF INSULIN RESISTANCE H. Harryanto*, M.L. Harland, P.A. Grant, M.J. De Blasio, J.S. Robinson, J.A. Owens. The University of Adelaide, Research Centre for the Early Origins Health and Diseases, The Robinson Intitute, Australia Introduction: Placental restriction (PR) is a major cause of intrauterine growth restriction (IUGR) which is associated with adult-onset type 2 diabetes. This is due to insulin resistance and down-regulation of gene and/or protein expression of insulin signalling molecules in skeletal muscle in human, rats and sheep. MicroRNAs (miRs) are small non protein coding RNAs that can down-regulate of multiple targets mRNAs. We hypothesised that PR and IUGR in the sheep alters expression of miRs in skeletal muscle of progeny, which able to alter insulin signalling expression and its action, in lambs (44 days old) and adults (18 months old). Methods: Placental growth in sheep was restricted by removal of the majority of endomentrial caruncles from non-pregnant Merino ewes, and hence placental size and function. Vastus lateralis was collected at postmortem, miR expression analysed by Exiqon microarray v8.1. Bioinformatic analyses used to identify the predicted gene targets for each differentially expressed miR. Ingenuity Pathway Analysis (IPA) was used to identify metabolic and signalling and other pathways that might be affected. Results: PR reduced skeletal muscle expression of miR-211 and miR-451 (p<0.05) in lambs overall and only miR-365 (p<0.05) in females and did not alter any in males. In adult sheep, PR increased miR expression in females only, they are miR-278, miR-376b, miR-175p, miR-142-3p, miR-21, miR-101 and miR-324-3p (p<0.05). Finally, PR reduced skeletal muscle expression of miRs in male progeny, regardless of age; including miR-663, miR-711, miR-720, miR-612, miR-197, miR-409-5p and miR-769-3p (p<0.05). Some of them were predicted to target mRNAs of proximal insulin signalling molecules, and other pathways; ERK/MAPK, GABA receptor and JAK/Stat signalling. Conclusion: The present study shows that early life perturbation by PR can alter the expression of miRs in skeletal muscle of progeny and lead to the altered expression of insulin signalling molecules and potentially other signalling pathways. Keywords: IUGR, microRNA, insulin resistance, skeletal muscle
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[P09.09]. HIF-1 TRANSCRIPTIONAL ACTIVITY IS INDUCED RAPIDLY BY HYPOXIA IN PRIMARY SYNCYTIOTROPHOBLAST N.P. Illsley*, E. Fik, S. Zamudio. UMDNJ-New Jersey Medical School, United States The HIF-1 transcription factor is a central mediator of the cellular response to hypoxia. We hypothesized that there would be a time-dependent increase in HIF-1 transcriptional activity in human syncytial cells in response to hypoxia. To dene the HIF-1 transcriptional prole we transfected trophoblast cells with a HIF-luciferase reporter and measured the response to a hypoxia-mimetic, dimethyloxallylglycine (DMOG), an inhibitor of HIF-1a degradation. Methods: Cytotrophoblast were allowed to plate for 18 hr before incubation with lentiviral particles (low or high titer) expressing the luciferase gene under the control of hypoxia response elements. After 48 hr incubation, cells were switched to medium in the absence (control) or presence (treated) of 1 mM DMOG. Triplicate samples were assayed for luciferase activity following 2, 6 or 18 hr of incubation. Results: Luciferase activity in the control cells was similar over the three time points however it was 3.0 0.3 fold higher in cells subjected to treatment with the lower viral titer; we report here the response in the low titer-treated cells. At 2 hr, luciferase activity was 3.7 0.6 fold higher in treated compared to control cells. After incubations of 6 hr and 18 hr, activity in treated cells was 2.6 0.3 and 2.1 0.2 fold higher. Conclusions: These results show the feasibility of lentiviral transduction although the reduced level of luciferase activity in cells transfected with a higher viral titer suggests that cytotoxicity may be an important consideration. The response to DMOG in these initial experiments was surprising in that there was a rapid response at the early time point followed by a decrease to level 2-fold higher than control by 18 hr. This data suggests that these cells can respond to hypoxia in both an acute and chronic manner. Supported by HD46982 (NI) and HD42737 (SZ). Keywords: HIF-1, Hypoxia, Syncytiotrophoblast, Human
[P09.10]. PLACENTAL ANTIOXIDANT PLACENTAL INSUFFICIENCY
EXPRESSION
IN
SHEEP
FOLLOWING
N.E. Marshall*, P. OTierney, S. Louey, K.L. Thornburg. Oregon Health and Science University, United States Introduction: Antioxidants play a key role in scavenging reactive oxygen species (ROS) generated during normal metabolic processes. Disruption of the balance between free radical production and antioxidant activity (e.g. by intermittent hypoxia and reoxygenation) leads to oxidative stress and cellular damage. Placental insufciency from daily umbilicoplacental embolization (UPE) in sheep leads to fetal hypoxemia which is intermittent during the rst 5-7d of embolization. We hypothesized that the cellular stress associated with embolization would stimulate the expression of antioxidant system protein. Methods: Fetal sheep underwent daily UPE from 115 days gestation (term w145d) to a level that leads to growth restriction. After 5 days of UPE, placental samples from UPE (n6) and control fetuses (n6) were snap frozen in liquid nitrogen. Superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase mRNA expression were measured in UPE and control cotyledons using real time quantitative PCR; expression was standardized to GAPDH as ratios, data are meanSEM. Results: UPE fetuses were mildly hypoxemic during the study (PaO2: 15.30.7 vs 20.70.4mmHg, p<0.01). No statistically signicant differences in antioxidant protein message expression were found. SODs catalyze the conversion of superoxide to hydrogen peroxide. SOD1 expression was 1.10.6 (UPE) vs 2.71.7 (control), p>0.40; SOD2 expression was 1.20.3 (UPE) vs 0.70.1 (control), p>0.10. GPx1, GPx4 and catalase convert hydrogen peroxide to oxygen and water. GPx4 expression was 1.90.6 (UPE) vs 1.10.2 (control), p>0.20 and even lower average expression of GPx1 1.10.3 (UPE) vs 2.00.6 (control), p>0.20; catalase expression was 2.00.9 (UPE) vs 4.42.8 (control), p>0.40. Conclusion: Despite signicant and intermittent fetal hypoxemia, placental antioxidant expression levels were not signicantly altered by placental insufciency. Differing levels of hypoxemia and recovery from embolization may account for the considerable variation in antioxidant expression. We are now studying the effects of a longer period of placental insufciency on placental antioxidant expression and activity. Keywords: oxidative stress, antioxidant, placental insufciency
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[P09.11]. THE ROLE OF NODAL IN THE UTERUS DURING PREGNANCY C. B. Park*, D. Dufort. McGill University, Canada Pre-term birth (PTB) is the leading cause of perinatal mortality, accounting for over 75% of perinatal death. Despite recent progress, PTB has continued to rise over the years and remains an important clinical dilemma in both developing and developed countries. Failure to decrease the rates of PTB is attributed, in part, to our lack of understanding of the causes and mechanisms that underlie pre-term delivery. In order to aid in the ongoing pursuit of elucidating these mechanisms, our laboratory has been characterizing the expression of the TGF-b superfamily member, Nodal, in the uterus and investigating the potential role this factor may play in facilitating the birth of healthy offspring. Utilizing the loxP-Cre recombinase system, we have generated a conditional knockout of Nodal in the female reproductive tract of adult mice, bypassing embryonic lethality. Interestingly, the Nodal decient mice exhibit various reproductive abnormalities, including reduced rates of establishing pregnancy, intrauterine growth restriction (IUGR) and fetal abortion late into development (d17.5). The placenta of the Nodal conditional knockout mothers exhibit signicant expansion of the labyrinth, and marked reduction of the spongiotrophoblast and maternal decidual layers. Furthermore, the trophoblast giant cells appear disorganized and signicantly reduced in number. We report here, the rst phenotypic characterization of a uterine Nodal knockout strain, implicating the Nodal signalling pathway in facilitating healthy pregnancy. Our observations indicate that Nodal ligand from a maternal source may play a crucial role in decidualization and proper placenta development and its absence leads to IUGR, PTB and aborted development. Understanding the mechanisms that underlie IUGR and preterm delivery are paramount in the ultimate goal of eliminating complications during pregnancy leading to pre-eclampsia, PTB and embryonic loss. Keywords: Nodal, Pre-Term Birth, Intrauterine Growth Restriction
[P09.12]. REDUCED VASCULARITY OF THE FETOPLACENTAL ARTERIAL TREE IN TRANSGENIC MICE WITH FETAL HEPATIC OVEREXPRESSION OF hIGFBP-1 MY Rennie*1,4, KJ Whiteley2, CS Watson2, VKM Han3, SL Adamson2,5, JG Sled1,4. 1Mouse Imaging Centre, Hospital for Sick Children, Canada, 2 Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Canada, 3 Childrens Health Research Institute, University of Western Ontario, Canada, 4Dept. of Medical Biophysics, University of Toronto, Canada, 5 Depts of Physiology and Ob/Gyn, University of Toronto, Canada Introduction: Insulin-like growth factors (IGFs) are critical to fetal and placental growth and their actions are inhibited by IGF binding proteins (IGFBPs). IGFBP-1 is elevated in the circulation of human IUGR newborns. As in human IUGR, transgenic mice (Tg) with elevated circulating hIGFBP-1 are growth-restricted and have abnormal umbilical artery Doppler waveforms, suggesting alterations in the fetoplacental vascular tree. The purpose of this study was to quantify the arterial fetoplacental vasculature in this model. Methods: Fetoplacental arterial trees from wild type (WT) and Tg mice were infused with X-ray contrast agent at embryonic day 15.5 and 3-D micro-CT images were obtained. Custom designed image processing software was used to determine length, diameter, and connectivity of each arterial segment more than 50 mm in diameter. Results: Fetal weight was reduced 21% in Tg fetuses. Tg vascular trees were more often asymmetric (5/10) compared to controls (0/12), with increased vascular depth but decreased span. Umbilical artery diameter was unchanged, however Tg trees had w20% fewer vessel segments in both small (50-100 mm) and large (>100 mm) vessel diameter ranges.
WT (n12) Fetal weight (g) Placental weight (g) Vascular span (mm) Vascular depth (mm) Umbilical artery diameter (mm) Segments 50-100mm Segments >100mm 0.41 0.01 0.15 0.01 6.79 0.11 1.13 0.02 0.49 0.06 1310 46 178 9 Tg (n10) 0.33 0.01* 0.14 0.01 6.45 0.10* 1.30 0.05* 0.46 0.03 1101 51* 152 6*
*p<0.05 (t-test). Values shown as mean SEM. Conclusions: Elevated hIGFBP-1 in Tg mice causes fetal growth restriction and an abnormally shaped arterial fetoplacental tree with signicantly fewer vessels. Therefore IUGR due to elevated circulating IGFBP-1 may be due in part to reduced fetoplacental vascularity and impaired placental function. Supported by Heart and Stroke Foundation of Ontario. Keywords: fetoplacental vasculature, IGFBP-1, micro-computed tomography, IUGR
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[P09.13]. PLACENTAL PLASTICITY IN A NON-HUMAN PRIMATE MODEL OF PLACENTAL INSUFFICIENCY: GESTATIONAL TIMING IS CRITICAL TO ADAPTIVE CAPABILITY VHJ Roberts*, JP Rasanen, MJ Novy, KE Sparks, S Louey, KL Thornburg. Oregon Health and Science University, United States Introduction: Structural abnormalities of the placenta or impaired blood supply lead to poor fetal outcomes including IUGR. The hemochorial placenta of the rhesus monkey is analogous to the human placenta with the added feature of a secondary lobe. Ligation of the bridging vessels to eliminate the functional capacity of the secondary lobe has been used as a model of placental insufciency (AJOG 2009; 199 suppl: 6A #430). Our aim was to determine the effects of ligation in early vs late gestation on placental growth regulating factors and volume blood ow (QPL). Methods: Inter-placental bridging vessels were surgically ligated at either 80 days gestation (Early, n3) or 110 days (Late, n3 term167 days). Doppler ultrasound measurements were conducted to correlate changes in QPL and fetal growth indices before and after ligation. Tissues were harvested from experimental animals and age-matched controls at 140 days, and placental characteristics recorded. Placental protein expression of mTOR, VEGF and 11bHSD2 was determined by western blot. Results: Functional placental mass (Control; combined lobes 85.04.0g SEM) increased with early ligation (primary lobe 96.975.5g) and decreased with late ligation (primary lobe 75.477.9g). Similarly QPL increased in the early group and was diminished in the late group. Fetal weights were reduced in both groups compared to control (Early: 3402g; Late: 3293g; Control: 4193g). A 30% increase in placental mTOR expression was demonstrated in the early group compared to control, whilst VEGF and 11bHSD2 expression remained unchanged in both groups. Conclusion: The diminished capacity of the placenta to compensate for a vascular insult in late compared to early gestation suggests a critical developmental window for plasticity. The mTOR pathway may play a key role in signaling the compensatory mechanisms which maintain the nutrient supply of the fetus. Keywords: Rhesus monkey, Placental insufcency, Placental plasticity, mTOR
[P09.14]. DOES PLACENTAL WEIGHT RELATIVE TO BIRTHWEIGHT AFFECT OUTCOME FOR STILLBIRTHS AND NEONATAL DEATHS? JMD Thompson*. University of Auckland, New Zealand Introduction: The placenta is an integral part of pregnancy and the growth of the fetus. The aim of this study was to determine whether so called placental insufciency is related to the outcomes of birthweight and placental weight, and whether the effect on these outcomes is proportionate in the presence of conditions resulting in stillbirth or neonatal death. Methods: Data on birthweight and placental weight was obtained for all deliveries at National Womens Hospital, Auckland from 1993-2000 (n69,680), and the ratio of the two calculated. These were converted to zscores according to population norms and were analysed according to cause of death (PSANZ denitions) for stillbirths (n437) and neonatal deaths (n410) in comparison to all live births. Results: Compared to normal controls, stillbirths due to congenital anomalies were signicant lighter whilst their placental weights were normal. For stillbirths associated with hypertension, and IUGR placental weight and birthweight were affected proportionately, whilst deaths caused by spontaneous preterm birth and unexplained deaths had relatively normal birthweights and placental weights. For neonatal deaths placental weights were generally proportionately higher than birthweights for most groups of death but particularly those deaths due to extreme prematurity, cardio-respiratory disorders, and gastro-intestinal disorders. Conclusions: Placental function as dened by size is often cited as a cause of poor outcomes despite the fact that one would expect a normal distribution of placental weights for any given birthweight. This study shows that whilst for some causes of death birthweight is affected proportionately to placental weight, for others birthweight is below that which would be expected from the placental weight. This suggests that placental insufciency may not be the issue in relation to poor outcomes but that other mechanisms such as utero-placental blood ow or compensatory growth may be important in some causes of death. Keywords: Placental weight, placental function, fetal death
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[P09.15]. MATERNAL SERUM LEVELS OF ANGIOPOIETIN-2 (ANG-2) IN IUGR PREGNANCIES, AND ANG-2 LOCALISATION IN REPRODUCTIVE TISSUES Y Wang*1, C Woolnough1,2, V Tasevski1,2, E Gallery1, J Morris1. 1Perinatal Research group, Kolling Institute of Medical Research, University of Sydney, Australia, 2Fetal Maternal Medicine (PaLMS), Royal North Shore Hospital, Australia Introduction: Reduced serum levels of Ang-2 were observed in pregnant women at 10-13 weeks gestation whose pregnancies were subsequently complicated by intrauterine growth restriction (IUGR)1. The aims of this study were: (1) to describe serum levels of Ang-2 throughout menstrual cycle and during normal pregnancy, (2) to compare serum Ang-2 levels between IUGR and normal controls at the time IUGR was rst diagnosed, and (3) to immunolocalise Ang-2 in placenta, placental bed, and decidua from both normal and/or IUGR pregnancies Methods: 29 non-pregnant and 68 normal pregnant women at distinct time points were recruited. Serum samples were also collected from 17 IUGR patients and their paired normal controls. Ang-2 was measured using an R&D systems ELISA assay. Chorionic villi were obtained from diagnostic chorionic villous sampling (CVS) at 10-13 weeks. Placenta and decidua were collected at the time of delivery from both normal and IUGR subjects. Protein localisation of Ang-2 was examined using immunohistochemistry. Results: Serum levels of Ang-2 were similar between the follicular and luteal phases. Levels of Ang-2 rose at 8 weeks, peaked at 13 weeks, and declined from 18 weeks of gestation. The median concentration of Ang-2 at the 13-week peak was 18.71 ng/mL, compared to 1.72 ng/mL in nonpregnant women (p<0.01) (Figure 1). Serum levels of Ang-2 were significantly lower in IUGR pregnancies than normal controls at diagnosis. (p<0.05). Ang-2 tissue expression was found in the syncytiotrophoblast of both the rst trimester (CVS) and the third trimester placentas. Ang-2 expression was also found in invasive cytotrophoblast, endothelial and decidual cells. (Figure 2).
Figure 2. Ang-2 localisation in a section of placenta.
Discussion: These ndings suggest that Ang-2 potentially plays important roles in early placental angiogenesis, with a 10-fold increase at 8 weeks of gestation. Placental angiogenesis may be compromised in IUGR pregnancy. Cells of fetal and maternal origin contribute to high Ang-2 serum levels in pregnancy. Reference 1. Wang et al., BJOG 2007. Keywords: Angiopoietin-2, IUGR
Figure 1. Serum levels of Ang-2 throughout transverse the menstrual cycle and during pregnancy.
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[P09.16]. EVIDENCE OF TRA-1-60 AND TRA-1-81 INVOLVEMENT IN L-SELECTIN MEDIATED ADHESION OF THE PORCINE EMBRYO E Oestrup, V Dantzer*, P Maddox-Hyttel. University of Copenhagen, Denmark L-selectin expression in the early human embryo is involved in trophoblast adhesion during implantation. The aim of this study was to investigate the potential role of the L-selectin adhesion-system in the adhesion and placentation of porcine embryos. Endometrial samples were collected from pregnant gilts and non-pregnant cycling sows at Days 10, 15 and 18 post insemination/oestrus. Embryos were collected from pregnant gilts at Days 9, 11 and 15 for expression studies and from Days 10 and 15 for immunohistochemistry. Expression analysis of L-selectin and its ligands was made using qPCR. Protein localization of L-selectin and the ligands PNAd, PEN5 as well as the podocalyxin epitopes Tra-1-60 and Tra-1-81 were investigated by immunohistochemistry. In pregnant gilts the mRNA expression of L-selectin was approximately 14 fold up-regulated at Days 15 and 18 compared to Day 10 p.i. No signicant changes were seen in the mRNA levels in the cyclic sows. Expression of mRNA for the potential L-selectin ligands, CSPG-2 and podocalyxin were 6 and 12 times up-regulated in tubular Day 11 blastocysts compared to Day 9 blastocysts. Staining for L-selectin was observed in the luminal epithelium of the endometrium. In the embryos, staining for MECA-79 and PEN5 were observed in trophoblast and hypoblast cells at day 15. Tra-1-81 and Tra-1-60 was conned to the epiblast at Day 10 but at Day 15 staining for both epitopes were observed in the trophoblast. Our results for the rst time demonstrate that key components of the L-selectin adhesion system are present in the porcine uterus and embryo around the time of initial placentation. Interestingly, the components are expressed in a pattern opposite to that found in man: In the pig, L-selectin is expressed in the endometrium and the ligands in the trophoblast with the reverse being true in man. Keywords: Porcine, Implantation, L-selectin
[P10.01]. EFFECTS OF INSULIN ON MEMBRANE LIPID COMPOSITION OF HUMAN SYNCYTIOTROPHOBLAST M Castro-Parodi*, A Reca, V Dietrich, C Rodrguez, MC Fernandez-Tome, AE Damiano. Catedra de Biologa Celular, Depto. de Ciencias Biologicas, Fac ultad de Farmacia y Bioqumica, Universidad de Buenos Aires, Argentina Introduction: Altered placental membrane lipid composition in pregnancy may affect the fetal-maternal exchange. With gestational progress, the composition, structure and functions of these membrane lipid bilayers are modied in order to meet the changing metabolic needs of the growing fetus. Previously, we reported that plasma membranes of syncytiotrophoblast (hST) are unusual in comparison to other cell types. Because of the increase of sphingomyelin we also informed that preeclamptic hST is more rigid than normal hST. Since we observed high serum levels of insulin in preeclamptic women, we hypothesized that insulin may be implicated in the changes observed in preeclampic hST. The aim of this study was to evaluate if the insulin may alter the lipid composition of hST. Methods: Explants from normal term placentas were cultured with different concentrations of insulin during 24 h. The biochemical viability of the explants was determined by estimation of b-hCG concentrations in the extracellular medium. Apical (MVM) and basal (BM) membrane vesicles were prepared by differential centrifugation. Lipid were extracted by Bligh-Dyer method and quantied by Fiske-Subarrow. Cholesterol was determined by enzymatic method. Results: Insulin treatment produced no changes on the total phospholipid concentration in BM. On the contrary, MVM showed an increase in total lipid content until reaching a constant value at 100 mUI/mL of insulin. There were no changes in the amount of cholesterol in both vesicles. Discussion: Our results suggest that insulin treatment only alter phospholipid concentration in MVM having no effect on BM vesicles. Further work is necessary to clarify the molecular mechanisms implicated in these changes and if they may play a role in the pathogenesis of preeclampsia. Keywords: insulin, lipids, syncytiotrophoblast, preeclampsia
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[P10.02]. PERFUSION OF THE HUMAN PLACENTA WITH FREE HEMOGLOBIN MIMICS PREECLAMPSIA IN-VITRO M Centlow*1, H Schneider3, S Hansson1. 1Department of Obstetrics & Gynecology, Lund University, Sweden, 2Division of Infection Medicine, Lund University, Sweden, 3Department of Obstetrics and Gynecology, Insel Spital, University of Bern, Switzerland Background: We have previously shown fetal hemoglobin (HbF) mRNA expression to be increased in the preeclamptic placenta and accumulated in the placenta blood vessels where it damages the feto-placental barrier, causing leakage of free HbF into the maternal blood circulation. The increased levels of HbF induce placental gene expression of alpha-1microglobulin (A1M) and increase maternal plasma levels of A1M. Alpha1-microglobulin is a heme and radical-scavenger, and has reductase- and anti-oxidative properties. Aims: We used the in vitro dual-placenta perfusion model to evaluate the molecular effects of free HbF on the placenta. Material and Methods: 20 placentas were collected at birth and perfused in the dual placenta perfusion model with perfusion medium only (n6), 2 mg/ml free Hb (n9) or 30 mM a1m (n3). Placental tissue samples were collected before and after a successful perfusion. Effects on the placenta were analyzed with Illumina whole-genome Beadarrays and the histological structure was evaluated with electron microscopy. Results: Hemoglobin perfusions resulted in increased gene expression of amongst other toll-like receptor adaptor molecule 1 (fold change (FC)8.5, p1.310-5) and oxidative stress induced growth inhibitor 1 (FC10.8, p1.910-4). A1M perfusions increased gene expression for g-coupled protein receptor 1 (FC11.6, p1.610-4) and collagen, type IV, alpha 5 (FC3.6, p1.610-4). Conclusions: Free Hb is well known to have pro-inammatory and prooxidative properties and interestingly, among the most increased genes several oxidative markers are seen. Thus, it is possible that free Hb not only oxidizes proteins and lipids but also induce oxidative changes on a gene expression level. Alpha-1-microglobulin seems to not only function as a scavenger, it may also up-regulate genes related to extra-cellular matrix, indicating that A1M could aid in extra-cellular matrix repair where oxidative damage has occurred. Keywords: Preeclampsia, Placenta perfusion
[P10.03]. ECCENTRIC CORD INSERTION AFFECTS BIRTH WEIGHT IN THE COLLABORATIVE PERINATAL PROJECT (CPP) CM Salaa2, DP Misra3, AK Charles*1, RK Miller4. 1University of Western Australia, Australia, 2Placental Analytics, United States, 3Wayne State University, United States, 4University of Rochester, United States Background: Fetal growth is a complex process depending on genetics, uterine environment and placental function. The placenta develops ideally with a near centrally inserted cord, an eccentric cord is due to non uniform radial growth of the placenta. This is likely to be due to less than ideal intrauterine environment. Previous (smaller) studies have shown a variable relationship between non central cord insertion and birthweight. The hypothesis was that there would be an association with an eccentric cord insertion with a low birthweight. Methods: Linear regression was used with product terms to model interactions between predictor variable pairs including cord eccentricity (the relative distance of cord insertion from placental margin), disk eccentricity (the ratio of the larger and smaller chorionic disk diameters), and abnormal disk shape (categorized as round/oval v. all other shapes), to fetal and placental weight in the 29475 infants of the NCPP cohort with the required measures. Results: A more centrally inserted cord was associated with a small but reliable direct (positive) effect on birthweight ( 7323g, , p0.001), but no effect on placental weight (p0.49). A regression of the three measures (umbilical cord insertion and chorionic disk eccentricities and normal v. abnormal disk shape) revealed that each of the three measures retained independent and signicant predictive effects on birthweight (cord centrality 6523, disk eccentricity -11421, and disk shape -5716, p0.004, p<0.0001 and p<0.0001 respectively). Conclusion: A non central cord insertion is associated with a reduced birth weight in this large cohort. The pathophysiology of this may be due to a suboptimal intrauterine environment leading to non uniform placental growth and affecting the birth weight, but also the less than optimal vascular branching on the chorionic plate may affect the efciency of the placenta. The ndings support the trophotropism theory of placental development. Keywords: Placenta, Birthweight, Growth
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[P10.04]. IDENTIFICATION OF DOWNSTREAM TARGET GENES OF THE HOMEOBOX GENE DLX3 IN THE HUMAN PLACENTA A Chui*1,2, B Kalionis1, N Pathirage1,2, SP Brennecke1,2, P Murthi1,2. 1The Royal Womens Hospital, Australia, 2The University of Melbourne, Australia Introduction: Fetal growth restriction (FGR) is a pregnancy disorder characterised by birth weight below the 10th percentile for gestational age with the likely presence of an underlying pathologic process that inhibits the expression of the normal intrinsic growth potential. In the human placenta, the homeobox gene DLX3 has been shown to be altered in FGR. Our recent work shows that DLX3 is implicated in the regulation of trophoblast cell differentiation. In this study, the aim was to determine the pathways by which DLX3 regulates trophoblast differentiation. Methods: BeWo cultured cells were treated with DLX3-specic siRNA to signicantly decrease DLX3 gene expression. cDNA was prepared from siRNA treated and control siRNA cells. A PCR-based low density Superarray system was used for gene proling to identify the downstream target genes of DLX3. Candidate genes from the array, which showed differential expression between siRNA treated and control siRNA cells, were chosen for further validation in FGR-affected human tissue (n25) compared with gestation matched controls (n25), by real-time PCR. Results: Decreased expression of DLX3 resulted in signicant increased (>50) fold changes in the expression of all the following genes: CEBPB, ESR1, GATA2, GATA3, TNFRS11A, PPARg, SMAD5, SP1, STAT2 and TP53. Of these genes, verication in placental tissue affected by FGR was performed for PPARg and SP1. The mRNA expression of both SP1 (2.811.138 control vs. 29.225.0 FGR, P0.0000343) and PPARg (4.90.56 control vs. 55.529.49 FGR, P0.00097) was signicantly increased in FGR-affected placental tissue compared with controls. Discussion: This study has identied downstream target genes of DLX3 including CEBPB, ESR1, GATA2, GATA3, TNFRS11A, PPARg, SMAD5, SP1, STAT2 and TP53. DLX3 may therefore play a key role in many aspects of differentiation, since these target genes affect multiple signal transduction pathways. Keywords: Homeobox gene, Trophoblast, Fetal Growth Restriction
[P10.05]. X-SHAPED FUSED-FORKED UMBILICAL CORD IN A MONOAMNIOTIC TWIN PREGNANCY AS A RARE VARIANT OF CONJOINED TWINNING S Dekan*, Y Bader, M Stammler-Safar, G Poschalko-Hammerle, D Prayer. Medical University Vienna, Austria Monomaniotic twinning is rare and accounts for 1 2 % of all monocygotic twin gestations, and is associated with high mortality rates of 10% to 20% due to entanglement of the cords or knotting, problems accounting to premature delivery, twin-to-twin-transfusion syndrome and malformations including conjoined twinning. Fusions of umbilical cords are rare, 9 out of 10 cases reported in literature occurred in monoamniotic twins, whereas only one case was reported in diamniotic twins. All of these cases showed y-shaped fused umbilical cords. The overall outcome was poor with only one case with 2/10 healthy survivors and 8/10 fetal demises. We show the rst case of a monochorionic monoamniotic pregnancy with a x-shaped fused umbilical cord. The fusion of the cords was detected in a routinely performed fetal MRI with 6 vessels in the fused part. At gestational age of 32+2 weeks two girls were delivered by caesarean section. The children were appropriate to gestational age in weight and maturity and developed normaly. The monoamniotic placenta weighting 384 gramms showed two umbilical cords inserting centrally at a distance of 0.8 cm, containing three vessels each. After 1.2 cm the cords Wharton jellies were fused over a distance of 6.3 cm, containing 6 vessels. After the separation of the two cords each had 3 vessels again. The placental size, weight and villous parenchyme maturity matched gestational age. The most popular theory of development of forked umbilical cords is due to defectional separation during embryonic ssion after developmental day 9/10 when the formation of the amnion is nished, but before day 12, when conjoined twinning would result. Another theory by Spencer is the fusion of two monovular embryos, which could be consistend with the monochorionic diamniotic case and the case presented here. Keywords: umbilical cord, fusion, twinning
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[P10.06]. PLACENTAL MORPHOMETRY RELATED TO MATERNO-FETAL BLOOD FLOW IN PRE-ECLAMPSIA JF Ducray*1, T Naicker2, J Moodley2. 1Durban University of Technology, South Africa, 2University of Kwa-Zulu Natal, South Africa Introduction: Adequate maternal, intervillous and fetal blood ow are all necessary for fetal wellbeing. Compromise to any part of this exchange would be detrimental to pregnancy outcome. Pre-eclampsia is associated with reduced maternal spiral artery ow, resulting in reduced placental perfusion. This in turn creates an ischaemic environment which may predispose morphological changes in placental villi. Methods: This study utilized morphometric image analysis to examine selected placental features associated with materno-fetal exchange in normotensive (NT) and pre-eclamptic (PE) groups. The features examined included: density of placental villi (expressed as percentage of eld area occupied by placental tissue); stem vessel carrying capacity (expressed as percentage of stem villus area occupied by vessel lumina); the relative thickness of the stem arterial walls (expressed as percentage of artery area occupied by arterial wall) and the extent of brosis associated with villi (expressed as percentage of eld area occupied by brosis). Results: Density of placental villus arrangement NT:51.896.19, PE:64.786.93 (P155232E-10); carrying capacity of stem villi NT:17.2011.78, PE:8.678.51 (P1.19097E-05); relative thickness of stem villi arterial walls NT:74.0812.92, PE: 86.8510.55 (P2.04099E-08); and extent of brosis NT:0.7270.310, PE:1.5820.707 (P1.66E-07). Discussion: It is well established that the maternal side of placental exchange is compromised in pre-eclampsia. However, precisely how placental development and function is altered as a result is not clear. One would expect possible morphological compensation or ischaemic changes. The signicant differences between normotensive and pre-eclamptic placentae observed in this study suggest possible fetal maladaptations in response to the intervillous ischaemia, compounding the existing maternal compromise to materno-fetal exchange. Keywords: Pre-eclampsia, materno-fetal exchange, placental villi, microscopic placental morphometry
[P10.07]. EXPRESSION OF THE RECEPTOR FOR ADVANCED GLYCATION END PRODUCTS (RAGE) AND ITS SOLUBLE FORM SRAGE IN PREECLAMPTIC PLACENTAS AND AGE-MATCHED CONTROLS S. Dekan, Y.-A. Chen, H. Uhrova, G. Poschalko-Hammerle, I. Ellinger*. Medical University Vienna, Austria Introduction: The receptor for advanced glycation end products (RAGE) binds a variety of ligands. The ligand-RAGE axis contributes to a wide spectrum of diseases, including diabetes mellitus or atherothrombosis. Soluble forms of RAGE (sRAGE) may counteract RAGE-mediated pathogenesis by acting as a decoy. Decreased levels of sRAGE may serve as a biomarker of ligand-RAGE axis hyperactivity, but also providing a target of therapeutic interventions. Severe pre-eclampsia (PE) was found associated with increased levels of RAGE ligands, but alterations of placental RAGE expression remain controversial. Levels of RAGE and sRAGE expression in healthy and pre-eclamptic placentas are unknown, but this knowledge is required to argue in favour or against a contribution of the ligand-RAGE axis to the development of PE. This study is concerned with the analysis of RAGE and sRAGE expression in healthy human term placenta, in pre-eclamptic placentas and their age-matched controls. Methods: RAGE/sRAGE expression was investigated by RT-PCR, western blotting and microscopy in term placentas. RAGE/sRAGE expression in moderate to severe pre-eclamptic placentas and their age-matched controls was analyzed by immunouorescence microscopy of parafnembedded sections applying RAGE and sRAGE specic primary antibodies. Results: RAGE and sRAGE transcripts were detected in healthy term placental tissue by RT-PCR. By western blotting, RAGE and sRAGE was detected using antibodies directed against either the C- or N-terminus of RAGE, respectively, but sRAGE was the predominant protein expressed in healthy term placental lysates. By immunouorescence microscopy, both sRAGE and RAGE were localized to STB but also endothelial cells in situ. Finally, the expression of RAGE in relation to sRAGE was signicantly increased in STB and endothelial cells of PE-derived placentas compared to their age-matched controls. Discussion: Our data indicate an up-regulation of full-length RAGE in PEderived placentas and suggest an involvement of the ligand-RAGE axis in the development/aggravation of PE. Keywords: RAGE, sRAGE, preeclampsia
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[P10.09]. EFFECT OF ABNORMAL MATERNAL PREGNANCY SUCCESS IN THE RAT
IMMUNE
ACTIVATION
ON
[P10.10]. THE DIFFERENT REGULATION OF HO-1 AND INOS IN PLACENTA OF PREECLAMPSIA Jongyun Hwang*, Jiyeon Lee, Youngmyeong Kim. Kangwon National University, Republic of Korea Background: Heme Oxygenase (HO) and nitric oxide synthase (NOS) have similarities in some aspects. HO and NOS play signicant role in placentation, placental angiogenensis and antioxidant protection from oxidative stress in normal pregnancy. Some researcher suggested that HO and NOS may be regulated by mutual isoform enzymes. However, the exact comprehension for the compensatory regulation between two enzymes was decient. Preeclampsia (PET) is a hypertensive disorder in pregnancy. It is hypothesized that the cause of PET is disturbance of spiral artery modication of trophoblast in the early placentation. The placenta hypoxia results in the initiation of maternal inammatory cascade with endothelial dysfunction in PET. We hypothesized that the deciencies of HO, HO-catalytic products, NOS and NOS-catalytic products may result in PET and there is a reciprocal compensatory system in two systems. Objects: The aim of this study was to identify the gene expression of HO-1 and iNOS in placenta of PET and to evaluate the potential reciprocal compensatory regulation between two systems. Methods: In this study, we designed Case-control study including fourteen women with PET. The placenta tissue samples were obtained from PET patients and normal pregnancy. We quantied gene expression of HO-1 and iNOS on placenta by RT-PCR, real time PCR. Results: In this study, it demonstrates that a signicant down-regulation of HO-1 gene expression in placenta when compared to normal placenta. However, we observed a signicant up-regulation of iNOS gene expression in placenta when compared to normal placenta. Conclusion: The present study demonstrates that there are different regulation to preeclampsia in two enzyme systems; the down-regulation of HO-1 gene expression and up- regulation of NOS gene expression. We suggest that HO-1 and iNOS system may be regulated by mutual gene expression between two enzymes in placenta. Keywords: Heme oxygenase, nitric oxide synthase, preeclampsia, placenta
SJ Renaud*, J Quirt, SK Macdonald-Goodfellow, CH Graham. Queens University, Canada Introduction: Aberrant activation of the maternal immune system is an important aspect of pregnancy-related diseases including pre-eclampsia, fetal growth restriction, and spontaneous abortion. Administration of lipopolysaccharide (LPS) has been used in rodents to model the aforementioned pregnancy-related diseases. However, the mechanisms by which LPS exerts negative effects on pregnancy are not fully understood. Therefore, we determined the outcomes of LPS administration on pregnancy success in the rat, in relation to changes in placental blood ow and levels of pro-inammatory and thrombotic molecules. Methods and Results: Administration of LPS (50, 75, or 100 mg/kg) to Wistar rats on gestational day 14.5 resulted in fetal loss in a dose-dependent manner; furthermore, surviving fetuses were signicantly growth restricted. These outcomes correlated with elevated levels of tumour necrosis factor-a (TNF) in maternal blood, as well as increased expression of cyclooxygenase-2 and inducible nitric oxide synthase (iNOS) in decidual tissues. Placental blood ow was impaired within 2 h of LPS administration as determined by Doppler ultrasound and there was hemorrhage in the decidua and the fetal-maternal interface. Evidence of disseminated intravascular coagulation was present within 1 h of LPS injection as determined by thromboelastography, concomitant with increased levels of plasminogen activator inhibitor-1 in maternal blood. LPS also caused placental oxidative stress as determined by immunoblotting for 4-hydroxy nonenal. Injection of interleukin-10 prior to LPS, but not the iNOS inhibitor aminoguanidine, improved fetal outcomes, concomitant with decreased TNF levels. Conclusion: We conclude that abnormal maternal immune activation negatively affects rat placental development and that LPS administration provides a useful model to study pathological pregnancies. Funding source: Canadian Institutes of Health Research and Heart and Stroke Foundation of Canada. Keywords: inammation, pre-eclampsia, IUGR, spontaneous abortion
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[P10.11]. THE INHIBITORY ROLE OF MCL-1 ON THE INDUCTION OF AUTOPHAGY IN THE HUMAN PLACENTA M Kalkat*1,2, J Garcia1, J Ray1,2, I Caniggia1,2. 1Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Canada, 2University of Toronto, Canada Introduction: Autophagy has recently been recognized both as an adaptive mechanism to cellular stress and an alternative cell death pathway. Placental development requires a balance between proliferation and death; however the relative contribution of autophagy in normal and pathological pregnancies remains elusive. Preeclampsia (PE), a complication of pregnancy, is characterized by oxidative stress and increased trophoblast cell death. Biochemical analysis implicated an essential autophagy inducer, Beclin-1, to interact with some Bcl-2 proteins. We have previously found Myeloid Cell Leukemia Factor-1 (Mcl-1), a Bcl-2 family member, to guide trophoblast cell fate following changes in oxygenation. Herein we sought to examine the role of Mcl-1 on autophagy in normal and pathological placentae. Methods: First trimester (4-15 weeks; n26), age-matched (AMC; n8) and term controls (TC; n9) and preeclamptic placentae with Hemolysis, Elevated Liver enzymes and Low Platelets (PE-HELLP; n14) were used. Immunoblotting was performed to determine protein expression of Mcl-1, Beclin-1 and the autophagosome marker LC3. Immunoprecipitation studies were conducted to test Mcl-1/Beclin-1 association. To examine the role of Mcl-1 in trophoblast autophagy, Mcl-1 was overexpressed in choriocarcinoma JEG3 cells. JEG3 cells were treated with sodium nitroprusside (SNP; 5 mM), a nitric oxide donor to mimic oxidative stress. Results: Immunoblotting revealed increased Beclin-1 and LC3 expression at 12-15 weeks gestation, correlating to decreased Mcl-1 protein levels. Overexpression of Mcl-1 in JEG3 cells decreased LC3 expression. Exposure of JEG3 cells to SNP resulted in decreased Mcl-1 followed by increased LC3 expression. Immunoprecipitation studies veried Beclin-1/Mcl-1 interaction. Increased expression of LC3 was observed in PE/HELLP placentae and it was associated with decreased Mcl-1 levels. Discussion: Herein we demonstrate that autophagy plays a role during placental development and that Mcl-1 is a direct inhibitor of autophagy in trophoblast cells. Increased autophagy in preeclampsia may be due to decreased Mcl-1 expression (Supported by CIHR and OWH/IGH). Keywords: Preeclampsia, Autophagy, Oxidative Stress, Bcl-2 Family
[P10.13]. THIRD TRIMESTER STILLBIRTHS: PATHOLOGY AND NEUROPATHOLOGY
CORRELATIVE
PLACENTAL
KTE Chang*1, P Shannon2, G Machin2, J Kingdom2, S Keating2. 1The Hospital for Sick Children, Canada, 2Mount Sinai Hospital, Canada Introduction: Placental pathology often identies the reason for stillbirth, providing important information for future obstetrical management. In this study, we aimed to describe the neuropathology of third trimester stillborn fetuses in relation to placental pathology and selected autopsy ndings and to identify specic placental lesions which correlate with neuropathological ndings. Methods: This was a retrospective review of third trimester stillbirth autopsies performed at Mount Sinai Hospital. Fetuses with congenital malformations and abnormal karyotypes were excluded. Placental pathological ndings were studied in the categories of maternal vascular, fetal vascular, inammatory and cord pathology. Meconium staining, the presence of increased nucleated red blood cells and thymic stress changes were also studied. Neuropathological ndings were categorized as follows: (i) neuronal injury (grade 0absent, 1early injury, 2pontosubicular necrosis, 3widespread neuronal injury), (ii) gliosis (0absent, 1present), and (iii) white matter injury (0absent, 1white matter edema, 2swollen axons and/or necrosis). The Fishers exact test was used to identify statistically signicant associations. Results: 37 cases of stillbirth were studied. The brains in 22 cases showed neuronal injury grades 2 and 3. 14 cases had gliosis. Two cases showed grade 2 white matter injury. There was signicant correlation of acute chorioamnionitis, meconium staining and thymic stress reaction with neuronal injury of grades 2 or 3, and with gliosis. Acute chorioamnionitis alone and placental inammation overall both showed signicant correlation with neuronal injury grades 2 and 3. There was no correlation of the placental parameters with grade 2 white matter injury. Discussion: Grey matter injury and gliosis were the most common neuropathological ndings in these third trimester stillbirths. White matter damage and germinal matrix hemorrhage were rare in this population. Of the categories of placental pathology examined, inammatory lesions showed the strongest correlation with neuronal injury of the fetal brain. Keywords: Stillbirth, Neuropathology, Acute chorioamnionitis
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[P10.14]. FREQUENCY OF PATHOLOGICAL CHANGES IN MORPHOLOGICALLY NORMAL PERINATAL DEATHS D.N. Kenwright*, N. J. Sidek, S. Jaffar, J. Zuccollo. University of Otago, Wellington, New Zealand Introduction: Placental pathology frequently contributes to perinatal death. In this study we examined a 12 year cohort of perinatal deaths in Wellington, New Zealand to determine which maternal and placental pathologies contribute to the deaths and the relationship between placental changes and maternal disease. Method: 725 reports of postmortems performed at Wellington Public Hospital on perinatal deaths from 20 weeks gestation to term were examined and the placental and maternal pathologies recorded in morphologically normal babies. These data were then analysed using ttest, Chi-square and Fisher test to determine the odds ratio (OR) and their signicance. Results: From 725 morphologically normal babies, 691 placentas were examined and placental pathology was found in 447 placentas. 139 pregnancies were complicated by maternal disease. Frequency of each placental pathology are as below:
Placental pathology Frequency (%) 18.5 10.1 10.1 18.4 9.1 Gestational age at which frequency peaks 20-23 wk 20-23 wk 22-23 wk 22-26 wk No specic age
[P10.15]. INSULIN-INDUCED VASODILATION IS REDUCED IN HUMAN UMBILICAL ARTERIES FROM GESTATIONAL DIABETES BJ Krause*, MJ Jo, P Casanello, L Sobrevia. Cellular and Molecular Physiology Laboratory (CMPL) and Perinatology Research Laboratory (PRL), Department of Obstetrics and Gynecology, Medical Research Centre (CIM), School of Medicine, Ponticia U, Chile Insulin increases the activity of nitric oxide synthases (NOS) and acts as vasodilator in several vascular beds. Gestational diabetes (GD) is associated with elevated fetal plasma insulin level compared to normal pregnancies, but fetuses from GD could exhibit either insulin resistance and low birth weight, or be responsive to insulin with high birth weight. In addition, placental blood ow correlates with birth weight in GD. We studied insulin vasoactive effect and the potential NOS role in umbilical arteries from normal and GD pregnancies. METHODS. Umbilical arteries from normal and GD pregnancies were dissected and vessel rings were mounted on a wire-myograph. Isometric force in response to insulin (0.001-10 nM) in the presence or absence of the NOS inhibitor N-nitro-Larginine (L-NA, 100 mM) was measured in KCl (37.5 mM) pre-contracted vessel rings. Responses were expressed as a percentage of relaxation relative to corresponding maximal effects of KCl. RESULTS. Insulin induced vasodilatation (18.21.3%) in a concentration-dependent manner (half-maximal effect10.80.2 nM) in vessels from normal pregnancies (n4), a response blocked by L-NA. However, insulin induced either vasodilatation, which was partially inhibited (w55%) by L-NA, or vasoconstriction in GD (n6), an effect that correlated (r20.9) with fetal weight. CONCLUSIONS. Human umbilical artery dilatation induced by insulin was dependent or partially dependent on NO in normal or GD pregnancies, respectively. Variability of the vasoactive response to insulin in umbilical arteries from GD could in part explain altered placental blood ow in fetuses small or large for the gestational age. Supported by FONDECYT 1070865/1080534. B.J.K. holds a CONICYT-PhD fellowship. Keywords: insulin, vasodilation, umbilical, vessels
Chorioamnionitis Funisitis Abruption Infarction Signicant/massive perivillous brin deposition Villitis of unknown aetiology Lymphohistiocytic intervillositis Decidual vasculopathy Fetal/maternal haemorrhage Rupture of fetal vessel Subchorial haemorrhage Fetal thrombotic vasculopathy Villous hypoplasia Villous dysmaturity
8.7 2.3 2.3 2.3 0.3 2.9 5.6 6.2 4.8
38-42 wk No specic age No No No No No specic specic specic specic specic age age age age age
22-26 wk 37-42 wk
Statistically signicant correlation was found between maternal diabetes and villous dysmaturity (OR: 5.91, p<0.05). Pregnancies complicated by maternal hypertension was found to correlate with chorioamnionitis (OR:3.127, p<0.05) and funisitis (OR: 2.76, p<0.05). Pregnancies complicated by PET have increased chorioamnionitis (OR:9.3, p<0.000), funisitis (OR:2.81, p0.05), decidual vasculopathy (OR:21.1, p<0.000), infarction (OR:2.21, p<0.05), and villous hypoplasia (OR:4.0, p<0.05). HELLP syndrome correlates with infarction (6.1, p<0.05) and villous hypoplasia (OR:12.1, p<0.05). IUGR correlates with maternal hypertension (OR:7.1, p<0.000), PET (OR:32.3, p<0.000), HELLP (OR:15.3, p<0.01). Conclusion: This is the rst comprehensive review of placental pathology in New Zealand. The high rate of placental morphologic abnormality found in morphologically normal infants shows the importance of placental examination in perinatal post mortems. Our data reects previous ndings in that high rates of chorioamnionitis and abruption are found in very early preterm deliveries. The histological ndings in the placenta with maternal diseases are similar to other reports, with the exception of maternal hypertension and chorioamnionitis/funisitis which was a surprise correlation. Keywords: placenta, pathology, perinatal, death
[P10.17]. UP-REGULATION OF THE IGF2 GENE EXPRESSION BY HYPOXIA IN PREECLAMPSIA AND INTRAUTERINE GROWTH RESTRICTION PLACENTAS C Louet*1,4, S Barbaux1, J Tost2, C Buffat3, D Vaiman1, H Jammes1,4. 1Institut pital La Cochin, France, 2Centre National de Genotypage, France, 3Ho Conception, France, 4INRA, France The paternally imprinted IGF2 gene encodes the Insulin-like growth factor 2 (IGF2), a key regulator of placental development whose dysfunctions are implicated in human pregnancy pathologies such as preeclampsia and/or intrauterine growth restriction. We rst performed a thorough update of the structure of IGF2 transcripts and promoters in human using extensive in silico data. We found an increase of IGF2 gene expression in all pathological placentas versus normal placentas. Analysing the methylation status at the IGF2/H19 locus (56 CpGs) by pyrosequencing, a tissue-specic hypomethylation of the IGF2 DMR2 was found in human placentas. Nevertheless, the imprinted status of IGF2 and H19 was conserved in all placental diseases. We investigated the use of the ve IGF2 promoters in order to elucidate the IGF2 gene up-regulation. It is mainly caused by the use of P0 promoter, correlated to the induction of YY1, a human P0 promoter specic transcription factor implied in the regulation of imprinted genes. Moreover hypoxia up-regulates the expression of the IGF2 transcript P0 in a human choriocarcinoma cell model. In the light of these results we hypothesise that, in addition to its primary imprinting regulation, the complex transcriptional regulation of the IGF2 gene permits an adaptation to the hypoxic environment present in placental diseases. Keywords: IGF2, hypoxia, placental pathologies
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[P10.18]. PLACENTAL BIGLYCAN EXPRESSION IS DECREASED IDIOPATHIC FETAL GROWTH RESTRICTION
IN
HUMAN
[P10.19]. VITAMIN C BUT NOT VITAMIN E INCREASES THE EXPRESSION OF PP13 AND BETA-hCG IN FORSKOLIN STIMULATED BeWo CELLS K Orendi*1, M Gauster1, G Moser1, H Meiri2, B Huppertz1. 1Institute of Cell Biology, Histology & Embryology, Medical University Graz, Austria, Austria, 2 Diagnostic Technologies Ltd, Yokneam, Israel, Israel Objectives: Maternal serum concentrations of placental protein 13 (PP13) have been shown to be altered in rst trimester pregnant women subsequently developing preeclampsia compared to controls. Vitamins C and E are putative therapeutics for prophylactic treatment of preeclampsia. Here we used the choriocarcinoma cell line BeWo as surrogate for primary trophoblast and investigated the inuence of vitamins C and E on the expression of PP13 and beta-hCG in these cells. Methods: BeWo cells were cultured for 48h with increasing concentrations of vitamin C (30 to 200 mM) and the vitamin E derivative Trolox (10 to 100 mM) in presence or absence of 20 mM forskolin. Culture supernatants and cell lysates were collected to determine PP13 and beta-hCG expression and release by Dela assays. Cell viability was estimated by LDH detection in supernatant. Immunouorescence was performed using antibodies against beta-hCG as a marker for differentiation and E-cadherin to visualize cell fusion. Results: Without forskolin stimulation vitamins C and E did not show any effects on the expression of PP13 in cell lysates. After forskolin stimulation, PP13 and beta-hCG concentrations in cell lysates signicantly increased with increasing vitamin C concentrations in a dose-dependent manner. Viability did not show any changes in any of the conditions. Morphological analysis of immunouorescent staining with beta-hCG indicated a higher rate of cell differentiation after vitamin C supplementation. Addition of vitamin E did not show to have an effect on the expression of PP13 but led to an increased expression of beta-hCG. Conclusions: Vitamin C but not vitamin E has a positive effect on the expression of PP13 and beta-hCG in differentiating BeWo cells compared to controls. It needs to be elucidated whether a similar effect is present in primary cells and in vivo as well. Keywords: Preeclampsia, Vitamin C+E, Placental Protein 13/PP13
P Murthi*1,2, FA F1,2, G Rajaraman1,2, NA Pathirage1,2, V Ignjatovic3,4, JM Said1,2. 1University of Melbourne, Australia, 2Royal Womens Hospital, Australia, 3Murdoch Childrens Research Institute, Australia, 4The Royal Childrens Hospital, Australia Introduction: Fetal growth restriction (FGR) is a leading cause of prenatal morbidity and mortality. The majority of FGR cases are idiopathic and are associated with placental insufciency, which can result from placental thrombosis. Evidence suggests that Dermatan Sulfate (DS) is an important anticoagulant in placentae of uncomplicated pregnancies. This study hypothesised that the expression of biglycan proteoglycan, a source of DS, is decreased in idiopathic FGR placentae compared with placentae from uncomplicated pregnancies. This study aimed to determine biglycan mRNA, protein expression and spatial distribution in idiopathic FGR placentae compared with the placentae from gestation-matched controls. Methods: This study investigated 26 placentae from idiopathic FGRaffected pregnancies and 27 placentae from uncomplicated, gestationmatched pregnancies (27 to 40 weeks gestation). Inclusion criteria for the idiopathic FGR group included a birth weight of less than the 10th percentile and at least two of the following; abnormal umbilical artery Doppler velocimetry, oligohydramnios or asymmetric fetal growth. Biglycan mRNA expression, protein expression and spatial distribution determined using real-time PCR, immunoblotting and immunohistochemistry, respectively. Results: Mean biglycan mRNA expression was signicantly decreased in FGR placentae compared with control placentae (2.871.93 (n26) vs 4.482.40, (n27) p0.01). FGR placentae demonstrated signicantly decreased mean biglycan protein expression compared with control placentae (23.97.7 vs, 59.310.7, n12, p<0.05). Biglycan immunoreactivity was detected in endothelial cells and smooth muscle cells lining the fetal capillaries. Semi-quantitative analyses demonstrated a signicant decrease in immunoreactive biglycan in FGR placentae compared with control placentae (51.119.3 vs, 500.7223, n6, p<0.001) Discussion: This is the rst study to demonstrate the association between decreased biglycan expression and idiopathic FGR placentae. Reduced biglycan expression may contribute to placental thrombosis within the fetal vasculature, and may contribute to the pathogenesis of idiopathic FGR. Keywords: fetal growth restriction, placenta, proteoglycans, thrombosis
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[P10.20]. TGIF EXPRESSION AND SIGNALLING IN HUMAN VILLOUS AND EXTRAVILLOUS TROPHOBLASTS NA Pathirage*1,2, B Kalionis1, SP Brennecke1,2, P Murthi1,2. 1Department of Perinatal Medicine, Pregnancy Research Centre, Royal Womens Hospital, Victoria, Australia, 2Department of Obstetrics and Gynaecology, The University of Melbourne, Victoria, Australia Introduction: Abnormal homeobox gene expression has previously been associated with the clinically signicant disorder fetal growth restriction (FGR) (1). Targeted disruption of transforming growth beta-induced factor (TGIF), a member of the TALE (three-amino-acid loop extension) superfamily of homeobox genes has been shown to result in placental dysfunction in the mouse (2). One aim of this study was to determine the expression patterns and expression levels of TGIF in FGR affected placenta compared with gestation matched controls. The other aim was to identify genes/regulatory networks modulated by TGIF in human trophoblast cells. Methods: To determine the spatial and temporal expression of TGIF immunohistochemistry was carried out on rst trimester, normal term and FGR affected placental sections. Relative TGIF mRNA expression of FGR placentae and gestation age matched normal placenta was determined using real-time PCR. Western blotting was conducted to assess TGIF protein expression. Signalling pathways regulated by TGIF were investigated by altering TGIF expression in extravillous trophoblast and villous trophoblast derived cell lines JEG3 and BeWo respectively and using siRNA and over-expression plasmids followed by PCR-array analysis to determine the transcriptional proles. Results: Immunohistochemistry localised TGIF protein expression in residual cytotrophoblast cells, syncytiotrophoblast cells, microvascular endothelial cells and stromal cells. TGIF mRNA levels were signicantly up regulated in FGR compared with normal placentae [1.290.06 (n25) FGR v. 0.780.04 (n25) normal, P<0.001]. Similarly, TGIF protein expression was signicantly increased [3970 1101 n6) FGR v. 2323644 n6) normal, (P<0.05)]. PCR array analysis revealed differentially regulated genes from several biological pathways including TGF-b /BMP, Wnt and MAPK signalling pathways. Furthermore, a subsets of genes was identied that were unique to BeWo and JEG3 cells, suggesting that TGIF may regulate different signalling pathways in villous and extravillous trophoblasts. Conclusion: We conclude that TGIF and its downstream target genes may be important for trophoblast function and may be a contributing factor to the developmental abnormalities seen in the FGR affected placentae. Keywords: Fetal Growth Restriction, Trophoblasts, Homeobox genes, Transforming growth beta-induced factor (TGIF)
and heterozygous forms of complete hydatidiform mole (CHM), as well as to prove origin of gestational choriocarcinoma. DNA analysis of 10 microscopically proven gestational choriocarcinomas (Fig. 1) appeared after CHM was done. DNA obtained from tumor cells and peripheral blood lymphocytes of patients and progenitors was isolated by QIAamp DNA Blood Mini Kit. For DNA analysis method of PCR amplication of particular VNTR sequences (ApoB, Col2A a MCT 118) was used. In 9 cases DNA analysis proved origin of gestational choriocarcinoma from heterozygous CHM (Fig. 2). Malignant transformation of homozygous CHM to choriocarcinoma happens only in 1 case. Obtained results show that diagnostics based only on the microscopic observations is insufcient and utilization of sophisticated methods of DNA analysis bring better perspectives in diagnostics of gestational choriocarcinomas after CHM pregnancy.
Fig. 1. Gestational choriocarcinoma (Hematoxylin Eosin, magnication 140x)
[P10.21]. ANALYSIS OF GESTATIONAL CHORIOCARCINOMA ORIGIN AT THE DNA LEVEL V. Repiska, L. Danihel, V. Sisovsky, L. Danisovic, S. Polak*, D. Bohmer. Comenius University, Faculty of Medicine, Slovakia Gestational trophoblastic disease (GTD) is a diverse group of trophoblast lesions with specic pathogenesis, morphological and clinical features. Gestational choriocarcinoma is a highly malignant tumor derived from the cells of trophoblast. It may arise from mola hydatidosa, after misscarriage or normal delivery, as well as after ectopic pregnancy. Its incidence is highest in Asia, Africa and Latin America (1:5001000 deliveries), in Europe, USA and Australia is lower (1:2000040000 deliveries). Choriocarcinoma may grow exophytic way in the uterine cavity or endophytic inside the uterus wall. It has grey-white colour with variable sized deposits of bleeding and necrosis. The microscopic picture shows heterogeneous population of trophoblast cells which inltrate and destroy tissue of uterus. Chorionic villi are not present. Moreover, extrauterine choriocarcinomas and very rarely also in placenta tissue, were described. Immunohistochemistry proved high expression of hCG, while presence of HPL in the cells of trophoblast is rare. DNA analysis enables to distinguish particular forms of GTD and unambiguously prove the origin of nuclear DNA. Currently it is unique possibility for identication of homozygous
Fig. 2. DNA analysis of gestational choriocarcinoma (1 heterozygous CHM, 2 progenitor, 3 choriocarcinoma from heterozygous CHM, 4 patient)
Keywords: Gestational trophoblastic disease, choriocarcinoma, DNA analysis, VNTR
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[P10.22]. PLACENTAL SYNDECAN-1 EXPRESSION IS SIGNIFICANTLY REDUCED IN HUMAN IDIOPATHIC FETAL GROWTH RESTRICTION G Rajaraman*1,2, P Murthi1,2, J Said1,2, V Ignjatovic3,4, PT Monagle3,4, SP Brennecke1,2. 1University of Melbourne, Dept of obstetrics and Gynaecology, Australia, 2Royal Womens Hospital, Australia, 3Murdoch Childrens Research Institute, Australia, 4Royal Childrens Hospital, Australia, 5 University of Melbourne Dept of Paediatrics, Australia Introduction: Fetal growth restriction (FGR) is a clinically signicant pregnancy disorder in which the fetus fails to grow to its full potential in utero. 70% of FGR pregnancies are idiopathic and associated with placental thrombosis. Syndecan-1 is a proteoglycan containing the heparan sulfate glycosaminoglycan, which binds growth factors and antithrombin. Previous studies have shown that placental syndecan-1 expression is restricted to the apical surface of villous syncytiotrophoblasts, suggesting its role in anticoagulation of the intervillous space. However, the potential role of placental syndecan-1 in idiopathic FGR is unknown. This study investigated syndecan1 expression in idiopathic FGR placentae compared with control placentae. Methods: Human placentae from pregnancies complicated by idiopathic FGR (n28) and gestation-matched control pregnancies (n28), were selected according to strict clinical diagnostic criteria. Placentae of 27 to 40 weeks gestation were obtained. Immunohistochemistry, western immunoblotting and real-time PCR were employed for analysis of syndecan-1 protein and mRNA expression, respectively. Results: Semi-quantitative analyses of immunohistochemistry showed signicantly reduced syndecan-1 protein expression in the villous syncytiotrophoblasts of idiopathic FGR placentae compared with controls (141.753.5 versus 362.5162.1, n6, t-test, p<0.01). Syndecan-1 mRNA expression relative to the housekeeping gene GAPDH, was signicantly reduced in idiopathic FGR placentae compared with controls (0.070.02 versus 4.480.91, n28, t-test, p<0.001). Immunoblotting using a mouse monoclonal antibody, demonstrated signicantly reduced syndecan-1 protein expression in idiopathic FGR placentae compared with controls (55.112.7 versus 132.345.7, n6, t-test, p<0.05). Conclusion: This is the rst study to demonstrate a signicant decrease in placental syndecan-1 mRNA and protein expression in idiopathic FGR, suggesting a potentially important role of syndecan-1 in the aetiology of placental thrombosis in human FGR. Keywords: Plaenta, Fetal growth restriction, Syndecan-1, Thrombosis [P10.23]. THE ABILITY OF ULTRASOUND TO DETECT PLACENTAL PATHOLOGY M Robertson*1,2, E Amyes1, D.A Ellwood1,2, J Dahlstrom1,3. 1Australian National University, Australia, 2Fetal Medicine Unit, The Canberra Hospital, Australia, 3Anatomical Pathology, The Canberra Hospital, Australia The placenta is generally under appreciated in the examination and monitoring of fetal health and well-being. Many problems during the antenatal period that lead to perinatal morbidity and mortality can be attributed to placental insufciency. The aim of this study was to examine the placenta in utero using ultrasound and compare the ndings with pathological features of the delivered placenta. Nineteen pregnant women whose antenatal ultrasound had demonstrated one or more intraplacental lesions together with evidence of IUGR or a previous history of IUGR or perinatal death where recruited. Placental ultrasound was performed before delivery. Measurements were taken to determine placental features including position and echogenicity of lesions. Following birth, placentas were examined macroscopically and microscopically for evidence of corresponding pathology. From the study, two groups were ascertained. The rst group were placentas with very small diameters but increased placental thickness that we have termed cup-cake. The second group were placentas that had hypoechoic lesions that were later determined to be intervillous thrombi. From the small number of participants recruited to the study, abnormalities of placental size and shape as well as discrete lesions from a number of placentas were identied on ultrasound. These were later conrmed at macroscopic and microscopic analysis indicating a role for ultrasound in detecting of placental pathology. Keywords: Ultrasound, Placental pathology
[P10.24]. METHODOLOGIC ISSUES IN THE STUDY OF THE RELATIONSHIP BETWEEN INFECTION AND PRETERM BIRTH CM Salaa*1,2, DP Misra3, J Miles4. 1Institute for Basic Research, United States, 2Placental Analytics LLC, United States, 3Wayne State University, United States, 4Rand Corporation, United States Goal: To determine the structure of the relationships of the histology scores for acute intraamniotic infection collected in the Collaborative Perinatal Project (CPP). Materials and Methods: 44,427 subjects of the CPP had complete histology scores available for the 9 measures related to acute intraamniotic infection (neutrophil inltrates in umbilical cord, amnion of membranes and chorionic plate, decidua, chorionic plate and fetal chorionic vessels). Conrmatory factor analysis was used to determine the relationships among the different markers of maternal inammatory responses (in amnion, chorion and decidua) and fetal inammatory responses (in cord and chorionic vessels). The conrmatory structure included both considerations of whose inltrates (maternal versus fetal) and tissue sites in which they were assessed (assuming that measures made from the same tissue sample would be more highly correlated that those made from different samples of the same placenta). Results: A single CFA model could not be developed across all CPP sites, indicating that the above assumptions that underlaid our CFA structure were not valid. A well-t CFA model conforming to our assumed structure of relationships was developed from the Boston site (N10,803). The factor loadings were then applied to the histology scores from the other CPP sites. The scores for the latent variables (maternal and fetal inammatory responses) were compared across sites. Factor loadings, intercorrelations of maternal and fetal inammatory scores and the signs of factor loadings were inconsistent across sites. Conclusion: Histopathology scores of neutrophil inltrates performed by different observers do not have the same interrelationships and, by extension, the latent variables they are supposed to reect (i.e., the measured maternal and/or fetal inammatory responses) may not be equivalent. The lack of measurement invariance renders the use of scoring neutrophil inltrates in placental tissues as indicators of the underlying processes of maternal and fetal inammatory responses problematic. Keywords: acute infection, neutrophils, structural equation modeling, Collaborative Perinatal Project
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[P10.25]. MODEL FOR OXYGEN TRANSPORT IN THE HUMAN PLACENTA JS Gill*1, MP Woods1, CM Salaa2,3, DD Vvendensky1. 1Imperial College, United Kingdom, 2Placental Analytics LLC, United States, 3Institute for Basic Research, United States Goal/Background: The mature placenta is a complex vascular network extending ultimately to the capillaries of the terminal villi, site of all oxygen and nutrient exchange between the mother and fetus. Respiratory transfer across the placenta to the fetus occurs in three steps: (i) maternal blood bathes the chorionic villi in oxygen, (ii) oxygen permeates the villus surface and diffuses into fetal capillaries, and (iii) oxygen is transported to the fetus via the bloodstream. Materials/Methods: Step (ii) has been modelled by the diffusion of oxygen from the villous membrane and the fetal capillaries. The stationary concentration c(x,t) of oxygen within each of the villi is the solution of the two-dimensional Laplaces equation, Dc 0, with a xed concentration cv at the villous surface and a Robin boundary condition at the capillary boundary: Dvc/vn Kc, where v/vn is outward normal derivative, D is the oxygen diffusion constant and K is the permeability of the capillary. These equations are solved in regions [panels (d,e,f)] determined by the villus and capillary boundaries obtained from digitized images [panels (a,b,c)]. Results: The solution for the oxygen concentration determines the diffusive current of oxygen across the capillaries. Many factors are expected to inuence this current, including the numbers and shapes of villi and capillaries. Our initial analysis ndicates that (c) yields the largest ux per unit perimeter length and area of the villi, followed by (b) and (a). Conclusions: The geometrical shapes and spatial distributions of the villi and capillaries are important placental characteristics for the transport of oxygen to the fetus. Once the main factors that determine oxygen transport have been identied, this approach, applied to digitized placental slides that allow analysis of many hundreds of villi per slide (and multiple slides per placenta) should provide a quantitative basis for measuring placental oxygen uxes.
[P10.26]. CENTRALITY OF THE UMBILICAL CORD INSERTION IN A HUMAN PLACENTA INFLUENCES THE BIRTH WEIGHT M Yampolsky1, O Shlakter1, CM Salaa*2,3, DH Mandel3. 1University of Toronto, Canada, 2Institute for Basic Research, United States, 3Placental Analytics LLC, United States Background: We hypothesize that trophotropism, considered to underlie eccentricity of umbilical cord insertion, results in a deformed placenta, less functionally efcient, and that the more eccentric the cord insertion, the less efcient the placenta. Materials and Methods: The model is based on a random fractal growth process (DLA). By placing the initial seed asymmetrically, the model produces placental vascular trees with a non-centrally placed umbilical insertion point, whereas the overall shape of the tree remains round to oval. To test this hypothesis, we calculated a measure of roundness by taking a mean radial distance to the perimeter. The sector was rotated in 2.5 increments to produce 24 radial measurements; the mean square deviation swas calculated for both real and model placentas. In the photographs of the UNC placentas the surface vascular branches were traced and, for each pixel in the chorionic surface, the minimal distance to a traced vessel was calculated. The resulting number is dimensionless (relative chorionic vascular distance D). A lower value means a better penetration of the chorionic surface by the blood vessels. Results: For a round placenta, the value of s is zero, the correlation of the value of s with the cord displacement variable in the UNC dataset is only 0.04. Thus, non-centrality of the cord insertion produces little effect on the shape. A round placenta with an asymmetrically placed umbilical cord has a signicantly lower vascular penetration, and hence reduced metabolic efciency. Conclusion: Trophotropism, the directional growth of the placenta due to variations in the intrauterine environment, is considered the most common basis for asymmetrical cord insertions. Our data suggest that even relatively mild eccentricity is associated with abnormal development of chorionic surface vessels. Compensation for a problematic intrauterine environment is incomplete, and does not restore the placenta to an optimally transporting structure.
Keywords: chorionic plate, placental growth, fourier analysis
Figure 1. Top row: the graph of the function r(q) for a round disk with cord displacement 5. Bottom row: two placental perimeters (P marked in blue, angular radius in red), with the umbilical insertion point placed at the origin.
Keywords: birth weight, Umbilical cord, placental growth, placental function
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[P10.27]. RELIABILITY OF AUTOMATED NEUTROPHIL QUANTITATION IN DIGITIZED H&E STAINED SLIDES: PILOT ANALYSIS OF CORRELATION WITH AMNIOTIC FLUID PROTEOMICS SCORE K Thomas*1, CM Salaa2,3, I Buhimschi4, CS Buhimschi4, E Zambrano4, M Sottile1. 1University of Oregon, United States, 2Placental Analytics LLC, United States, 3Institute for Basic Research, United States, 4Yale University School of Medicine, United States Background: The diagnosis of intraamniotic infection is considered clinically relevant to maternal, fetal, neonatal and childhood morbidity and mortality. However, intraobserver variability, even among expert pathologists, remains problematic, apart from 0.96 agreement on present v absent (Pediatr Dev Pathol. 2003;6(5):435-48), with consensus (rather than valid biomarkers) being the gold standard. An automated and reliable method for quantitation of neutrophils would be a useful diagnostic tool. Methods: We sampled 10 cases with amniotic uid proteomics (AFMR) scores, 2 each of scores 0-4 as per Buhimschi et al, BJOG. 2009 Jan;116(2):257-67. Two images were taken at random of extraplacental membranes; each image was analyzed at both 10x and 20x magnication. The image analytic technique begins with scanned slides in the form of color (RGB) images. We rst segment the image to separate tissue from cells of interest by using the difference in color that results from staining. After this initial segmentation, we then must lter out image features that are the correct color but fail to t the expected shape of a neutrophil, such as [carrie insert name of elongated cells here].This requires the computation of the area, perimeter, and eccentricity (a measure of how circular a shape is) of each connected component identied by the segmenter. Three segmentations were done:1. based on color threshold alone; 2. based on color and then rejecting anything bigger or smaller than the area threshold interval; 3. based on color, rejecting groups based on area threshold as well as rejection based on eccentricity (rejecting cells such as broblasts that have cigar shaped nuclei). Results: Using Method 1, only the number of pixel groups was associated with AF MR (r0.494), with Method 2, AF MR was associated with both positive pixel count (r0.544), and percent positive pixels (r0.544). With Method 3, positive pixel count (r0.546), percent positive pixels (r0.546), and the number of pixel groups (r0.505) were high correlated with AF MR. These counts also signicantly correlated with histologic grading of neutrophils in amnion, chorionic and decidua, and in umbilical cord. Magnication at analysis did not modify the strength of the associations. Conclusion: Pilot data suggests that reproducible and reliable automated segmentation and quantitation of neutrophils can be performed, with strong correlations with amniotic uid proteomic markers of infection and inammation. We anticipate that a larger image sample per tissue will result in improved correlation with AFMR score.
[P10.28]. EFFECTS OF GLUCOSE ON THE EXPRESSION OF VEGF SPLICE VARIANTS IN NORMAL AND DIABETIC PLACENTA F. Sciota*, L. Leach. University of Nottingham, United Kingdom Hyperglycaemia is a main characteristic of diabetes and may affect placental vascular development and permeability. In Type1 diabetes, the placental vasculature displays increased angiogenesis and permeability. The main form of VEGF165 is pro-angiogenic and pro-permeability, whilst VEGF165b, a recently discovered splice variant of VEGF165a, is thought to be anti-angiogenic, but pro-permeability. Whether glucose can affect the expression of these variants in the human normal and diabetic placenta is not known and is the aim of this study. Using chorionic villous explants, 15mM glucose was administered to normal (N15) and Type1 diabetic (D15) study groups (n3 placentae per group) for 4h. Euglycaemic controls (n3 per group, N5 and D5 respectively) contained 5mM glucose. VEGF-165a and VEGF-165b were localised and counted by immunocytochemistry and selective random sampling. D5 showed downregulation of VEGF165b compared to N5 (p<0.01). Hyperglycaemic insult resulted in a decrease (p<0.01) in vessels expressing VEGF165b in N15 compared to N5. This aggravation was not seen in D15. The values of N15 were similar to those in D5 and D15. VEGF165a staining showed increased expression in diabetic explants compared with normal, with further increases seen with 15mM glucose. VEGF165a upregulation was also seen between N5 and N15 (p<0.001). There was a negative correlation between VEGF165a and VEGF165b (p<0.001;R20.6852). These changes indicate that glucose is able to affect the expression of both splice variants of VEGF, with downregulation of the anti-angiogenic splice variant being a predominant feature. Hyperglycaemia can induce the diabetic phenotype in normal explants, upregulating VEGF165a and downregulating VEGF165b. In diabetics, with pre-existing high levels of VEGF165a and low levels of VEGF165b, hyperglycaemia induced further increases in the pro-angiogenic VEGF165a whilst expression of the splice variant VEGF165b remain damped. The ratio of the two VEGF splice variants may be an important predictor of vascular dysfunction in diabetes. Funded by ASGBI. Keywords: Diabetes, VEGF-A, VEGF165b
Keywords: image analysis, chorioamnionitis, neutrophil, amniotic uid proteomics
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[P10.29]. TGFb SIGNALLING VIA PAR6 REGULATES TROPHOBLAST CELL POLARITY T Sivasubramaniyam*1,2, I Caniggia1,2. 1Samuel Lunenfeld Research Institute of Mount Sinai Hospital, Canada, 2University of Toronto, Canada Introduction: Cell polarity plays an important role in cell differentiation shaping proper organogenesis. Loss of cell polarity is partly mediated through the TGFb Smad-independent signalling pathway. Activation of Par6, a key regulator of cell polarity, by TGFb leads to its association with Smurf1 (Smad ubiquitin regulatory factor 1) and to the dissolution of tight junctions. The contribution of the Smad-independent signalling pathway via Par6 to trophoblast cell polarity and differentiation remains elusive. Herein we sought to examine the expression/role of Par6 in normal and pathological placentae. Methods: Expression of Par6 and Smurf1 were examined in placentae throughout gestation (6-39 weeks) and in preeclamptic (24-31 weeks) and age-matched control tissues (25-34 weeks) using immunoblotting. Par6/ Smurf interaction was assessed by co-immunoprecipitation and duallabeled immunouorescence. To establish a role for Par6 in regulating trophoblast cell polarity Par6 siRNA was employed in choriocarcinoma JEG3 cells and cell polarity markers including Zona occludin-1 (ZO-1) and E-cadherin were tested. Par6 and Smurf1 expression was examined in JEG3 cells cultured at 3% and 20% oxygen. Results: Par6 and Smurf1 protein expression and interaction peaked at 1014 wks of gestation, when oxygen tension increases. Par6/Smurf1 expression also increased in JEG3 cells with increasing oxygenation. Early on in gestation, Par6 localized mainly to the nuclei of cytotrophoblast cells while, with advancing gestation, it shifted to the cytoplasm and it was found at the interface between cytotrophoblasts and syncytium, where it associated with Smurf1, ZO-1 and E-cadherin. Silencing of Par6 resulted in decreased ZO-1 and E-cadherin expression. Of clinical signicance, both Par6 and Smurf1 protein expression levels were decreased in preeclampsia. Discussion: Par6 plays a role in regulating trophoblast cell polarity during placental development. These ndings provide novel insights into the role of Smad-independent signalling with respect to cell polarity in the pathogenesis of preeclampsia. (Supported by CIHR and OWH/IGH). Keywords: cell polarity, preeclampsia, oxygen regulation, development
[P10.30]. EVIDENCE OF ABNORMAL PLACENTAL LYMPHATIC DEVELOPMENT IN A CASE OF PLACENTAL MESENCHYMAL DYSPLASIA SD Smith*1, N Sahasrabudhe2, EA Martindale3, AEP Heazell1. 1Maternal and Fetal Health Research Group, United Kingdom, 2Department of Histopathology, Royal Blackburn Hospital, Blackburn, United Kingdom, 3Department of Obstetrics and Gynaecology, Royal Blackburn Hospital, Blackburn, United Kingdom Placental mesenchymal dysplasia (PMD) is a rare disorder affecting 0.02% of human pregnancies. PMD is associated with stillbirth, intrauterine growth restriction (IUGR) and Beckwith-Wiedemann syndrome (an imprinting disorder characterised by elevated insulin-like growth factor (IGF)-II expression). 86 cases of PMD have been described and its morphology is characterised by placentomegaly and the presence of vesicles, similar to those seen in molar pregnancies. The underlying cellular origin of this condition is unclear. We investigated the placental cell type involved in a case of PMD associated with a live born female infant with IUGR. In this case, intermediate and terminal villi contained cisternae, lined by non-proliferative cells, as veried by immunohistochemistry for Ki67. Immunostaining for cytokeratin-7 and CD-34 revealed that these cisternae were not of trophoblast nor vascular endothelial origin. However, the cellular lining of the cisternae exhibited positive immunostaining using antibodies specic for lymphatic endothelial markers: CCL21, vascular endothelial growth factor (VEGF) receptor 3 and D2-40. No staining was detected in the intermediate or terminal villi of normal third trimester pregnancies. This aberrant lymphangiogenesis is in accordance with current hypotheses regarding the potential role of VEGFD and IGF-II in the aetiology of PMD, both of which can induce lymphangiogenesis in vitro. Furthermore, such observations suggest that placental villous mesenchyme has the potential to differentiate into various cell types, including those not normally present in the term human placenta. Keywords: Placenta, IUGR, Mesenchyme, Lymphangiogenesis
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[P10.31]. CATCH-UP GROWTH OF PLACENTAS IN LATE-ONSET PREECLAMPTIC PREGNANCIES? D.U. Ulrich*1, G.D. Desoye1, C.W. Wadsack1, U.W. Lang1, J.H. Haas1, D.S. Schlembach1,2. 1Department of Obstetrics and Gynecology, Medical University of Graz, Austria, 2Prenatal Center Muenchen, Germany Background: Placental weight has been correlated with fetal outcome, morbidity and mortality and even with diseases in later life. Although the dynamics of placental growth as reected by weight changes has been documented in few studies and placental weight percentile curves were established for uncomplicated singleton pregnancies, similar data for pregnancy pathologies have not been available. The aim of our study was to establish placental weight percentiles for pregnancies complicated by preeclampsia (PE) to calculate weight increases and to compare these results with normal pregnancies. Methods: In this retrospective analysis the data of 4834 singleton pregnancies were analysed with deliveries !28 week of gestation (wks). Among these 197 (4%) were PE pregnancies (dened by RR >140/90, proteinuria >0,3 g/ 24 hrs). All placentas were weighted within 10 minutes after their delivery and after cutting the umbilical cord. Growth dynamics was calculated as difference in placental weights along 4-week intervals. Results: In both groups the mean placental weight showed a steady increase throughout pregnancy. As expected the preeclamptic placenta weight was signicantly lower compared to the control group. Both absolute and relative weight difference between normal and preeclamptic placental weight decreased from early to late pregnancy: at 32 wks -134 g (-31%), 36 wks -90 g (-17%) and at 40 wks -62 g (-11%). Placental weight difference between wks 28-40 and 32-40 were higher in PE (+65%, +46%) than in normal pregnancies (+35%, +28%), whereas fetal weight differences were virtually similar in both groups. Conclusion: Although placental weight is lower in PE than in normal pregnancies in the third trimester of pregnancy, placentas in PE gain more weight in this period than in normal pregnancies. This is not accounted for by a similar catch-up growth of the fetuses. Keywords: placenta weight percentile, preeclampsia, placenta weight gain
[P10.33]. IMPAIRED SYNCYTIALISATION IN SEVERE PRETERM IUGR K Widdows*1, S Drewlo2, J Kingdom2, T Ansari1. 1Dept. of Surgical Research, NPIMR, Harrow, London, United Kingdom, 2Dept. Obstetric & Gynaecology, SLRI, Mount Sinai Hospital, Canada The small placental phenotype of severe preterm IUGR is characterized by signicant reductions in fetoplacental blood ow, resulting from increased vascular impedance from poorly developed peripheral villous capillaries secondary to a defective angiogenic drive in the late second trimester of pregnancy. Molecular evidence suggests this diminished growth potential arises at the level of villous trophoblast differentiation. We previously reported using stereological tools that the poorly developed villi of severe IUGR with abnormal umbilical artery Doppler contain 30% fewer proliferating villous cytotrophoblast cells (vCT), reecting premature loss of villous progenitor cells that can differentiate, fuse and regenerate the overlying syncytium throughout gestation. This depletion however did not reduce the relative numbers of vCT, indicating an imbalance in the relative proportions of proliferating vs. differentiating vCT- favouring a differentiating vCT phenotype. We therefore tested the hypothesis that in severe IUGR, impaired vCT differentiation leads to the focal depletion of syncytiotrophoblast nuclei, using stereological analysis. The morphological basis of syncytial fusion was investigated by estimating (i) the relative and total number of syncytiotrophoblast nuclei and (ii) the mean individual (size) and overall volume of syncytial knots as an index of syncytial extrusion and apoptosis, in ve preterm normotensive IUGR and nineteen IUGR with pre-eclampsia (PET) cases with documented abnormal umbilical artery Doppler. Whilst total numbers of SCT were signicantly reduced in comparison to preterm controls (n12), the relative numbers were further reduced by 50% in both IUGR and IUGR-PET. This focal depletion in syncytial nuclei alongside normal numbers of vCT (non-fusing) implies focal impairment of syncytial fusion. Furthermore, syncytial knot volume, size and apoptosis were increased. Our data indicates that impaired syncytial fusion indicative of defective vCT differentiation results in insufcient numbers of syncytiotrophoblast nuclei needed to maintain adequate nutrient transport to the severely growth restricted fetus independent of the small placental phenotype.
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[P10.34]. A CASE OF TWIN PREGNANCY WITH COMPLETE HYDATIDIFORM MOLE AND COEXISTENT FETUS: MORPHOLOGICALLY NORMAL PLACENTA WITH TRIPLE TRISOMY M. Yamaguchi*, K. Uchino, J. Miyoshi, H. Tashiro, T. Ohba, H. Katabuchi. Kumamoto University, Japan A 27-year-old nulliparous woman at 11 weeks of gestation was referred to our University Hospital because of unusual ultrasound ndings of the conceptus. Ultrasonography showed multiple cysts within the placenta in the presence of the living fetus. The patients serum hCG level was 462,200 mIU/ml. On the basis of these ndings, partial hydatidiform mole, and placenta with hydropic change were considered in the differential diagnosis. The hCG level was continually elevated and peaked at 900,000 mIU/ ml. Each placental cystic lesion enlarged in size during the follow-up period. We performed MRI scan at 15 weeks of gestation. The MRI ndings showed a clear distinction between the normal placenta and the multivesicular tissue. A diagnosis of hydatidiform mole coexisting with a fetus was made. We informed the patient about the high risk of maternal complications and sequelae. Consequently, she decided to terminate pregnancy at 15 weeks of gestation. The evacuated vesicular tissue and morphologically normal placenta were histophathologically examined. The diagnosis of hydatidiform mole was conrmed and the placenta was conrmed as normal. The karyotype of the molar tissue was 46,XX and that of the histophathologically normal placenta was 49,XX,+2,+12,+16. Cytogenetic examination showed that the genome of the complete hydatidiform mole were androgenetic homozygous diploid. It is postulated that a dispermic triploid zygote may divide into a normal biparental cell and a cell with a haploid set of paternal chromosomes. The latter cell could develop into a complete mole by diploidization. A tripolar spindle may be formed at the time of the rst cleavage division, resulting in an aberrant chromosomal distribution, which may affect the diploid fetus. This case can support the hypothesis of postzygotic diploidization of triploids. Keywords: hydatidiform mole and coexistent fetus, postzygotic diploidization of triploids, chromosomal aberration, molar pregnancy
[P11.01]. CONDITIONED MEDIUM OF PLACENTAL MULTIPOTENT MESENCHYMAL STROMAL CELLS PROTECTS ENDOTHELIUM FROM OXIDATIVE INJURY VIA STAT3 AND MANGANESE SUPEROXIDE DISMUTASE ACTIVATION C.-P. Chen*1, J.-P. Huang1, Y.-Y. Chen1, Y.-H. Wu2, C.-Y. Chen2, S.-H. Liu2. 1 Division of High Risk Pregnancy, Mackay Memorial Hospital, Taiwan, 2 Department of Medical Research, Mackay Memorial Hospital, Taiwan We hypothesized that endothelial cells undergone oxidative injury induced by reactive oxygen species could be repaired by the paracrine factors of human placental multipotent mesenchymal stromal cells (hPMSCs). The alterations of antioxidant enzyme activities and mechanisms of antiapoptotic effects on endothelial cell induced by the conditioned medium of hPMSCs were studied. hPMSCs were isolated from term placentas and endothelial cells from umbilical veins. A conditioned medium of hPMSCs was harvested. Medium conditioned by hPMSCs supported endothelial cell survival and enhanced endothelial cell against tert-Butyl hydroperoxide induced intracellular peroxides and apoptosis. RT-PCR revealed hPMSC expressed cytokines of IL-6 family and the receptors of these cytokines in endothelial cells. The conditioned medium activated the expression and transcriptional activity of signal transducer and activator of transcription 3 (STAT3) in endothelial cells. The endothelial cell STAT3 expression and transcriptional activity was inhibited by gp130 neutralizing antibody added in the conditioned medium. The anti-apoptotic effect of conditioned medium was further inhibited when the endothelial cells was transfected by STAT3 small interfering RNA. Manganese superoxide dismutase, but not copper/zinc superoxide dismutase, catalase or glutathione peroxidase of endothelial cell was signicantly up-regulated by conditioned medium both at mRNA transcript and protein levels, which was mediated by the activation of STAT3 and was inhibited by the gp130 neutralizing antibody or STAT3 small interfering RNA. Thus, the paracrine factors secreted by hPMSCs may support endothelial cell survival. The activation of STAT3 and manganese superoxide dismutase induces a protective effect on oxidative stress-induced endothelial cell damage. Keywords: conditioned medium, multipotent mesenchymal stromal cells, manganese superoxide dismutase, STAT3
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[P11.02]. MESENCHYMAL STEM CELLS DERIVED FROM HUMAN PLACENTA ISOLATION, IN VITRO EXPANSION AND CHARACTERIZATION L. Danisovic*1, M. Ulicna1, I. Varga1,2, V. Repiska1, D. Bohmer1, S. Polak1. 1 Comenius University, Faculty of Medicine, Slovakia, 2Slovak Medical University, Slovakia Background: Stem cells are generally characterized as clonogenic, undifferentiated cells with unique self-renewal potency and plasticity. Over the past few years, stem cells have been derived from various tissues of embryonic, fetal and adult origin. Embryonic stem cells are considered pluripotent, but their utilization is restricted by the ethical consideration. For that reason, multipotent adult stem cells also referred as mesenchymal stem cells (MSCs) represent exclusive tool for regenerative medicine. Human placenta also belongs to promising source of MSCs. The main goal of present work was isolation, in vitro expansion and morphological as well as biological characterization of human placentaderived MSCs. Material and Methods: Human placentas (n5; normal pregnancies) were obtained from healthy donors, always following patients informed consent. Samples of tissue (1cm3) from placenta lobules were cut into small fragments, vigorously rinsed in phosphate buffered saline, hemolyzed and digested with 0.25% trypsin for 15 min at 37 C. Obtained cell suspension with residual fragments was ltered through 70 mm cell strainer. After centrifugation (1200 rpm for 10 min), collected cells were suspended in a-minimum essential medium with 10% fetal bovine serum and gentamicin (80 mg/ml). Cultures were expanded up to third passage at 37 C in humidied atmosphere with 5% CO2. Cell culture medium was refreshed twice a week. Cells from third passage were analyzed by light and transmission electron microscope (TEM), the analyses of surface markers were performed by FACS. Moreover, the multilineage potential was examined as well. Results and Conclusion: Light microscopy showed that MSCs derived from placenta had broblast-like morphology. Subsequent TEM observation showed typical ultrastructure of MSCs. Almost all of the analyzed cells were CD29, CD44, CD90, CD105, CD166 positive and CD34, CD45 negative. They did not express antihuman broblast surface protein. Moreover, the chondrogenic and osteogenic differentiation was proved. In conclusion, human placenta represent high throughput source for mesenchymal stem cells. Keywords: placenta, mesenchymal stem cell, in vitro expansion, characterization
[P11.03]. IN VITRO CHARACTERIZATION OF YOLK SAC MEMBRANE FROM EQUINE ALR Franciolli*1, BM Cordeiro2, AC Morini1, JC Morini-Junior1, CV Wenceslau1, MA Miglino1. 1Department of Surgery, Faculty of Veterinary Medicine, University of Sao Paulo, Sao Paulo, Brazil, 2Presbiteriana Mackenzie University, Sao Paulo, Brazil, 3Department of Morphology, UNIfeob, Sao Joao da Boa Vista, Sao Paulo, Brazil The yolk sac is the rst of the fetal membranes that develops in eutherian mammals. It is attached to the wall of exocelom and extra-embryonic mesoderm. The aim of this study was to characterize the morphology of the equine yolk sac cells in culture with 20, 30 and 40 days of gestation in order to demonstrate its multipotentiality and its ability to differentiate in different lineages. The yolk sac explants were cultivated in DMEM-H with 20% FBS Hyclone and 1% penicillin/streptomycin. The culture asks were maintained at 37 C in a humidied environment containg 5% carbon dioxide. After expansion, the cells were morphologically analyzed by inverted microscopy (NIKON ECLIPSE, TS100). The cultures of equine yolk sac cells with 20, 30 and 40 days of gestation were composed of numerous undifferentiated cells oating, with circular format at the rst 24 hours of culture. Followed the rst ve days of culture, the medium of culture was discarded along with the oating cells, leaving only cells that have capacity for adhesion to substrate. Among the different cell types in culture by fragments released yolk in both ages, we found cells with broblast-like characteristics and with characteristic epithelial and less frequently oval cells. Morphologically the cells with broblast-like appearance were small format with elongated, fusiform and reduced cytoplasm. The epitheliallooking cells were large and rounded, with regular cytoplasmic membrane, cytoplasm, and large sparse, small, circular core. As preliminary conclusions, we believe that in the three gestational ages (20, 30 and 40) the yolk sac exhibits two distinct types of cell populations, but new knowledge related to the capacity of differentiation are under experiment to conrm the pluripotential cells of the yolk sac. Keywords: Yolk sac, Equine, Stem cells, Culture
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[P11.05]. AMNION EPITHELIAL CELL ISOLATION FOR USE IN CLINIC S Murphy*, S Rosli, R Acharya, R Lim, G Jenkin, E Wallace. Monash University, Australia Introduction: Human amnion epithelial cells (hAECs) are a heterogeneous population positive for stem cell markers and display multi-lineage differentiation potential, differentiating into cells of the endoderm (liver, lung epithelium), mesoderm (bone, fat), and ectoderm (neural cells). They have a low immunogenic prole and possess potent immunosuppressive properties. Hence hAECs may be a valuable source of cells for cell therapy. Aim: Demonstrate feasibility of animal product-free methods of isolation and culture according to current guidelines on cell preparation for clinical use. Methods: An animal product-free cell isolation method was developed and compared to traditional animal product-containing methods. Total cell yield and viability determined by cell counts and trypan blue exclusion. Purity was established through FACS analysis of epithelial (EpCAM) and mesenchymal (CD90, CD105) marker expression. Animal product-free cryopreservation and growth media were developed and compared to conventional serum-based media. Post-thaw viability, recovery of cell metabolism, and proliferation rates were determined. hAECs were analysed after 5 passages by karyotype analysis, cell cycle distribution and changes to telomere length. hAEC were differentiated into lineages of the three primary germ layers and specic markers analysed using PCR, immunocytochemistry and histology. Results: The method developed was comparable to established animal product-containing methods, producing an average yield of 12040x106 hAECS with average viability of 834%. Isolated populations were 92% EpCAM positive with <1% mesenchymal cell contamination. After 5 passages hAEC displayed normal karyotype, cell cycle distribution and long telomeres, suggesting that hAEC are unlikely to be tumorigenic. Multipotent differentiation potential of hAEC was demonstrated by induction of neural (MAP2, GFAP, Nestin), bone (osteocalcin, osteonectin), fat (PPARg LPL), and lung epithelial (SP-C, CC10, Nkx2.1) gene expression as well as by immunocytochemical and histological methods. Discussion: We have now optimised an animal product-free method for efcient isolation and cryopreservation of hAECs suitable for clinical therapies. Keywords: amnion, epithelial, isolation, differentiation
[P11.06]. Ly6e EXPRESSION IS RESTRICTED TO SYNCYTIOTROPHOBLAST CELLS OF THE MOUSE PLACENTA M Hughes, DRC Natale*. University of Calgary, Canada The labyrinth layer of the mouse placenta is primarily derived from cells of the chorion and is the site of nutrient and gas exchange between maternal and fetal blood. Organized in a xed orientation, three layers of trophoblast followed by a layer of fetal endothelia separate the maternal and fetal blood spaces. A mononuclear trophoblast giant cell layer lines the maternal blood spaces, followed by two multi-nucleated syncytiotrophoblast layers (SynT I and II respectively). It has been shown previously, that SynT I and II cells can be identied by the layer-restricted expression of Syncytin a and Gcm1, respectively. In addition, these genes are expressed in a similar, spatially related manner in the chorion, suggesting prepatterning of labyrinth development. In the present study, we characterized the expression of lymphocyte antigen 6, locus E (Ly6e), in the mouse placenta. Ly6E is a membrane-associated protein that is expressed in the hematopoietic lineage and is a marker of T-cell precursors. We identied Ly6e mRNA expression in trophoblast stem (TS) cells in a gene expression screen. Northern blot analysis conrmed that Ly6e was expressed in both undifferentiated and differentiated TS cell cultures as well as placental RNA from embryonic day (E) 10.5 to 18.5. FACS analysis indicated that in vitro, Ly6e was expressed in 33% of undifferentiated TS cells and increased following differentiation. Interestingly, in vivo, Ly6e was rst detectable by mRNA in situ hybridization in a subset of cells in the chorion beginning at E8.5. This pattern of expression differed from Gcm1 but was similar to that of Syncytin a, and at later stages of gestation, Ly6e expression was restricted to syncytiotrophoblast cells in the labyrinth. To date, Ly6e represents a novel marker of syncytiotrophoblast cells. Ongoing experiments, utilizing siRNA knockdown of Ly6e in TS cells will determine if it has a role in syncytiotrophoblast differentiation. Keywords: syncytiotrophoblast, trophoblast stem cell, labyrinth, mouse
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[P11.07]. INACTIVATION OF INTEGRIN ALPHA 1 (ITGA1) IN MESENCHYMAL STEM CELLS DERIVED FROM THE HUMAN PLACENTA RA Pace*1,2, P Murthi1,2, N Castrechini1,2, SP Brennecke1,2, B Kalionis1. 1 University of Melbourne, Department Obstetrics & Gynaecology, Australia, 2Department of Perinatal Medicine, Pregnancy Research Centre, Royal Womens Hospital, Parkville 3052, Australia Introduction: Cultured mesenchymal stem cells derived from human placenta (PMSCs) are intensely studied because of their potential utility in regenerative medicine [1]. Our interest is in the potential contribution of PMSCs to the signicant pregnancy disorder of fetal growth restriction (FGR). In preliminary work (Castrechini et al, unpublished data), the gene ITGA1 showed altered expression in PMSCs from FGR-affected placentae compared with control term PMSCs. ITGA1 is an important cell adhesion molecule. In MSCs, ITGA1 regulates proliferation and is used as a colony forming unit (CFU) marker [2,3]. Here, we used siRNA technology to reduce expression of ITGA1 in PMSCs. Methods: Term placentae were obtained from healthy mothers with informed consent. PMSCs were obtained by mechanical and enzymatic digestion of chorionic villi, and plating onto plastic in specialised cell culture medium [4]. FACS analysis was performed on PMSCs with positive (CD105, CD73) and negative (CD45) markers [5]. Validated short interfering RNAs (siRNA) were used to reduce ITGA1 expression in PMSCs. Relative ITGA1 mRNA levels were determined by real-time PCR analysis. Results: FACS revealed 95-98% of PMSCs were positive for CD73 and CD105, and negative (<1%) for CD45. PMSCs were transfected with two siRNAs (ITGA1-si1 and ITAG1-si2). Real-time PCR analysis showed a significant relative reduction of ITGA1mRNA with both siRNAs [99.55%0.0018 SEM (si1) and 99.34%0.0013 SEM (si2)]. Apoptosis markers Bcl2 and Bax were tested by real-time PCR and there were no signicant differences between the siRNAs (ITGA1-si1 and ITAG1-si2) and the negative control suggesting that reduced ITGA1 expression did not alter expression of Bcl2 and Bax. Discussion: This is the rst study to successfully knockdown gene expression in PMSCs. Current studies are investigating the role of ITGA1 in cell migration, proliferation and adhesion. Bibliography: 1. Wulf, G.G., et al., Tissue Eng, 2004. 10(7-8): p. 1136-47. 2. Rider, D.A., et al., J Mol Histol, 2007. 38(5): p. 449-58. 3. Stewart, K., et al., Cell Tissue Res, 2003. 313(3): p. 281-90. 4. Battula, V.L., et al., Differentiation, 2008. 76(4): p. 326-36. 5. Brooke, G., et al., Br J Haematol, 2009. 144(4): p. 571-9. Keywords: Placental stem cells, FGR, Integrin
[P11.08]. PPARg REGULATES DIFFERENTIATION AND sFLT EXPRESSION DOWNSTREAM OF HYPOXIA IN MOUSE TROPHOBLAST STEM CELLS V Tache*1, A Ciric1, DS Milstone2, MM Parast1. 1University of California San Diego, United States, 2Brigham and Womens Hospital, United States PPARg is a ligand-activated transcription factor involved in many cellular processes, including inammation, metabolism, and differentiation. PPARg-null embryos die at midgestation due to placental abnormalities, the most severe of which is lack of formation of the labyrinth. We have previously derived trophoblast stem (TS) cells from both wild-type (WT) and PPARg-null mouse embryos. PPARg-null TS cells differentiate prematurely, and exclusively, into trophoblast giant cells (TGC), showing that PPARg is necessary for differentiation into labyrinthine trophoblast. In the current study, we investigated the relationship between oxygen tension and PPARg expression, since hypoxia inhibits trophoblast differentiation and is a causative factor in many obstetric complications involving placental dysfunction. Using qPCR analysis of a panel of markers, we evaluated differentiation patterns of WT and PPARg-null TS cells under both normoxia and hypoxia (2% oxygen). Our results show that PPARg expression is turned on when TS cells are switched to differentiation media, and is downregulated under hypoxia. In addition, PPARg was not required for hypoxia-induced inhibition of giant cell differentiation; however, when reintroduced into WT-TS cells differentiating under hypoxia, PPARg induced differentiation specically into labyrinthine trophoblast, as shown by upregulation of Gcm1 and syncytin-A. PPARgagonist (rosiglitazone) treatment of WT-TS cells inhibited giant cell differentiation under normoxia, and also specically inhibited hypoxiareoxygenation-induced expansion of spongiotrophoblast. Finally, PPARgnull TS cells showed highly upregulated sFlt expression, and rosiglitazone treatment of TS cells decreased sFlt secretion in a PPARg-dependent manner. We propose a model where HIF-induced downregulation of PPARg leads to inhibition of labyrinthine trophoblast differentiation and enhanced expression of sFlt, which has been associated with development of pre-eclampsia. We suggest PPARg as an excellent therapeutic target for hypoxia-associated obstetric complications, including pre-eclampsia and fetal growth restriction. Keywords: PPAR-gamma, Hypoxia, sFlt, trophoblast stem cells
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[P11.09]. HOMEOBOX GENE EXPRESSION IN PLACENTAL MESENCHYMAL STEM CELLS S Qin*1,2, RA Pace1,2, P Murthi1,2, SP Brennecke1,2, B Kalionis2. 1University of Melbourne, Department Obstetrics & Gynaecology, Australia, 2Department of Perinatal Medicine, Pregnancy Research Centre, Royal Womens Hospital, Parkville 3052, Australia Introduction: Mesenchymal stem cells (MSCs) are widely studied for their potential in regenerative medicine due to their ability to differentiate into multiple cell types and avoid allograft rejection. In embryonic stem cells, many important regulatory genes including the homeobox genes NANOG and OCT4 have been identied. Few studies of regulatory genes in MSCs have been reported. Previously in our laboratory, we screened for homeobox genes in placental MSCs (PMSCs) and identied 5 novel homeobox genes, DLX5, TGIF, MEIS2, HLX, and HEX, by real-time PCR. The objective is to examine the protein expression of these genes in PMSCs using immunocytochemistry. Method: The MSCs for this study were isolated by enzymatic digestion from normal term placentae (37-41 weeks, n5). Expression of stem cell markers on cultured PMSCs was veried by ow cytometry using the MSC positive markers CD105, CD73 and negative marker CD45. Immunocytochemistry was carried out with the appropriate primary antibody and colour detection was by AEC red. Results: PMSC preparations showed characteristic broblast-like morphology and were capable of forming CFU-F colonies. Greater than 90% of the cells expressed the markers CD105 and CD73, and less than 2% of the cells express CD45. DLX5 and TGIF show expression exclusively in the nucleus whereas in MEIS2, HLX, and HEX were predominantly expressed in the nucleus but also showed cytoplasmic expression. Discussion: We have conrmed at the protein level that novel PMSC homeobox genes are expressed in the nuclei of PMSCs. The role of the homeobox genes in PMSCs is not known, however TGIF and MEIS2 play roles in MSCs in other tissues. HLX is of particular importance since we have studied this gene extensively in the placental trophoblast cells where it regulates cell proliferation and migration but its role in PMSCs has not been determined. We are optimizing conditions for HLX inactivation in PMSCs and will investigate the effect on PMSC functions. Keywords: Placental stem cells, homeobox genes, FGR
ECFC:3.089x10-38.963x10-3 vs. 2.760x10-44.438x10-3, medianSD, p<0.05, n10). In perfused chorionic vessels, trackered ECFCs were shown to incorporate into the endothelial layer, whilst CACs were shown to invade the vessel wall and embed behind the endothelial lining and vascular intima. Discussion: These results suggest that fetal-derived EPCs (both ECFCs and CACs) migrate into the human placenta, where they play a potential role in placental vascularisation. (1) Duda et al. Nat Protoc. 2007;2:805. (2) Lin Y et al. J Clin Invest. 2000;105:71. This study was supported by the Wellcome Trust. Keywords: Endothelial progenitor cell, sequestration, placenta, vasculogenesis
[P12.02]. THE EFFECTS OF PLACENTAL MALARIA PARASITE AND MONOCYTE PRODUCTS ON SYSTEM A-MEDIATED AMINO ACID TRANSPORT IN BeWo CELLS EH Aitken*1, P Boeuf1, J Glazier2, S Rogerson1. 1Department of Medicine (RMH), University of Melbourne, Parkville, Australia, 2Maternal Fetal Health Research Group, University of Manchester, St Marys Hospital, Manchester, United Kingdom The pathogenetic mechanisms underlying the fetal growth restriction associated with placental malaria are unknown. However, in a pilot exvivo study of malaria-infected and control placentas from Malawi, we showed that low transcript levels of SNAT-1 (an isoform of amino acid system A transporter) were associated with placental-malaria, intervillositis and low birth weight (LBW). We therefore hypothesized that placental malaria infection and subsequent intervillositis leads to a decreased activity of system A amino acid transporters in the syncytiotrophoblast resulting in fetal growth restriction and LBW. To model the effect of placental malaria and intervillositis on system A activity, BeWo choriocarcinoma cells (as a model of placental trophoblast) were exposed to supernatant from a monocyte/malaria parasite co-culture or to cytokines previously reported to be associated with placental malaria. System A activity was measured as the uptake of 14C-labelled methylaminoisobutyric acid (MeAIB). As IL-1b has been associated both with decreased system A activity and LBW in placental malaria, we measured IL1b levels in placental serum from malaria-infected and uninfected women by ELISA, and related these to birth weight and pregnancy outcome. BeWo cells exposed to supernatant from a monocyte/parasite co-culture had decreased system A activity compared to the control. Exogenous IL-1b was effective in decreasing system A activity in BeWo cells, whereas other cytokines associated with placental malaria and LBW, including IL-8 and TNF-a, were not. IL-1b was increased in placental serum of women with placental malaria and intervillositis and IL-1b concentration was negatively associated with birth weight in this group. Taken together, these ndings suggest a pathogenetic mechanism for fetal growth restriction in placental malaria whereby malaria parasites and monocytes sequestered in the placenta lead to reduced transplacental amino acid transport, partly due to IL-1b secretion by monocytes, thereby reducing amino acid provision to the fetus. Keywords: malaria, low birth weight, intervillositis, amino acid transporter
[P11.10]. FETAL-DERIVED ENDOTHELIAL PROGENITOR CELLS ARE SEQUESTERED BY THE HUMAN PLACENTA P Sipos*, M Wareing, I Crocker, P Baker. Maternal and Fetal Redearch Center, The University of Manchester, United Kingdom Introduction: Endothelial Progenitor Cells (EPCs) are circulating cells produced by the bone marrow in the adult, which contribute to vascular repair and neovascularisation. There are two subtypes: Endothelial Colony Forming Cell (ECFCs), which are highly proliferative and develop into mature endothelial cells, and Circulating Endothelial Cells (CACs), haemopoietic cells which migrate into the intima of the forming vessel and regulate ECFC function. It is unknown whether EPCs play a role in placental vasculogenesis. It is also unknown whether EPC are produced by the placenta or the fetus. We therefore aimed to investigate whether EPCs migrate to the placenta from the fetus and become incorporated into placental vessels. Methods: We counted CAC and ECFC numbers in both arterial and venous umbilical blood from 10 newborns from uncomplicated pregnancies by 5 colour ow cytometry(1). ECFCs were recorded as CD31bright/CD45-/KDR+/CD34+ and CACs as CD31+/CD45/CD133+/CD34+. FcR blocker was used to prevent nonspecic binding and 7AAD to exclude non-viable cells. ECFCs and CACs were further expanded from fetal blood(2), trackered and perfused ex vivo into placental chorionic arteries, before culturing for 48 hours. Snap frozen, OCTembedded vessel sections were subsequently cut to determine EPC-localisation. Results: Numbers of CACs and ECFCs (as percent of mononuclears) were signicantly higher in the umbilical artery than vein (CAC:0.3750.388 vs. 0.2930.306, medianSD, p<0.01 Wilcoxon-paired t-test, n8;
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[P12.03]. EXPRESSION, LOCALISATION AND REGULATION OF ATP-BINDING CASSETTE TRANSPORTERS A1 AND G1 IN HUMAN GESTATIONAL TISSUES AND DIFFERENTIATING TROPHOBLASTS IL Aye*, BJ Waddell, PJ Mark, JA Keelan. University of Western Australia, Australia Objectives: The ATP-Binding Cassette (ABC) transporters ABCA1 and ABCG1 maintain cellular lipid homeostasis by transporting cholesterol, phospholipids and sphingolipids, as well as limiting the entry of toxic oxysterols. As major lipid regulators, these transporters have been implicated in diverse cellular processes including cell differentiation and apoptosis. Loss of ABCA1 function in mice leads to placental malformation, perturbed steroidogenesis, intrauterine growth restriction and neonatal death. Recently, efux of cholesterol via ABCA1 and ABCG1 was demonstrated in fetal endothelial cells of the human placenta. In this study, the expression of ABCA1 and ABCG1 was investigated in human term placental and extra-embryonic tissues. Furthermore, the expression and regulation of these transporters was also investigated in differentiating primary trophoblasts in vitro to determine their role in placental development. Methods & Results: Using quantitative PCR and immunoblotting analyses, ABCA1 and ABCG1 expression was detected in placenta and fetal membranes (amnion and choriodecidua). ABCA1 and, to a lesser extent, ABCG1 were localised by immunouorescence to the apical syncytiotrophoblast membrane. Endothelial cells of terminal villi also stained for ABCA1, while ABCG1 was prominent in vessels of stem villi. Both transporters were also present in amniotic epithelium, chorion and decidual tissues. ABCA1 and ABCG1 mRNA and protein expression increased with differentiation after 5 days in culture (approximately 2-6 fold), consistent with either an involvement in the syncytialisation process or a phenotypic expression marker. In culture, expression of both transporters in placental trophoblasts was stimulated by THE liver X receptor ligand T0901317 whereas peroxisome proliferator-activated receptor (PPARa and PPARg) ligands (GW7647 and rosiglitazone) and proinammatory cytokines (IL-1b and TNF-a) were ineffective. Conclusion: These ndings suggest that ABCA1 and G1 mediate cholesterol delivery to both the fetal and maternal circulation, in addition to as-yet undened roles in fetal membranes. Keywords: Transporter, Cholesterol, Differentiation, Lipid
[P12.04]. DIFFERENTIAL IMPACT OF MATERNAL FOOD RESTRICTION ON AQP1 AND AQP8 EXPRESSION IN RAT PREGNANCIES AT E16 L Belkacemi*, MH Beall, JT Lin, Q Liu, M Desai, MG Ross. Dept. of Obstetrics and Gynecology, David Geffen School of Medicine at UCLA and LABIOMED Research Institute at Harbor-UCLA Medical Center, CA, United States Introduction: Transplacental water ow, essential for maintenance of fetal body water and amniotic uid (AF), may be regulated in part by aquaporin (AQP) channels. Maternal food restriction (MFR) during pregnancy results in intrauterine growth restriction (IUGR) and IUGR is associated with oligohydramnios. Given the potentially crucial role of water ow in the placenta, we determined the effect of MFR during pregnancy on AF volume (AFV) and the placental expression of AQP1 and AQP8. We focused our study on the two placental positions (proximal and mid-horns) with the extremes of nutrient/oxygen supply, and the two distinct placental zones (basal, site of hormone production; and labyrinth, site of feto-maternal exchange). Methods: Pregnant rats were fed an ad libitum diet (AdLib) or were 50% MFR beginning at E10. At E16 rats were euthanized and gestational sacs from left and right mid- and proximal horns dissected. AFV was quantied as the difference in sac weight before and after drainage. AF osmolality was determined by freezing point depression. The placenta and fetus were separated and weighed. Six placentas from right mid- and proximal horns were separated into basal and labyrinth zones and analyzed for AQP1 and AQP8 protein expression. Results: MFR fetal weights from the proximal and mid-horns were not signicantly different from their respective AdLib. MFR placental basal zone weights from mid-horn placentas were signicantly decreased compared to AdLib. In contrast, MFR placental labyrinth zone weights from both mid- and proximal horns were not signicantly different from their respective AdLib. AFV was signicantly lower in the MFR from mid- and proximal horn sacs. Inversely, AF osmolality was signicantly increased in MFR from mid- and proximal horn sacs. AQP1 protein decreased signicantly in MFR basal zone from proximal horn and MFR labyrinth zone from mid- and proximal horn placentas. Inversely, AQP8 was increased in MFR basal and labyrinth zones from mid- and proximal horn placentas. Conclusion: MFR results in decreased AFV and increased osmolality prior to signicant fetal growth restriction. This change was accompanied with a reduction in AQP1 protein in the placenta suggesting a reduction in water ow. The increase seen in AQP8 expression may result in increased placental ow of solutes rather than water since AQP8 transport both water and ammonia. Keywords: Aquaporin, pligohydramnios, placenta, water
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[P12.05]. ZONAL AND POSITIONAL IMPACT OF MATERNAL FOOD RESTRICTION ON PLACENTAL AQP1 AND AQP8 EXPRESSION AT TERM GESTATION IN RAT L Belkacemi*, MH Beall, JT Lin, Q Liu, M Desai, MG Ross. Dept. of Obstetrics and Gynecology, David Geffen School of Medicine at UCLA and LABIOMED Research Institute at Harbor-UCLA Medical Center, CA, USA, United States Introduction: Transplacental water ow, essential for the maintenance of fetal body water and amniotic uid (AF), may be regulated in part by certain aquaporin (AQP) channels. Maternal food restriction (MFR) during pregnancy results in intrauterine growth restriction (IUGR) and IUGR is associated with oligohydramnios. Therefore, we hypothesize that impaired placental water ow in MFR results from dysregulation of placental AQP proteins. We assessed the effect of MFR during pregnancy on AF volume (AFV) and placental expression of AQP1 and AQP8 at E20. We focused our study on two uterine positions (proximal and mid-horns) with the extremes of nutrient/ oxygen supply. We also separately studied the basal placenta, site of hormone production and the placental labyrinth, site of feto-maternal exchange. Methods: Pregnant rats were provided either an ad libitum (AdLib) diet or were restricted to 50% of the food of ad libitum-fed animals (MFR) beginning at E10. At E20 rat dams were euthanized and gestational sacs from left and right mid- and proximal horns dissected. AF volume was quantied as the difference in sac weight before and after drainage and AF osmolality determined by freezing point depression. The placenta and fetus were separated and weighed. Six placentas from right mid- and proximal horns were separated into basal and labyrinth zones and analyzed for AQP1 and AQP8 protein expression (Western blot). Results: MFR fetuses from mid- and proximal horns weighed signicantly less than their respective AdLib. Additionally, MFR mid- and proximal horn placentas were signicantly smaller in the basal and labyrinth zones. AFV was signicantly decreased and osmolality was signicantly increased in the MFR as compared to AdLib from both mid- and proximal horn sacs. In MFR, AQP1 protein decreased signicantly in the placental basal zone from midhorn placentas and increased signicantly in the labyrinth zone from both mid- and proximal horn placentas. AQP8 was increased in MFR placental basal and labyrinth zones from both mid- and proximal horn placentas. Conclusion: The differential effects of MFR on AQP1 and AQP8 suggest that altered AQPs expression may contribute to reduced AFV and increased AF osmolality. Keywords: AQP, IUGR, placenta, transport
[P12.06]. THE EFFECT OF REDUCED SYSTEM BETA TRANSPORTER ACTIVITY ON CYTOTROPHOBLAST CELL DIFFERENTIATION IN VITRO L Parsons, SL Greenwood, M Westwood, M Desforges*. University of Manchester, United Kingdom Trophoblast cell turnover maintains a functional syncytiotrophoblast, capable of sufcient nutrient transfer from mother to fetus. Fetal growth restriction (FGR) is associated with aberrant trophoblast cell turnover1 and reduced activity of certain amino acid transporters, including system beta2. The system beta substrate, taurine, has a role in cell volume regulation3. In other tissues, cellular hydration state inuences proliferation, differentiation, apoptosis, and hormone secretion4. We therefore hypothesise system b-mediated taurine transport is important for trophoblast cell turnover. The objective of this study was to investigate cytotrophoblast cell differentiation following knockdown of system beta (TauT) using siRNA technology. Methods: Cytotrophoblast cells isolated from human term placenta were maintained in primary culture for 66 hours. Cells were transfected with siRNA at 18 hours of culture as described previously5. Knockdown of TauT mRNA (SLC6A6) was conrmed using QPCR and effects on system beta activity was determined by measuring sodium-dependent 3H-taurine uptake. Immunouorescent staining of desmosomes (allowing visualisation of multinucleation) and measurement of hCG secretion were used to assess morphological and biochemical differentiation respectively in cells with TauT knockdown compared to untransfected control cells. Results: Transfection of cytotrophoblast cells with 50nM TauT-specic siRNA signicantly reduced SLC6A6 mRNA expression and system beta activity (p<0.05 Wilcoxon signed rank, n5) whereas transfection with non-targeting siRNA, included as a negative control, had no signicant effect. Multinucleation was reduced by 20-60% (mean:37%, n4) in cytotrophoblast cells with TauT knockdown compared to untransfected controls, however there was no effect on hCG secretion (p0.6, Wilcoxon matched pairs, n5). Conclusion: Morphological, but not biochemical, differentiation of cytotrophoblast cells is compromised when system beta activity is reduced. This suggests that, as well as being directly important for fetal development, placental taurine transport is important for trophoblast cell turnover and normal placental development. References 1. Crocker et al (2004). J Pathol 204: 11-18. 2. Norberg et al (1998). Pediatr Res 44: 233-238. 3. Shennan DB (2008). Cell Physiol Biochem 21: 15-28. 4. Lang et al (1998). Physiol Rev 78: 247-306. 5. Forbes et al (2009). Placenta 30: 124-129. Funded by The Wellcome Trust. Keywords: taurine, syncytiotrophoblast, fetal growth restriction, placenta
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[P12.07]. MOLECULAR EXPRESSION OF NHE-3 IN HUMAN PREECLAMPTIC PLACENTAS V Dietrich*1, A Reca1, M Castro-Parodi1, B Maskin2, AE Damiano1. 1 Laboratorio de Biologa de la Reproduccion, Catedra de Biologa Celular, Facultad de Farmacia y Bioqumica. Universidad de Buenos Aires, Argentina, 2Hospital Nacional A. Posadas, Buenos Aires, Argentina The exchange barrier between maternal and fetal circulation in the human placenta consists, essentially, of two cellular layers, the syncytiotrophoblast (hST), and the fetal capillary endothelium. The hST, a multinucleated true syncytium, is the transporting epithelium of the placenta. Transport activity of the hST is essential for a supply of a range of solutes required for fetal growth, as well as homeostasis of the cell itself. The sodium hydrogen exchanger isoform 3 (NHE-3) plays an important role in electrolyte and water homeostasis. These functions are compromised in pregnancies complicated with preeclampsia. At present it is not known whether NHE-3 expression is altered during preeclampsia. We investigated the molecular expression of the NHE-3 in hST term placentas obtained from uncomplicated (n6) and preeclamptic pregnancies (n6). RT-PCR and Western blot assays showed that the expression of NHE-3 was signicantly reduced in hST from preeclamptic placentas. In normal placentas NHE-3 was localized in the apical and basal membranes and in the cytoplasm. However, in preeclamptic placentas NHE-3 was almost undetectable. Further studies are needed to determine whether the diminished expression of NHE-3 in preeclamptic placentas may compromise placental function and may contribute to the development of this syndrome. Keywords: NHE-3, syncytiotrophoblast, preeclampsia, human placenta
[P12.09]. CALCIUM AND LIPIDS ASSISTED TRANSPORT OF LEAD IN PLACENTA J. Foltinova*1, V. Foltin2, E. Neu3. 1Comenius University, Slovakia, 2Slovak Technical University, Slovakia, 3Umweltmedizin Institut, Germany Introduction: In recent years great attention has been paid to effect of heavy metals on the human organism from various points of view. Particularly, relations between lead, placenta and fetus are of special importance, since they are connected with the rise of hyperkinetic syndrome (ADHD) in children. Material and Methods: In this work we prepared and evaluated sections from excisions of placentas of 104 healthy patients. We carried out proof of Ca by the method after Koss, proof of Pb by the new methodical approach after Foltinova [1] and proof of lipids by histochemical methods. For evaluation we used light microscope Reichart Polyvar (Germany), scanning electron microscope PHILLIPS CM (Holland), infrared spectrostrometer SPECORD M80, Carl Zeiss, Jena (Germany) and JEOL JXA 840A (Japan) EDAX electron probe micro-analyzer. Results: Our results show that: a) calcium and lipids play a role of lead carriers, b) lead transport through placenta participates in the rise of hyperkinetic syndrome, c) expelling of iron from haemoglobin by lead leads to insufcient transport of oxygen through transplacental barrier, d) nding lead in placenta may help in preventing this disease that has increasing occurrence in the world. Histochemical results were confronted with the results of EDAX analysis and infrared spectroscopy. Conclusions: Signicant occurrence of lipids in syncytiotrophoblast and inside the villi of placenta in the places of lead occurrence shows that also lipids are carrier of lead in placenta. This explains why in case of hyperkinetic syndrome in children lead is cumulated in the striatum of brain which contains myelin in neurons. Finding of lead in placenta and umbilical cord blood can be utilized for prevention of the hyperkinetic syndrome. Relevance of this nding has the same value as the relevance of nding IgE for revealing allergic terrain of the newly born child. Reference [1] J. Foltinova, V. Foltin, E. Neu: Occurrence of lead in placenta important information for prenatal and postnatal development of child. Neuroendocrin. Letters 28 (2007) 335-340. Keywords: lead in environment, transport of lead in placenta, hyperkinetic syndrome
[P12.08]. ENDOSOMAL Ph REGULATION IN POLARIZED BeWo CELLS I. Ellinger*, R. Fuchs. Medical University Vienna, Austria Introduction: IgG transcytosis and apical IgG recycling in polarized BeWo cells are mediated by hFcRn. Following apical uptake by a uid phase mechanism, IgG enters apical early endosomes (AEE) from where it is predominantly recycled (40-50%), either directly or via transferrin receptorpositive recycling endosomes, as well as transcytosed (30-35%). High-afnity FcRn-IgG interaction is pH-dependent and requires mildly acidic endosomal pH (<6.5). As the pH of distinct endosomal subpopulations (AEE and basolateral early endosomes (BEE), late endosomes (LE)/lysosomes, recycling endosomes (RE)) in BeWo cells is completely unknown, but expected to vary, this was analysed using dextran (uid phase marker) and transferrin. Methods: Endosomal subcompartments were marked with FITC (pHdependent) and Cy5 (pH-independent)-conjugated dextran or transferrin. Single-organelle ow analysis (SOFA) was applied to determine the pH of labelled endosomes. Results: Following pulse-chase (15/5min) internalization from apical or basolateral side, the uid-phase marker dextran was delivered to perinuclear endosomes (common LE) with an average pH of 5.6-5.7. When internalized from the basolateral side for 5 min, dextran entered peripheral endosomes that exhibited an average pH of 6.7 (BEE). In contrast, following apical internalization for 5 min, dextran-marked endosomes had an average pH of 7.3 (AEE). This difference in pH among AEE and BEE might be explained by a larger fraction of apically internalized uid phase markers entering rapid recycling through more neutral compartments. Supportive to this concept, apically applied transferrin passed peripheral AEE and was recycled via perinuclear recycling compartments (RE). All endosomes labelled continuously for 20 min exhibited an average pH of 6.1; in subsequent pulse/chase experiments transferrin-labelled early (5 min) and late (20 min) endosomes exhibited low pH (below 6.0). This supports the existence of a sufciently low pH in AEE to allow for IgG-FcRn interaction. DISCUSSION: Our data for the rst time addressed pH-regulation in endosomal subpopulations of BeWo cells. Keywords: IgG, hFcRn, endosomal pH, BeWo
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[P12.10]. PLACENTAL EXPRESSION OF INSULIN-LIKE GROWTH FACTORS (IGFs), SYSTEM A AMINO ACID AND GLUCOSE TRANSPORTERS ARE DIFFERENTIALLY REGULATED BY PREGNANCY AND NUTRITION IN THE GUINEA PIG PA Grant*1, KL Kind1, A Sohlstom2, CT Roberts1, JA Owens1. 1University of Adelaide, Australia, 2Linkoping University, Sweden The placenta is a major determinant of fetal growth and development through the delivery of substrates and secretion of hormones. Maternal plasma IGFs increase during pregnancy and promote placental transport and nutrient partitioning near term. The effect of maternal food restriction in the guinea pig, on placental expression of IGFs, glucose transporters Slc2a1, Slc2a3 and system A amino acid transporters Slc38a2 and Slc38a4, fetal and placenta weight were examined at D30 and D60 of gestation (term w70 days). Guinea pigs were fed either ad libitum (AL) D30 (n10), D60 (n6) or restricted (R) D30 (n10), D60 (n10) (70% AL from 2 weeks before pregnancy to D34, then 90% AL to D60). Placental Slc2a1, Slc2a3, SLC38a2 and Slc38a4 mRNA were quantitated by real-time PCR. Placental Slc2a1 mRNA in AL(14x) and R(21x) (p<0.0001), IGF-I mRNA in AL(78x) and R(48x) and IGF-II mRNA expression in AL(95x) and R(85x) (p<0.0001) were decreased at D60 compared with D30 regardless of nutrition. Slc38a2 mRNA (3x) (p<0.05) was increased at D60 compared to D30 in AL fed animals. Feed restriction reduced Slc38a2 mRNA (2x) (p<0.05) at D60. Placental weight correlated negatively with placental Slc2a3 (p<0.01) and positively with Slc38a4 (p<0.01) in late pregnancy in AL animals. Placental and fetal weight were correlated positively with placental IGF-II mRNA (p<0.05) and Slc38a4 mRNA (p<0.01), respectively in late pregnancy in R animals. Placental weight correlated positively with placental Slc2a1 mRNA (p<0.05) and negatively with Slc38a2 mRNA (p<0.02) in early pregnancy in AL animals. This shows there are opposing gestational changes in expression of IGFs and glucose transporters compared to amino acid transporters in the placenta and that their associations with placental weight change with increasing gestation. Keywords: Insulin-like Growth factors, Glucose transporters, System A amino acid transporters, Nutrition
[P12.11]. CORRELATION BETWEEN hCG, cAMP AND AQP9 IN HUMAN PLACENTA G Marino*1, M Castro-Parodi1, E Zotta2, AE Damiano1. 1Laboratorio de Biologa de la Reproduccion, Catedra de Biologa Celular, Facultad de Farmacia y Bioqumica. Universidad de Buenos Aires., Argentina, 2Labo ratorio de Fisiopatogenia, Departamento de Fisiologa, Facultad de Medicina, Universidad de Buenos Aires., Argentina Since trophoblastic abnormalities play a central role in the pathophysiology of preeclampsia, it is conceivable that some placental hormone proles such as an increased secretion of human chorionic gonadotropin (hCG) are modied in the maternal circulation, which could affect the placental function. We have described in preeclamptic pregnancies that there are elevated levels of serum hCG with an increased aquaporin-9 (AQP9) expression in the syncytiotrophoblast (hST). Several reports indicate that AQP9 expression may be mediated by some factors induced by cAMP. In addition hCG signal transduction usually involves cAMP signaling. Here, we are focused on the hypothesis that there is a correlation between the increased levels of hCG and AQP9 expression in preeclamptic placentas, involving a cAMP dependent pathway. Normal placental explants were cultured at different times with 20 mIU/ml of recombinant hCG and 100-500 mM 8-bromo-cAMP (8-Br-cAMP), a potent cAMP analogue. Semiquantitative Western blot and immunohistochemistry were performed to evaluate AQP9 expression and localization. In Western blots we observed a stimulatory effect on AQP9 protein expression after hCG treatment. AQP9 was localized in the apical membrane of hST and in the cytoplasm, and similar results were obtained with placental explants incubated with 8-Br cAMP, this effect being time and dose dependent. Our results suggest that hCG may be implicated in the regulation of AQP9 expression involving a cAMP dependent pathway in hST. Keywords: Aquaporin 9, hCG, syncytiotrophoblast, cAMP [P12.12]. THE DISTRIBUTION OF CAVEOLAE AND CAVEOLIN-1 IN THE MOUSE PLACENTA AND YOLK SAC S. Mohanty, C.L. Anderson, J.M. Robinson*. The Ohio State University, United States Introduction: Caveolae are specialized microdomains in the plasma membrane and are found in several cell types. Caveolae are characterized biochemically by the presence of members of the caveolin protein family. We have studied the mouse placenta and yolk sac to determine if caveolin-1 is expressed in these tissues and if so what cell types express this protein. Methods: The methods employed in this study were immunoblotting of tissue lysates and immunouorescence microscopy of cryostat sections of intact tissue to determine if caveolin-1 is expressed and which cell types contain this protein. Additionally, we have employed electron microscopy of plastic-embedded tissue samples to study the distribution of caveolae. Results: Immunoblot analysis showed that both placental and yolk sac lysates contain caveolin-1. Immunouorescence analysis showed that endothelium, smooth muscle cells, and mesothelium contain caveolin-1 in yolk sac but endoderm lacks this protein. The labyrinth of the mouse placenta is enriched in caveolin-1. However, the distribution of this protein is restricted to endothelial cells. Caveolin-1 was not detected in any of the three trophoblast layers. Yolk sac and placenta were also examined by electron microscopy. Ultrastructural analysis showed morphologicallydened caveolae were present in both yolk sac and placenta. The cells types expressing caveolae were the same cells as those identied as being positive for caveolin-1 in the immunouorescence experiments. Discussion: We have identied the cells types expressing caveolae and caveolin-1 in mouse yolk sac and placenta. Morphologically-dened caveolae were present only in cell types expressing the protein caveolin-1. These data on caveolin-1 and caveolae in the placental labyrinth parallel those from the human placenta since trophoblasts are negative while endothelial cells are positive. Keywords: Caveolae, Caveolin-1, Yolk sac, Placenta
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[P12.13]. MODEL FOR OXYGEN TRANSPORT IN THE HUMAN PLACENTA JS Gill*1, DS Grebenkov2, CM Salaa3,4, DD Vvendensky1. 1Imperial College, United Kingdom, 2Ecole Polytechnique, France, 3Placental Analytics LLC, United States, 4Institute for Basic Research, United States Goal/Background: The mature placenta is a complex vascular network extending ultimately to the capillary beds of the terminal villi, the sites of all oxygen and nutrient exchange between the mother and fetus. Respiratory transfer across the placenta to the fetus occurs in three steps: (i) maternal blood bathes the chorionic villi in oxygen, (ii) oxygen permeates the villus surface and diffuses into the fetal capillaries, and (iii) oxygen is transported to the fetus via the fetal bloodstream. Materials/Methods: Step (ii) has been modelled by the diffusion of oxygen from the villous membrane and the fetal capillaries. The stationary concentration c(x,t) of oxygen within each of the villi is the solution of the two-dimensional Laplaces equation, Oc 0, with a xed concentration cv at the villous surface and a Robin boundary condition at the capillary boundary: Dvc/vn Kc, where v/vn is outward normal derivative, D is the oxygen diffusion constant and K is the permeability of the capillary. These equations are solved in regions [panels (d,e,f)] determined by the villus and capillary boundaries obtained from digitized images [panels (a,b,c)]. Results: The solution for the oxygen concentration determines the diffusive current of oxygen across the capillaries. Many factors are expected to inuence this current, including the numbers and shapes of villi and capillaries. Our initial analysis for the sections below indicates that panel (c) yields the largest ux per unit perimeter length and area of the villi, followed by panel (b) and then panel (a). Conclusions: The geometrical shapes and spatial distributions of the villi and capillaries are important placental characteristics for the transport of oxygen to the fetus. Once the main factors that determine oxygen transport have been precisely identied, this approach, applied to digitized placental slides that allow analysis of many hundreds of villi per slide (and multiple slides per placenta) should provide a quantitative basis for discriminating between normal and abnormal.
[P12.14]. INSULIN INCREASES EXPRESSION AND ACTIVITY OF EQUILIBRATIVE NUCLEOSIDE TRANSPORTER 2 IN HUMAN PLACENTA MICROVASCULAR ENDOTHELIAL CELLS FROM GESTATIONAL DIABETES Carlos Salomon*, Francisco Westermeier, Paola Casanello, Luis Sobrevia. Ponticia Universidad Catolica de Chile, Chile Human endothelial cells from placenta microcirculation (hPMEC) express functional equilibrative nucleoside transporters 1 (hENT1) and 2 (hENT2) in culture. ENTs-mediated adenosine transport is regulated by insulin in human umbilical vein endothelium from normal or gestational diabetic (GD) pregnancies, but no information is available regarding the effect of this hormone in hPMEC. We studied insulin effect on hENT2 expression and activity in hPMEC from GD pregnancies. Methods: Primary cultures of hPMEC (passage 2) from normal or GD pregnancies were cultured under standard conditions. hENT2-mediated adenosine transport (0-500 mM adenosine, 4 mCi/ml [3H]adenosine, 22 C), hENT2 protein abundance and mRNA level were determined. Results: Insulin increased hENT2 mediated adenosine transport by w1.5fold, but only w1.2-fold in hPMEC from normal compared with GD pregnancies, respectively. Basal expression of hENT2 (protein and mRNA) was not signicant different in cells from normal or GD pregnancies. Insulin increased in a concentration-dependent manner hENT2 protein abundance (half-maximal effect (SC50) w1 nM), mRNA level (SC50 w1.2 nM) and transport activity (SC50 w1.1 nM) in hPMEC. However, all insulin effects were higher (w1.4-fold) in cells from normal compared with GD pregnancies. Conclusion. Human placenta microvascular endothelium is less responsive to insulin regarding hENT2 expression and activity. This phenomenon could partially explain the insulin resistance exhibited by some fetuses from gestational diabetes. Supported by FONDECYT 1070865/1080534. C.S. holds a Faculty of Medicine, PUC-PhD fellowship. F.W. holds a CONICYT-PhD fellowship. Keywords: Adenosine, Gestational Diabetes, hPMEC, ENTs
Keywords: diffusion, oxygen ux, placental capillaries, terminal villi
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[P12.15]. HIGH D-GLUCOSE INHIBITS EQUILIBRATIVE NUCLEOSIDE TRANSPORTER 1 (hENT1) ACTIVITY AND EXPRESSION BY ACTIVATION OF TYPE II RECEPTOR FOR TRANSFORMING GROWTH FACTOR 1 (TGF-1) IN HUMAN FETAL ENDOTHELIUM Jose Luis Vega*, Carlos Puebla, Paola Casanello, Luis Sobrevia. Cellular and Molecular Physiology Laboratory (CMPL) and Perinatology Research Laboratory (PRL), Department of Obstetric and Gynaecology, Medical Research Centre (CIM), School of Medicine, Ponticia U, Chile Introduction: The transforming growth factor 1 (TGF-1) inhibits expression and activity of hENT1 in a nitric oxide (NO)-dependent manner in human umbilical vein endothelial cells (HUVEC). High extracellular Dglucose (25 mM) also increase the release of TGF-1 from primary cultures of HUVEC leading to altered transport of L-arginine and NO synthesis, most likely due to activation of type II TGF-1 receptors (TRII). We have studied whether TGF-1 is involved in the inhibitory effect of high D-glucose on hENT1 activity and expression in HUVEC. Methods: Cells were exposed to D-glucose (5-25 mM, 6-24 h) and/or TGF1 (2 ng/ml, 6 h) and [3H]adenosine transport (4 mCi/ml, 20 sec, 22oC) was measure in absence or presence S-(4-nitrobenzyl)-6-thio-ionosine (ENT1 inhibitor). hENT1 mRNA was estimated by semi-quantitative PCR and protein level by western blot. The TGF-1 or high D-glucose effect on reporter activity of plasmid constructs containing a promoter region of SLC29A1 (-1114 bp to ATG, pGL3-hENT1-1114) was also analyzed. All assays were performed in cells transduced with an adenovirus to induce expression of a truncated form of TRII (Ad-tTRII) or with a control adenovirus (Ad-control, empty vector). Results: D-glucose reduced in a dose-dependent manner adenosine transport via hENT1, mRNA expression, protein abundance, and pGL3hENT1-1114 transcriptional activity. TGF-1 effects were absent in cells transduced with Ad-tTRII. Conclusion: High D-glucose reduced hENT1 activity and expression requires activation of native type II receptor for TGF-1 in primary cultures of HUVEC. FONDECYT 1070865/1080534, CONICYT AT-24090199 (Chile). JL Vega and C Puebla hold CONICYT-PhD fellowships. Keywords: High D-glucose, hENT1 , TGF-1, Human Fetal Endothelium
[P12.16]. INSULIN RECEPTOR ISOFORMS -11 (IR-A) AND +11 (IR-B) ARE EXPRESSED AND COULD MEDIATE INSULIN-INCREASED EXPRESSION AND ACTIVITY OF THE EQUILIBRATIVE NUCLEOSIDE TRANSPORTER TYPE 2 IN HUMAN UMBILICAL VEIN ENDOTHELIAL CELLS FROM PREGNANCIES WITH GESTATIONAL DIABETES Francisco Westermeier*, Carlos Salomon, Paola Casanello, Luis Sobrevia. Ponticia Universidad Catolica, Chile Adenosine is an endogenous nucleoside that is taken up by the human umbilical vein endothelial cells (HUVEC) via equilibrative nucleoside transporters (predominantly hENT1 and hENT2). Both the expression and activity of hENT1 are decreased in HUVEC from gestational diabetes (GD), as well as in HUVEC exposed to insulin. However, insulin increases adenosine transport in HUVEC from normal pregnancies and regulates SLC29A2 (for hENT2) gene expression. We studied insulin effect on adenosine transport mediated by hENT2 in HUVEC from pregnancies with GD. Methods: HUVEC from normal and GD were exposed (8 hours) to insulin (0.1-10 nM). [3H]Adenosine transport (4 mCi/ml, 20 s, 22 C) was measured in absence or presence of nitrobenzylthioinosine (NBTI, 0.01-10 mM, 30 min). SLC29A2 transcriptional activity was estimated by rey and renilla luciferase activity from two upstream sequences (1500 and 600 bp from the ATG) subcloned into pGL3-basic vector. Insulin receptors isoforms -11 (IR-A) and +11 (IR-B) mRNA level were determined. Results: Insulin increases hENT2-adenosine transport (SC50 w0.53 and w0.51 nM), protein abundance (SC50 w0.78 and w0.82 nM) and mRNA level (SC50 w3 and w3 nM) were increased by insulin in cells from normal and GD pregnancies, respectively. pGL3-hENT2-1500 and pGL3-hENT2-600 luciferase promoter activity was similar in cells from normal and GD pregnancies. Only the pGL3-hENT2-1500 promoter activity was increased by insulin in a similar pattern in cells from normal and GD pregnancies. IRA mRNA level was higher (w1.3-fold, P<0.05) in GD compared with normal pregnancies. However, IR-B mRNA level was similar in both conditions. IR-A was higher that IR-B mRNA levels in normal (w1.5-fold) and GD (w1.8-fold) pregnancies. Conclusion: Insulin could increase adenosine transport via hENT2 by increasing expression of this transporter isoform in HUVEC from GD and normal pregnancies. In addition, GD is a pathological condition where the placenta/fetal tissue-predominat isoform IR-A, could play a critical role in insulin signalling. Supported by FONDECYT 1070865/1080534 (Chile). F. Westermeier holds a CONICYT-PhD fellowship. C. Salomon holds a Faculty of Medicine, PUC-PhD fellowship. Keywords: Gestational Diabetes, Insulin receptor, Human equilibrative nucleoside transporter, Adenosine
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[P13.01]. SINGLE NUCLEOTIDE POLYMORPHISMS IN ANGIOGENESIS REGULATING GENES ARE ASSOCIATED WITH PREGNANCY COMPLICATIONS PH Andraweera*, SD Thompson, RC Novak, VJ Zhang, GA Dekker, CT Roberts. University of Adelaide, Australia Introduction: Angiogenesis is crucial for normal placentation and when deranged is implicated in pregnancy complications. Vascular endothelial growth factor (VEGF) is a potent pro-angiogenic factor and thrombospondin1 (THBS1) an anti-angiogenic factor. We aimed to determine whether functional polymorphisms in VEGF (VEGF C2578A and VEGF C936T) and THBS1 (THBS1 A2099G) genes are associated with pregnancy complications. Methods: 1380 nulliparous pregnant women and their partners were recruited prospectively at the Lyell McEwin Hospital and women monitored throughout pregnancy. Gestational hypertension (GHT), Preeclampsia (PE), Small for gestational age (SGA) and PE+SGA pregnancies were classied using strict guidelines. Uncomplicated pregnancies were selected as controls. Peripheral blood was collected from couples and cord blood collected at delivery. DNA extraction from buffy coats and genotyping were performed at the Australian Genome Research Facility using the Sequenom MassARRAY system. To date the rst 700 mother, father, baby trios have been genotyped. Genotyping results for GHT (n58), SGA (n53), PE (n37), PE+SGA (n11) were compared with controls (n250). Data were analysed using ANOVA and Chi Square. Likelihood Ratios (LR) were calculated. Results: Neonatal VEGF C2578A was associated with PE (p0.007, LR 10.3) and neonatal VEGF C936T with SGA (p0.029, LR6.3). Maternal THBS1 A2099G was associated with GHT (p0.027, LR6.0), paternal THBS1 A2099G was associated with PE+SGA (p0.036, LR6.3) and neonatal THBS1 A2099G was associated with SGA (p0.038, LR6.2). Neonates of fathers with THBS1 A2099G GG genotype were 701g lighter (p0.026) and 4.8cm shorter (p0.001) than those of GA and 785g lighter (p0.008) and 4.9cm shorter (p0.001) than those of AA. Maternal VEGF C2578A polymorphism was associated with the urinary protein:creatinine ratio. Conclusion: Our results suggest that VEGF C2578A, VEGF C936T and THBS1 A2099G gene polymorphisms are associated with pregnancy complications. Ongoing research will determine the role of these polymorphisms in placental angiogenesis and function. Keywords: angiogenesis, vascular endothelial growth factor, thrombospondin1, polymorphisms
[P13.02]. PREDICTION OF PREECLAMPSIA WITH ELEVATION IN ERYTHROBLAST COUNT IN MATERNAL BLOOD Fatemeh.* MD Davari Tanha*, Jalleh Mohammad pour, MD , Mahbod Kaveh. Tehran University Medical Sciences, Republic of Islamic, Iran Introduction: The predominant etiologic theory of preeclampsia is that reduced uteroplacental perfusion is the unique pathogenic process in the development of preeclampsia. Maternal and fetal erythroblast counts are elevated in the peripheral blood of pregnant women with preeclampsia. The purpose of this study was to examine whether this elevation actually occurs before the clinical onset of the disorder. Study Design: In a prospective cohort survey erythroblasts were enumerated in 599 maternal blood samples obtained in 19-26 weeks with singleton pregnancy.After complete blood count a peripheral blood smear was done and erythroblast was counted , and results were subsequently correlated with pregnancy outcomes. The data were analyzed by SPSS 13.0.Independent sample t-test and Fishers exact test was used. A p value of <0.05 was considered statistically signicant. Results: Signicantly higher quantities of erythroblasts (mean 2.461.23 vs 0.440.55;p0.009)were detected in blood samples obtained from women who later acquired preeclampsia (n50) than in blood samples from the control Cohort (n549). Intrauterine growth restriction was accompany by a similar rise in erythroblast count(mean NRBC 0.820.8 in preeclamptic group vs 0.590.85 in normotensive group;p0.009). Mean gestational age was less in preeclamptic group(37.581.45 vs 39.070.94, p0.009). On the basis of 1.5 erythroblast as point of convergence there was sensitivity 61.45, specicity93.02, NPV98.16, accuracy91.65 Conclusion: Because a large proportion of the erythroblasts in maternal blood are fetal origin, our data suggest that fetal-maternal cell trafc is affected early in pregnancies that are later complicated by preeclampsia. Keywords: erythroblasts, preeclampsia, IUGR, fetal cell trafc
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[P13.04]. PLASMINOGEN, THE PLACENTA AND PREGNANCY OUTCOME RC Nowak*, SD Thompson, J Zhang, GA Dekker, CR Roberts. Research Centre for Reproductive Health, Australia Introduction: Plasminogen (PLG) has been shown to play a crucial role in placental development. Poor placental development is associated with several potentially life-threatening pregnancy complications, including preeclampsia (PE) and small-for-gestational-age (SGA) babies. Aim: To determine if two single nucleotide polymorphisms (SNP) within PLG, rs28598789 and rs425114, are associated with pregnancy outcome. Method: First trimester placental tissue was collected from women undergoing termination of pregnancy to determine PLG expression in placental villous tissue. To examine genotype with pregnancy outcome, blood was collected prospectively from pregnant women at 15 weeks gestation, their partners and neonates at birth. DNA, extracted from blood, was genotyped by the Australian Genome Research Facility with the use of Sequenom MassArray technology. Pregnancies were fully characterised and classied into control, PE or SGA after delivery by an experienced obstetrician. Detailed clinical information was collected from each participant. Results: PLG is expressed in placenta throughout rst trimester , with no difference in mRNA levels across 6-12 weeks gestation or between sexes. In women, the frequency of the PLG rs28597879 TT genotype was increased in those who developed PE (n41) compared to controls (n285, p0.011, LR 6.43 p0.016). This genotype was also positively associated with the maternal birthweight (p0.024). Likewise there was an increase in the frequency of the rs425114 C-allele in women (n41, p0.007 LR 7.9) and men (n30, p0.019 LR 6.2) who parented a PE pregnancy compared to controls (n282 and n240 respectively). This same increase was also observed in neonates who were SGA (n51, p0.014 LR 6.2) compared to controls (n241). Conclusion: This is the rst study to show an association between these polymorphisms of PLG and pregnancy outcome. PLG is expressed in placenta during rst trimester and functional SNPs within PLG may be associated with altered placental development and subsequent pregnancy outcome. Keywords: Genotype, Plasmiogen, Pregnancy outcome
[P13.06]. EXPRESSION OF ANGIOGENIC FACTORS IN PREECLAMPSIA Ji Kwon Park*, Jong Chul Baek, Jeong Kyu Shin, Won Jun Choi, Soon Ae Lee, Jong Hak Lee. Gyeongsang National University, Republic of Korea Elevated expression of soluble vascular endothelial growth factor receptor1 (sFlt-1), an antiangiogenic protein, in preeclampsia plays a major role in the pathogenesis of this serious disorder of human pregnancy. Although reduced placental oxygenation is thought to be involved in the pathogenesis of preeclampsia, it is unclear how oxygen regulates placental sFlt-1 expression. The aims herein were to investigate sFlt-1 expression in in vivo and in vitro physiological and pathological models of human placental hypoxia and to understand the role of phosphatidylinositol-3-kinase (PI3K) pathway in regulating the expression of this molecule. I would like to examine placental sFlt-1 and VEGF expression in preeclamptic pregnancies (in vivo pathological hypoxia) and models of placental hypoxia (Human choriocarcinoma trophoblast cells with hypoxiainducing chemical agents). Additionally, using models of placental hypoxia, we have investigated the mechanisms by which reduced oxygenation (in vitro hypoxia) regulate sFlt-1 expression. Finally, we have determined whether PI3K pathway, in particular PI3K, Akt, HIF-1, plays a direct role in regulating the expression of sFlt-1 in the models of placental hypoxia. Through this study, exploration of some agents that neutralize the excess sFlt-1 may also be promising therapeutic modalities in patients with preeclampsia. Keywords: Preeclampsia, Human choriocarcinoma trophoblast cells, sFlt, PI3K
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[P13.08]. COLLAGEN INTEGRITY OF THE UTERINE CERVIX CORRELATES WITH AMNIOTIC FLUID CYTOKINE LEVELS, PROVIDING A NOVEL METHOD TO DETECT INTRA-AMNIOTIC INFLAMMATION SM Keeler*1, O Rust2, E Bornstein1, M Sottile3, CM Salaa4. 1New York University, United States, 2Lehigh Valley Hospital, United States, 3University of Oregon, United States, 4Placental Analytics LLC, United States Objective: To investigate the correlation between the histological staining characteristics of cervical collagen and amniotic uid cytokines in women with midtrimester short cervix. Study Design: Amniotic uid (AF) and cervical biopsy samples were obtained from women with a midtrimester transvaginal cervical length .25mm. AF cytokine concentrations were quantied. From a routine stained slide, one 40x photomicrograph was selected from each specimen by an observer blinded to the amniotic uid cytokine levels. The eld was selected to be at the apparent mid point of the biopsy and to ll the 40x eld of view. The photomicrographs were analyzed using RGB channels; the G and B channels were discarded due to the predominance of R channel variance given the normal eosinophilia of collagen. The red channel histogram was extracted and aspects of its distribution, including mean, standard deviation, skew and kurtosis, were calculated. Since these variable would reasonably be intercorrelated, a single collagen staining factor score (CFS) was extracted from these data by principal components analysis and compared to the cytokine concentrations using Spearmans correlation. A high score represents impaired cervical integrity with the biopsy containing loose, poorly interconnected collagen brils and has an increased amount of white space in the image. A low score represents normal dense, closely interconnected collagen brils, with very little white space in the image. Results: Thirty-three paired AF and cervical biopsy specimens were analyzed. A signicant association was detected between the collagen score and AF IL-6 (p0.04), IL-8 (p0.03), Eotaxin (p0.04), IP-10 (p0.04) and MCP-1 (p0.02) levels. Conclusion: Analysis of collagen integrity on H&E stained slides by a semiquantitative scoring system had previously proved fruitless in this data set (Salaa CM, unpublished observations). A simple R channel analysis allows extraction of collagen structural features that highly correlate with inammatory cytokines, suggesting that intra-amniotic inammation and the structural integrity of the cervix are related. This allows an aspect of cervical integrity to be reliably and reproducibly quantied, that previously could not be assessed on histology slides.
[P13.09]. JELLY-LIKE HETEROGENEOUS PLACENTA AS PREDICTOR OF ADVERSE PREGNANCY OUTCOME. SOME CLINICAL, ULTRASOUND, AND MORPHOLOGICAL FEATURES O. Solovyov*1, T. Zadorozhna2, I. Sudoma1,3, Y. Goncharova1. 1Reproductive Medicine Clinic Nadija, Ukraine, 2Institute of Pediatrics, Obstetrics and Gynecology, Academy of Medical Sciences of Ukraine, Ukraine, 3National medical Academy of Postgraduate Education named after P.L. Shupyk, Ministry of Health of Ukraine, Ukraine Objective: to investigate outcome of pregnancies with sonographic ndings of thick and heterogeneous placenta, comparing histopathological, clinical and Doppler data. Methods: a series of 13 cases were analyzed. A jelly-like placenta was dened as a thick heterogeneous placenta with a layer of decreased echogenecity with visible slow swirling movements of contents, which quivered like jelly to ultrasound probe jerks. Doppler evaluation of the placenta and uteroplacental circulation was performed. The pregnancy course and neonatal outcome of present cases were reviewed. Morphological evaluation of placenta after delivery was done. Results: Perinatal death occurred in 5 cases (38,5%). In all these cases the uterine artery Doppler was abnormal or secondary normalized after previously increased resistance for uterine arteries blood ow. In 4 cases (30,8%) neonatal outcome was good. In these patients uterine arteries Doppler was normal during pregnancy. In 4 women with boundary uterine Doppler indices neonatal outcome was satisfactory but with low-birthweight babies. Morphologically typical vascular changes were noticed, namely plasmorragia, strokes and thrombosis, local blood stasis in the vessels of decidua, villi and chorionic lamina, full arteries obliteration of some stem villi of II and III line. The presence of distally located parts of villi xed in brinoid, pushed aside from intervillous blood ow, narrowing of intervillous space due to intervillous strokes, thrombosis, conglomerates of villi xed by brinoid and presence of thin bres structures with severe edema were seen. Conclusions: the sonographic presence of thick heterogeneous jellylike placenta is strongly associated with an adverse pregnancy outcome, especially in case of abnormal uterine artery Doppler or secondary normalisation after previously abnormal one. Keywords: jellylike, placenta
Keywords: short cervix, cervical collagen, cytokines, intra-amniotic infection
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[P13.10]. DYNAMICS OF BIOPYRRINS (BPn) AS A MARKER OF PSYCHOLOGICAL STRESS IN PREGNANT WOMEN ACCORDING TO THE MODE OF DELIVERY M.S. Suzuki*, R.K. Kawaguchi, T.T. Tanemoto, N.U. Umehara, S.W. Wada, K.O. Ohura, T. Onda, S. Isonishi, K. Ochiai, T. Tanaka. Jikei University School of Medicine, Japan Objectives: BPn, one of the bilirubin oxidative metabolites, are generated from bilirubin as a result of this scavenging action against free radicals. The level of BPn in urine reects oxidative stress and has been reported to serve as a marker of psychological stress. We focused on the dynamics and signicance of BPn in intra-partum women with special attentions to the manner of delivery. Methods: Subjects were composed of full-term pregnant women without any complication, and classied into 4 groups : normal vaginal delivery without any intervention (ND), delivery with the administration of oxytocin (OX), cesarean section (CS), and delivery with epidural anesthesia plus oxytocin (EOx). Spot urinary samples were collected before and after the delivery, and the BPn levels were compared. This study was approved by the Institutional Review Boards of The Jikei University, and the Clinical Study in Jikei-Aoto Hospital. And the informed consent was obtained from all patients registered. Results: Cases of 57(ND), 34 (OX), 37(CS), and 11(EOx) were analyzed. The mean age and gestational week of those were 31.95.4 (meanSD) years and 38.52.1. No signicant difference was found in age, gestational week and other factors, such as BMI, intra-partum hemorrhage, duration of the delivery among the group. The BPn levels before the onset of delivery were not signicantly different. Although signicant elevation after the delivery was found in ND-group and OX-group (1.3 and 2.5 fold), no statistically different was seen between before and after the delivery, in CS- and EOxgroup. Moreover, BPn levels in EOx-group were clearly elevated compared with other delivery-modes with the signicant difference. Conclusion: These data suggested that BPn is also useful marker in pregnancy. Researchs concerning the existence of BPn in placenta and their clinical applications for predicting obstetrical disorders are in progress. Keywords: biopyrrin, delivery, oxidative stress
[P13.11]. STATUS OF ESSENTIAL FATTY ACID, LONG CHAIN POLYUNSATURATED FATTY ACID AND TRANS FATTY ACID IN MATERNAL AND FETAL BIOLOGICAL COMPARTMENTS OF PREGNANT ADOLESCENTS ORC Oliveira, MG Santana, FC Domingues, FLC Sardinha, GV Veiga, MG Tavares do Carmo*. Universidade Federal do Rio de Janeiro, Brazil Introduction: In Brazil, 21% of the babies born in the public health system come from adolescent mothers. Studies of the fatty acids composition of maternal and fetal placental tissues have been conducted in adult pregnant women, but there is a remarkable lack of similar studies in pregnant adolescents. In this study, we investigated the fatty acids composition of maternal and fetal placental tissues, as well as in maternal and umbilical cord plasma, in 30 Brazilian adolescent healthy mothers. Methods: Levels of Trans fatty acids (tFA), essential fatty acids (EFAs), arachidonic (20:4,n-6 AA) acids, eicosapentaenoic (20:5, EPA) and docosahexaenoic (22:6, DHA) acids of the n-3 family were measured by gas-liquid chromatography. Results were expressed as a percentage of total fatty acids. Results: We found that the total concentrations of AA, EPA and DHA were higher in cord than in maternal plasma. In addition, we observed a decrease in AA and EPA concentration in cord plasma compared to fetal placental tissues. The percentage of EFAs and tFAs were lower in cord than in maternal plasma. Furthermore, the level of DHA was inversely related to linoleic acid concentration in fetal placental tissues. Discussion: We found higher accumulation of AA, EPA and DHA in cord plasma. This fact provides evidence of a selective long chain polyunsaturated fatty acid transfer across the placenta. We speculate that the increased conversion of AA to the eicosanoids and the DHA transfer to the fetal circulation for neurodevelopment may be responsible for the decreased concentrations of of these fatty acids in cord plasma compared to fetal placental tissues. In a future study, we intend to compare the placental fatty acid transfer in adolescent and adult mothers. Keywords: fatty acids, maternal anda fetal placental, pregnant, adolescents
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[P13.12]. IGF2 AND IGF2AS POLYMORPHISMS ASSOCIATED WITH ADVERSE PREGNANCY OUTCOMES S.D. Thompson*1,2, R.C. Nowak1,2, J.V. Zhang1,2, G.A. Dekker1,2, C.T. Roberts1,2. 1Research Centre for Reproductive Health, Australia, 2Discipline of Obstetrics and Gynaecology, University of Adelaide, Australia Objectives: IGF-II is an important determinant of trophoblast invasion, differentiation and subsequent placental function and thereby promotes fetal growth. IGF2 antisense (IGF2AS) overlaps IGF2 in the opposite orientation and is highly expressed in rst trimester chorionic villi. IGF2 and IGF2AS are imprinted and expressed from the paternal allele. Aim: To determine whether maternal, paternal or fetal polymorphisms of IGF2 and IGF2AS are associated with common pregnancy complications. Methods: 586 nulliparous caucasian couples who required no fertility treatment were recruited prospectively from the Lyell McEwin Hospital, Adelaide, Australia. Pregnancy outcomes were classied into healthy (n261), preeclampsia (PE, n38), gestational hypertension (GH, n51), small-for-gestational-age (SGA, n60) or preterm birth (PTB, n32) by an experienced obstetrician. Four single nucleotide polymorphisms were investigated, IGF2 rs680 and rs3741204 and IGF2AS rs1004446 and rs1003484. DNA was extracted then genotyped by Australian Genome Research Facility using the Sequenom massARRAY System. Data were analysed using Chi Square and Odds Ratios (OR). Results: In partners, both IGF2 rs680 and IGF2AS rs1003484 were associated with GH (P<0.00096 OR3.10, P<0.0073 OR2.58, respectively). In neonates, IGF2 rs680 was associated with PE (P<0.0061 OR3.22), SGA (P<0.01367 OR2.35) and PTB (P<0.033 OR2.71), while IGF2AS rs1003484 was associated with PTB (P<0.035 OR2.77). In mothers IGF2 rs680 was associated with PTB (P<0.025, OR2.41). Discussion: We are the rst to identify associations between paternal and neonatal genotype for paternally expressed genes and adverse pregnancy outcome. For these paternally expressed genes, a strong association was seen between paternal genotype and GH, as well as neonatal genotype with many adverse pregnancy outcomes. These data suggest that neonatal IGF2AS and particularly IGF2 genotype confers a greater risk of developing pregnancy complications, with a 2.4-3.2 fold higher risk of PE, SGA and PTB. Future investigations will identify a functional role in the placenta for these polymorphisms. Keywords: Association, Polymorphism, IGF2, Genetic
[P14.01]. PREDICTORS OF PREGNANCY COMPLICATIONS IN WOMEN WITH SYSTEMIC LUPUS ERYTHEMATOSUS D Chirico*, I N Bruce, P N Baker, I P Crocker, C L Tower. University of Manchester, United Kingdom Introduction: Systemic lupus erythematosus (SLE) is an autoimmune disease associated with pregnancy complications (notably pre-eclampsia) in which dysregulation of CD4+/CD25+/Foxp3+ T-cells (T-regulatory cells) is considered a contributory factor. Transforming Growth Factor beta-1 (TGF-b1) is a cytokine involved in both T-reg cell activation and recognised alterations in arterial stiffness, a potential predisposing factor for pre-eclampsia. In this preliminary study, we have measured these components in the peripheral circulation of women with SLE and healthy pregnant and non-pregnant individuals. Methods: T-reg cells were assessed by ow cytometry (CD4+/CD25+/ Foxp3+), TGFb1 by ELISA and arterial stiffness, expressed as stiffness index (SI), by pulse wave analysis. Results: Preliminary results show that CD4+/CD25+ T-reg cells (as a proportion of total lymphocytes) increased in early pregnancy (12 weeks gestation) [15.4% median (11.8-20.9) interquartile range, N9] compared to non-pregnant healthy controls [8.9% (7.5-9.3), N11, p0.001]. Similarly, Foxp3 positivity of these T-reg cells was also elevated [25.2% (18.232.1), N6 vs. 12.1% (10.9-15.8), N3, p0.02]. CD4+/CD25+ T-reg cells were signicantly decreased in SLE early pregnancy [10.43% (8.4-11.1)], compared with healthy early pregnant controls (p0.03). TGFb1 was signicantly higher in non-pregnant healthy individuals [1.6 activation index (1.5-1.9), N8] compared with non-pregnant SLE patients [1.0 (0.9-1.2), N3, p0.02], but there was no difference in early healthy pregnancy [1.81 (1.5-2.3), N4]. SI was non-signicantly elevated in women with SLE [9.0m/s (7.2-13.2), N6, p0.06)] compared to non-pregnant healthy women [7.1m/s (6.78.3), N9], and no further differences were dened in early pregnancy [7.1m/s (6.5-8.2), N8]. Discussion: In conclusion, increased levels of CD4+/CD25+/Foxp3+ T-reg cells, but not TGFb1, may be a component of uncomplicated pregnancy. Arterial stiffness may not be a predisposing factor for pregnancy complications in SLE, but a failure to acquire necessary T-reg cells may impact upon pregnancy outcome. Work is ongoing to conrm this suggestion. This study was funded by the Wellcome Trust.
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[P14.02]. THE RELATION BETWEEN THE INCIDENCE OF PLACENTA PREVIA ACCRETA AND THE METHODS OF UTERINE CLOSURE AT PREVIOUS CESAREAN SECTION C Sugiyama*, S Sumigama, Y Mano, T Kotani, H Hayakawa, F Kikkawa. Nagoya University Graduate School of Medicine, Japan In placenta previa, history of cesarean section (CS) is a risk factor for placenta accreta. In this study, we carried out to survey the relationship between the two methods of uterine closure (continuous vs interrupted) at previous CS and the incidence of placenta accreta.We reviewed records of patients in 11 tertiary hospitals in Japan between 1994 and 2008. In total 94, 968 deliveries, 867 patients had placenta previa, and 119 of whom had one or more histories of previous CS and 80 records were obtained. Four patients with classical incision or closure by unabsorbable material were excepted and 76 patients with lower transverse incision and closure by absorbable material were included for this study. In this sample, 31 patients had been diagnosed as placenta previa with accreta pathologically. 31 patients had single-layer closure include 7 with continuous suture (2 placenta accreta) and 24 with interrupted suture (10 placenta accreta). 45 patients had double-layer closure include 17 with continuous suture (12 placenta acreeta) and 28 with interrupted suture (7 placenta accreta). We record that placenta accreta occurred in the patients with single-layer is 39% and 42% of double-layer group, which showed no signicant difference (p0.76). However, placenta accreta occurred in 52% of the patients with continuous group and 26% of interrupted group, which showed a signicant difference with p0.034. When limited to double-layer closure, placenta accreta occurred in 71% of continuous group and in 25% of interrupted group, which showed strong difference (p<0.01). In addition, multivariable logistic regression analysis adjusted for confounder (gravid, history of induced abortion, placental location, number of previous CS) also showed signicant difference (p0.023, OR8.15).In conclusion, we showed the possibility that continuous suture for the closure of uterine incision at previous CS could be a risk factor for accreta in placenta previa with history of CS. Keywords: placenta accreta, cesarean section, uterine incision, suture method
[P14.05]. DO PREGNANCIES WITH PREECLAMPSIA HAVE SMALLER PLACENTAS? A POPULATION STUDY OF 317 688 PREGNANCIES WITH AND WITHOUT GROWTH RESTRICTION IN THE OFFSPRING A Eskild*, LJ Vatten. 1Dep. of Obstetrics and Gynecology and Medical Faculty Division Akershus University Hospital, Norway, 2Inst. of Public Health. Norwegian University of Science and Technology, Norway Background: Preeclampsia is thought to be caused by placental dysfunction. A small placenta may serve as an indicator of dysfunction. Aim: To study whether pregnancies with preeclampsia have smaller placentas than normotensive pregnancies. This was studied in pregnancies with and without small for gestational age (SGA) offspring, dened as birth weight lower than the 2.5 percentile. Study population: All singleton births in Norway from 1999 through 2004, a total of 317 688 pregnancies. Outcome measures: Proportion of pregnancies within placental weight tenths (based on z-scores of placental weight). Results: Among pregnancies with SGA offspring, close to 60% were in the lowest tenth of placental weight, with similar proportions of preeclamptic (59.9%) and normotensive pregnancies (61.4%). Less than 1% of pregnancies with SGA offspring, regardless of preeclampsia status, were in the highest tenth of placental weight. Pregnancies without SGA offspring were evenly distributed across placental weight tenths, but in these pregnancies, the proportion with preeclampsia was slightly higher both at the lowest (9.5% versus 8.8%) and the highest tenth (11.9% versus 10.2%) of placental weight. Conclusion: We found that pregnancies with and without preeclampsia were similarly distributed across placental weight tenths, with consistent ndings in pregnancies with and without SGA offspring. Our ndings suggest that a small placenta is not associated with peeclampsia, but with SGA in the offspring. Keywords: Placental weight, Preeclampsia, Fetal growth restriction, Population study
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[P14.06]. DECREASED ANTIOXIDANT STATUS OF HUMAN CERVICO-VAGINAL FLUID (CVF) WITH IMPENDING LABOUR: DEVELOPING A PREDICTIVE CAPACITY HM Georgiou*1,2, YJ Heng1,2, MKW Di Quinzio1,2, M Permezel1,2, GE Rice1,3. 1 University of Melbourne, Australia, 2Mercy Hospital for Women, Australia, 3 Baker IDI Heart & Diabetes Institute, Australia Introduction: Proteomic analysis of CVF by 2D electrophoresis revealed signicant differential expression of four major antioxidant enzymes during late pregnancy and labour. Cu,Zn superoxide dismutase (Cu,Zn SOD) and thioredoxin-1 (Trx-1) were signicantly decreased in spontaneous term labour while glutathione-s-transferase and peroxiredoxin-2 were signicantly increased with impending labour [1]. The differing expression of these antioxidants prompted the investigation of the total antioxidant capacity (TAC activity assay) of CVF, both prior to and in term labour. The study further investigated the temporal changes of Cu,Zn SOD and Trx-1 (ELISA) with impending labour and the predictive potential of these biomarkers was determined. Methods: Serial CVF samples were collected weekly from 73 healthy, multiparous women with singleton pregnancies beginning from 36 weeks gestation until the onset of spontaneous labour (before membrane rupture). Data was analysed after correction for total protein. Results: 193 samples were assayed for TAC (Fig 1A). TAC was 8-fold signicantly lower in labour than >29 days prior to labour onset (ANOVA, p<0.001). TAC was about 2-fold signicantly lower at 0-7, 8-14, 15-21 and 22-28 days compared to >29 days before labour (ANOVA, p<0.001). Cu,Zn SOD was 1.5 to 1.9-fold signicantly decreased in labour compared to samples collected at 0-7, 8-14, 15-21 and 22-28 days before labour onset (Fig 1B, n170, ANOVA, p<0.001). Regression analysis indicated that Cu,Zn SOD signicantly decreases with impending term labour (p0.003). Trx-1 was 2.8 to 5-fold signicantly lower in labour compared to 0-7, 8-14, 15-21 and 22-28 days before labour onset (n163, ANOVA, p0.002). There was a signicant correlation between Cu,Zn SOD and Trx-1 concentrations (r0.433, p<0.001), and between Trx-1 and TAC (r0.349, p<0.001). Receiver Operator Characteristic (ROC) curves were produced for each biomarker and Binary Logistic Regression analysis was performed to determine the efcacy of these biomarkers (singly and in combination) to predict labour within 3 days of onset (Table 1). The combination of TAC and Cu,Zn SOD produced the best predictive efcacy with good sensitivity and very high specicity. Positive and negative predictive values were superior compared to each biomarker alone. Discussion: These ndings suggest that labour is associated with increased oxidative stress even before its onset. These biomarkers could serve as potential therapeutic targets as well as predictors of labour onset. Studies are in progress to determine the role of these biomarkers in the preterm labour setting.
Table 1
TAC Cu,Zn SOD Trx-1 TAC and Cu,Zn SOD 0.928 61.3 97.0 82.6 91.4 3.1 38.7
Cut off values ROC - Area under curve Sensitivity (%) Specicity (%) +ve Predictive Value (%) -ve Predictive Value (%) False +ve (%) False -ve (%)
30 uM/mg total protein 0.646 51.6 69.5 28.6 85.9 30.5 48.4
0.5 ug/ml/mg total protein 0.687 54.8 78.6 37.8 88.0 21.4 45.2
160 ng/ml/mg total protein 0.646 51.6 76.3 34.0 87.0 23.7 48.4
Reference [1] Di Quinzio MKW et al, 2008, J Proteome Res, 7:1916-1921. Keywords: Labour, Oxidative Stress, Prediction, Cervico-vaginal uid
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[P14.08]. INVOLVEMENT OF OXIDATIVE AND ANTI-OXIDATIVE STRESSES STRESS MECHANISMS IN PREGNANCY-INDUCED HYPERTENSION Mitsuyo Matsumoto*, Osamu Akutagawa, Hirotaka Nishi, Chinatsu Higuma, Keiichi Isaka. Department of Obstetrics and Gynecology, Tokyo Medical University, Japan Objective: The body can clear reactive oxygen species and free radicals generated under normal conditions through its antioxidative system. DNA damage occurs when generation exceeds antioxidative capacity and is associated with various disorders. It is becoming increasingly clear that the pathogenesis of pregnancy-induced hypertension (PIH) is associated with the inammatory response, oxidative stress, and anti-oxidative stress that results from reduced uteroplacental circulation. The purpose of this study was to clarify the role of antioxidative stress in PIH pathogenesis. Methods: We explained the purpose of the study to participants, obtained consent, and examined amniotic uid from the following tissues at various stages: rst trimester chorionic villi (18 cases), second trimester chorionic villi (10 cases), normal placenta from Caesarean sections (12 cases), normal placenta from vaginal births (10 cases), and placenta from PIH patients (15 cases). We used Cu/Zn superoxide dismutase (SOD) and Mn-SOD as antioxidant indicators, and 8-OHdG as an oxidative stress indicator. Measurements were performed by ELISA, microplate assays, and real-time RT-PCR. Results: SOD mRNA levels did not change by delivery method, but were signicantly higher in rst trimester chorionic villi compared to second and third trimester chorionic villi. The PIH group had signicantly lower SOD mRNA and protein levels compared to the normal group, and the PIH group with intrauterine growth retardation showed an even more significant decrease. In contrast, 8-OHdG levels were signicantly higher in the PIH group compared to the normal group. Conclusion: Oxidative and anti-oxidative stresses are associated with various pathologies, including PIH. Our ndings suggest the possibility that placental oxidative stress inuences embryonic development through an antioxidative mechanism. Keywords: pregnancy, pregnancy-induced hypertension, anti-oxidative stress, oxidative stress
[P14.09]. MARKERS OF ENDOTHELIAL ACTIVATION IN HIGH RISK PREGNANCIES Jana Prochazkova*1, Martin Prochazka2, Ludek Slavk2, Jana Ulehlova2, Ondrej Simetka3, Alena Mechurova4. 1Dept. of Hemato-oncology, Medical Faculty of Palacky University, Czech Republic, 2Dept. of Obstet. and Gynaecol, Medical Faculty of Palacky University, Czech Republic, 3Dept. of Obstet. and Gynaecol, Faculty Hospital Ostrava, Czech Republic, 4Institute fo the Care of Mother and Child, Prague, Czech Republic Introduction: The hypertension and preeclampsia in pregnancy are multisystemic diseases characterized by hypertension, proteinuria and generalized systemic vasoconstriction.The ischaemia of the fetoplacental unit cause the release of specic factors into maternal vessels and subsequent activation of the endothelium and vasoconstriction. There is a rush development of the laboratory tests and a new markers of the endothelial activation have been found. Eg. t-PA, PAI-1, vWF, EPCR, thrombomodulin and endothelial microparticules with procoagulant activity. The aim of the study: To detect of above mentioned markers of endothelial activation in healthy pregnant women compared to those with pregnancy complicated by hypertension, diabetes mellitus and preeclampsia. The work hypothesis: We suppose that plasma specimens of the women with preeclampsia and diabetes mellitus will contatin a higher levels of endothelial activation markers compared to healthy pregnant. Methods: All included patient have to assign an informed constent. The blood samlping will be taken by the routine way at the time of the rst blood pregnancy sampling the end of the rst trimester. The second specimen will be taken between 24.- 28. weeks of gestation. The following tests will be performed: t-PA ELISA, PAI-1 ELISA, vWF.Ag EIA (immunologic detection by imunoturbidimetry), ePCR ELISA, MMP-2,9 ELISA (uorogenic detection), endothelial microparticules Flow cytometry. Results: The levels of vWf Ag and vW activity are signicantly different (P 0,02 resp. 0,01) in group patietns with diabetes in comparison with control group. The levels of TRM are signicantly different (P 0,03) in group patiens with hypertension as well as in group patietns with diabetes (P 0,02) in comparison with control group. The levels of ePCR are signicantly different (P 0,04) in group patiens with hypertension as well as in group patietns with diabetes (P 0,01) in comparison with control group. The levels of tPA and PAI are not signicantly different in group patiens with hypertension as well as in group patietns with diabetes in comparison with control group. Conclusion: As expected, there were stat. Signicant differences in the diabetes group in the case of vWf Ag, vWact., thrombomodulin and ePCR. Signicant diference was found in the hypertension group compared to controls in ePCR. Whereas EMP, MMP-9 were not different in all groups. Supported by the Grant of Min.of Health, Czech republik NR 9282-3/2007 Keywords: Endothelium, Preeclampsia, Diabetes mellitus, Thrombosis
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[P14.10]. MATERNAL-FETAL DISTRIBUTION COMPARED WITH ADULT WOMEN
OF
MINERALS
IN
TEENAGERS
[P14.11]. POTENTIAL MAGNETIC RESONANCE IMAGING (MRI) BIOMARKERS OF PLACENTAL STRUCTURE C Wright*1, DM Morris2, PN Baker1, IP Crocker1, GJ Parker2, CP Sibley1. Maternal & Fetal Health Research Centre, University of Manchester, United Kingdom, 2Imaging Sciences & Biomedical Engineering, University of Manchester, United Kingdom, 3Magnetic Resonance Centre, University of Nottingham, United Kingdom
1
MI Moraes, RF Barbosa, RE Santo, FS Santos, EFO Jesus, MG Tavares do Carmo*. Universidade Federal do Rio de Janeiro, Brazil Introduction: Healthy intra-uterine development of the fetus requires adequate amounts of minerals, which are only obtainable from maternal blood via placenta. Teenagers are still in growth and development and often present inadequate nutrition habits, therefore their mineral intake is usually insufcient. The aim of this study was to compare calcium, iron, copper and zinc levels in maternal and cord plasma and in maternal and fetal placenta of teenagers and adult pregnant women. Methods: In Forty teenagers aged between 15 19 years and forty adult women, aged between 20 35 years samples were collected of maternal blood at day 2 postpartum and umbilical cord blood and maternal and fetal placental tissues at birth. X-Ray Total Reection Fluorescence Spectrometry was used for mineral analysis. The Mann-Whitney test was used to compare plasma and placental concentrations of minerals. Results: Calcium, iron, copper, and zinc levels in maternal placenta presented no difference between teenagers and adult women. However, these levels in fetal placenta were higher in adult women than in teenagers. In maternal and cord plasma, calcium, copper and zinc levels presented no difference between teenagers and adult women. Only iron levels were higher in maternal and cord plasma of teenagers than in adult women. Discussion: Our results suggest that the distribution of minerals in the fetal placenta is different for teenagers and adult women. If there is a competition between mother and fetus in pregnancy in adolescence deserves better elucidation in future research. We recommend studies on the placental transport of these minerals. Keywords: minerals, teenagers, nutrition, maternal anda fetal placenta
Placental insufciency is a major cause of fetal growth restriction (FGR) and accumulating evidence indicates that several aspects of placental structure are altered in this condition (Sibley et al. Paed Res 2005;58:827). MRI provides quantitative indices that may be used in the non-invasive assessment of the human placenta, such as relaxation time measurements, T1 and T2. In previous studies (at 0.5 T magnetic eld strength) these have been found to be reduced in FGR (Gowland et al. Mag Res Imag 1998;16:241). We hypothesised that these observed differences may relate to alterations in placental tissue structure and be more readily dened at increased eld strengths. Here, we report on the rst phase of testing this hypothesis, in a study of women having normal pregnancy. Objectives: 1) To assess T1 and T2 relaxation time measurements and correlate these with morphometric analyses of the human placenta following delivery. 2) To assess placental heterogeneity by comparing relaxation times and morphometric analyses in central and peripheral placental regions. Methods: 16 women with uncomplicated pregnancies underwent MRI examination (1.5 T) between 20 and 40 weeks gestation. T1 and T2 measurements were acquired with whole placental coverage, co-localised with a structural scan. T1 and T2 values were calculated by combining 8 points (3 central, 5 peripheral) across the placenta, central and peripheral regions were also analysed independently. Placental biopsies were taken at 8 randomly sampled points (3 central, 5 peripheral). Formalin-xed, waxembedded sections were stained with hematoxylin and eosin and subjected to image analysis. The areas occupied by villi and brin were quantied. Results: 7 of the 16 women (28-38 weeks at time of scan) had placentas available for analysis. A signicant correlation was observed between villous area and T2 (p0.048), but not T1 (p0.302) relaxation times. Correlations between relaxation times and brin area or brin: villous area ratio were not signicant, although trends for a fall in relaxation times with increasing brin were seen. Placentas were heterogeneous, with no signicant differences between central and peripheral regions in villous or brin area, T1 or T2.
Conclusions: Villous area is known to be reduced in FGR (Daayana et al. J Soc Gynecol Investig. 2004;11(8):545). These early data suggest relaxation times may be a useful biomarker of villous area and potentially useful in identifying FGR in utero. We will continue to examine placentas from normal and FGR pregnancies, making comparisons with placental heterogeneity, morphometry and function. Funded by the MRC. Keywords: MRI, IUGR, fetus, morphometry
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[P14.12]. A CASE OF GESTATIONAL TRANSIENT THYROTOXICOSIS IN TWIN PREGNANCY COMPLICATED BY PANHYPOPITUITARISM K. Takaishi*, M. Yamaguchi, K. Uchino, R. Honda, T. Ohba, H. Katabuchi. Kumamoto University, Japan In a minority of pregnant women, the concentration of thyroxine increases above the upper normal value because of excess stimulation of the TSH receptor by hCG. Here we report a case of gestational transient thyrotoxicosis in twin pregnancy complicated by panhypopituitarism. A 29-yearold woman was referred to our University Hospital with complaint of infertility. She had been treated for a craniopharyngioma at 25 years of age. Postsurgical hormone replacement was carried for panhypopituitarism with diabetes insipidus. A diagnosis of pituitary amenorrhea was made after gynecological evaluations, and the patient underwent treatment for infertility. The patient became pregnant with dichorionic diamniotic twins during hMG-hCG treatment cycle with AIH. The patient was complicated by frequent vomiting and weight loss after 5 weeks of gestation and the patient was admitted to our hospital. A diagnosis of hyperemesis gravidarum was made at 8 weeks of gestation. Laboratory tests showed elevated free T3 and T4, and with absent TSH and thyroid autoantibodies. Adrenal failure was excluded because hydrocortisone administration was ineffective. A diagnosis of gestational transient thyrotoxicosis was made. The patients symptoms improved after the discontinuation of thyroxine supplementation and the decrease in serum hCG. The patient had a vaginal twin delivery at 38 weeks of gestation, and thyroxine supplementation was resumed on the third postpartum day. The patients serum hCG strongly correlated with the serum free T4 under conditions of low TSH secretion. In conclusion, gestational thyrotoxicosis was triggered despite the lack of endogenous TSH. This case proves that TSH receptor is capable of being stimulated by endogenous hCG. Keywords: gestational transient thyrotoxicosis, human chorionic gonadotropin, thyroid stimulating hormone receptor
[P14.13]. PATERNAL RENIN ANGIOTENSIN SYSTEM POLYMORPHISMS ASSOCIATED WITH PREGNANCY COMPLICATIONS
ARE
A Zhou*, S Thompson, R Nowak, J Zhang, G.A. Dekker, C.T. Roberts. Research Centre for Reproductive Health, Discipline of Obstetrics and Gynaecology, University of Adelaide, Australia Introduction: Preeclampsia (PE), small for gestational age (SGA) and preterm birth (PTB) together affect 20% of rst pregnancies. Currently there is no reliable way to identify women at risk. Polymorphisms in genes in the renin angiotensin system (RAS) may be associated with impaired placentation and poor maternal response to pregnancy and hence predict risk for pregnancy complications. We aimed to determine if three functional polymorphisms in RAS genes, namely, AGT T174M, ACE A11860G and AT1R A1166C are associated with pregnancy complications. Methods: Pregnancy trios were prospectively recruited from Lyell McEwin Health Service in Adelaide. Pregnancies were classied into normal (n258), PE (n38), SGA (n60), gestational hypertension (GH, n51), PTB (n32) and gestational diabetes (GD, n13). Parental blood and maternal blood pressure were sampled or measured at 15 weeks gestation. Cord blood was sampled after delivery. DNA was extracted from buffy coats and genotyped by the Australian Genome Research Facility utilizing the Sequenom MassARRAY system. Maternal plasma [ACE] was measured by ELISA. Data were analysed by ANOVA, Chi-square and Fishers exact test. Likelihood ratios (LR) were calculated. Results: None of the RAS maternal and neonatal genotypes were associated with pregnancy complications. For maternal ACE A11860G, plasma [ACE] in women with GG was 24% and 74% higher than AG (p0.03) and AA (p0.001), respectively. A positive correlation between plasma [ACE] and mean arterial pressure (r0.378, p0.003) was observed in normal pregnancy. However, maternal ACE genotype did not correlate with blood pressure at 15 weeks gestation. Of great interest, paternal AGT T174M, AT1R A1166C and ACE A11860G were associated with GD (p0.028, LR5.0), PTB (p0.032, LR7.3) and PE (p0.038, LR6.7), respectively. Discussion: Our data suggest that paternal genotype for genes in the renin angiotensin system may be important in determining risk for pregnancy complications, consistent with the role of paternity in their aetiology. Keywords: Placenta, Pregnancy complications, Renin angiotensin system, Paternal genotype
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[P14.14]. THE EFFECTS OF FETAL HYPERINSULINAEMIA ON VEGF-165 AND VASCULAR PERMEABILITY IN THE PERFUSED HUMAN PLACENTA J Lucas, SP Walker, PEB Rodgers, F Sciota, L Leach*. University of Nottingham, United Kingdom Insulin is known to upregulate vascular endothelial growth factor (VEGFA), a potent enhancer of vascular permeability. In diabetic pregnancies, fetal hyperinsulinaemia may be one of the factors leading to elevated levels of VEGF observed in the placenta, with its resultant leaky vascular phenotype. The aim of this study was to assess levels of VEGF-165a and its splice variant VEGF 165b and the integrity of junctions in fetal vessels after a brief hyperinsulinaemic insult. Using an established method, term human placenta from normal pregnancies (obtained by elective Caesarean section) were perfused for 20 minutes with (n3) or without (n3) 25mU/mL insulin in the fetal circulation. In the last 10 min, TRITC-dextran (76Mr) was added to the fetal circulation in both groups. After perfusion xation, tissue biopsies were subjected to immunocytochemistry to localise VEGF isoforms, junctional b-catenin and occludin and tracer hot spots. Selected random sampling was used to count the % of vessels in terminal, intermediate or stem villi displaying positive immunoreactivity. In the insulin group, analysis of large vessels in stem villi and microvessels in intermediate and terminal villi revealed increases in percentage of all VEGF-positive vascular proles (p<0.05), whilst there were decreases in VEGF165b-positive large vessels. Increased vascular leakage was seen in both large and microvessels in the insulin group. This was accompanied with a downregulation of b-catenin in all vessels and downregulation of the tight junctional molecule, occudin in large vessels. These combined data suggest that brief fetal hyperinsulinaemia is capable of affecting both adherens and tight junctions, altering the phenotype of chorionic vessels throughout the villous tree. Beyond implications in placental barrier function, the results raise the possibility that chronic high fetal insulin may alter the fetal vascular phenotype, with implied potentially long-lasting effects on the infant. Funded by ASGBI Keywords: fetal insulin, VEGF, tracer leakage, feto-placental vessels
[P15.01]. SCANNING ELECTRONE MICROSCOPY IN TIME IMPLANTATION BY ENDOMETRIAL PATHOLOGY
OF
PROPOSED
O. ILINA*, T. ZADOROGHNA, I. SUDOMA, I. ILYIN. 1Institute of Paediatrics, Obstetrics and Gynecology, Ukraine, 2Clinic "Nadiya", Ukraine, 3Institute of Genetic Reproduction, Ukraine Aim of the study: to investigate peculiarities of cilliar cells and pinopodes formation in endometrial luminal epithelia in women with pathological statements of endometrium. Object: double Pippelle biopsies of endometrium from 360 women in time of implantation window with different types of endometrial pathology: hypotrophy, hyperplasia, chronic endometritis, asynchrony of endometrial maturation. Methods: Scanning electrone microscopy (JEOL Superprobe 733) and routine histological investigation of endometrial samples of every woman on two terms. Results: Defects of pinopodes (uterodomes) formation and development was more signicant by endometrial hypotrophy. Pathological changes of cilliar cells in quantity and structure were observed by endometrial hyperplasia. Summary: Signicant changes in luminal epithelia in time of implantation window in women with endometrial pathology were revealed by scanning electrone microscopy that may cause infertility in these women and should be taken into consideration by diagnostic and treatment. Keywords: Scanning electron microscopy, implantation, endometrial pathology, infertility
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[P15.02]. CHANGES IN VEGF-C, VEGF-D, VEGF-R2 AND VEGF-R3 IN NONPREGNANT HUMAN ENDOMETRIUM DURING THE MENSTRUAL CYCLE AND IN WOMEN WITH RECURRENT MISCARRIAGE GE Lash*1, RK Adcock1, BA Innes1, JA Drury2, S Quenby2, JN Bulmer1. 1 Newcastle University, United Kingdom, 2University of Liverpool, United Kingdom Introduction: Endometrial vascular development is a critical process. Spiral arteriole vascular smooth muscle cell (VSMC) numbers and differentiation state increase throughout the menstrual cycle. In some women with recurrent miscarriage (RM) vascular development is enhanced. VEGFC and -D have both angiogenic (via VEGF-R2) and lymphangiogenic (via VEGF-R3) properties. Aim: Determine the temporal and spatial expression of VEGF-C, -D, -R2, and -R3 in endometrium throughout the menstrual cycle and in women with RM. Methods: Endometrial biopsies taken at the time of hysterectomy (n7 proliferative (P), n4 early secretory (ES), n7 mid-late secretory (LS)) and endometrial pipelle biopsies from women with RM at LH+7 (n14 LS) were immunostained for VEGF-C, -D, -R2, and -R3 and assessed using quickscore. Stroma, VSMC, endothelial cells (EC) and glandular epithelium (GE) were scored separately. Scores for all cell types were added together to give a total score. Results: Endometrium immunostained positive for all factors. VEGF-C immunostaining was reduced in LS compared to P (total P0.009; EC P0.0009; GE P0.001) and ES (total P0.02; EC P0.005). No changes in VEGF-D expression were observed, and VEGF-D was not detected in VSMC. VSMC immunostaining for VEGF-R2 was increased in ES (P0.003) and LS (P0.01) compared to P. VEGF-R2 was decreased in RM compared to LS (total P0.003; stroma P0.009; VSMC P0.0003). Stromal immunostaining for VEGF-R3 was reduced in ES (P0.03) and LS (P0.03) compared to P. GE immunostaining for VEGF-R3 was decreased in LS compared to P (P0.004), but was increased in RM compared to LS (P0.02). Discussion: VEGF-C, -R2 and -R3 are temporally and spatially regulated during the menstrual cycle. These factors are likely involved in endometrial regeneration. VEGF-R2 and VEGF-R3 expression was widespread in endometrium suggesting importance of their ligands in endometrial function. The signicance of reduced VEGF-R2 expression in RM needs to be further explored. Keywords: angiogenic growth factors, non-pregnant endometrium, recurrent miscarriage
[P16.01]. PLACENTAL TROPHOBLAST DYNAMICS IN SUDDEN INFANT DEATH SYNDROME (SIDS) T Ansari*1, B ONeill2, JE Gillan2. 1Dept. of Surgical Research, NPIMR, Harrow, London, United Kingdom, 2Dept. of Histopathology, Rotunda Hospital, Dublin, Ireland Introduction: SIDS occurs postnatally but the origins are hypothesised to occur in utero resulting from delayed or arrested developmental homeostasis. The degree of maternalfoetal exchange in utero is paralleled by an increased developmental complexity of the placenta. Specically, during the third trimester placental villous growth experiences an exponential increase in transfer capacity to match the growing demands of the foetus. It is during this period that perturbations in villous trophoblast kinetics are likely to have the greatest impact on foetal organogenesis. Methods: SIDS placentae were retrieved from archived storage and subdivided into two groups based on a birth weight above (SIDS NBW n12) or below (SIDS LBW n12 the 10th centile for gestational age and compared with term controls (n12). Uniform random sampling was employed and tissue samples used to produce 5mm H&E stained sections. Stereologically derived volumes of the following features were estimated: cytotrophoblast (CT), syncytiotrophoblast (ST) and syncytial knots (SK)both apoptotic and non-apoptotic for all villous types. Results: Total trophoblast volume was increased (P0.007) in SIDS NBW placenta, originating from signicant increases in both CT (P0.004) stem and terminal villi) and ST (P0.031; stem villi) volumes. In contrast, SIDS LBW placentae showed no signicant change in total trophoblast volume or its subcomponents; however, CT volume showed a trend towards an increase (stem villi). An increase in total apoptotic SK volume was observed in SIDS NBW (intermediate and terminal villi) and SIDS LBW (intermediate villi). Histologically, the CT nuclear size appeared larger and existed in places as a multi-layered arrangement. Discussion: Although the trophoblast layer is thicker in SIDS NBW the increased volume of focal apoptotic SK suggests increase extrusion to maintain a minimum diffusion distance for oxygen and nutrient transfer. Trophoblast kinetics in SIDS NBW and SIDS LBW differ with the former initiating an adaptive mechanism to maintain foetal growth. Keywords: SIDS
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[P16.02]. CYTOKERATIN AND CEA EXPRESSION IN VILLOUS CHORION T. Archakova*, T. Zadoroghna. Institute of Paediatrics, Obstetrics and Gynecology, Ukraine Aim of the study was to investigate Cytokeratin and CEA expression peculiarities in villous chorion by physiological pregnancy (II trimester). Object: 20 biopsies from healthy women with physiological pregnancy, who had a chromosomal pathology were observed. Methods: immunohistochemical investigation with monoclonal antibodies to Cytokeratin Clones AE1/AE3 and CEA (DAKO) on parafn embded cuts. Results: Cytokeratin expression was high in cytotrophoblast, vascular endothelium, stromal cells and trophoblastic proliferates; moderate in sincitiotrophoblast. CEA expression was insignicant and was revealed as weak or at times - moderate in sincitium and cytotrophoblast of isolated intermediate differentiated villi. Summary: Strongly marked Cytokeratin expression and weak expression of CEA were observed in II trimester placenta, that characterize one of placental histogenesis way. Keywords: Cytokeratin, CEA, chorion, placental histogenesis
[P16.05]. IS MATERNAL PROGESTERONE ACTUALLY INDEPENDENT OF THE FETAL STEROIDS? Antonin Parizek*, Andrea Paskova, Martin Hill, Michaela Klimkova, Marta Kalousova, Luboslav Starka. Department of Obstetrics and Gynecology of the First Faculty of Medicine and General Teaching Hospital, Prague, Czech Republic Progesterone and estradiol are foremost steroid hormones in human pregnancy; however, the origin of maternal progesterone has not been still satisfactorily explained despite the generally accepted opinion that maternal LDL-cholesterol is a single substrate for placental synthesis of maternal progesterone. There is still a question: Why the levels of progesterone are substantially higher in fetal and not in maternal blood? Hence, the role of the fetal zone of the fetal adrenal (FZFA) in the synthesis of progesterone precursors was addressed. The FZFA may be directly regulated by placental CRH inducing an excessive production of sulfated 3b-hydroxy-5-ene steroids like sulfates of dehydroepiandrosterone (DHEAS) and pregnenolone (PregS). To our hypothesis, besides the necessity to synthesize de novo all the maternal progesterone from cholesterol, it may be more convenient to utilize the fetal PregS. The activities of sulfatase and 3b-HSD are substantially higher than those ones of cytochrome P450scc and are not rate-limiting for the placental progesterone synthesis. Keywords: steroids, labor, progesterone, metabolome
[P16.04]. CORD BLOOD CYTOKINE AND CHEMOKINE PROFILE IN PREGNANCIES COMPLICATED BY ASTHMA A Osei-Kumah*, N Hodyl, V Clifton. University of Adelaide, Robinson Institute, Australia Introduction: Fetal exposure to an adverse intrauterine environment can increase susceptibility towards disease in adult life. Furthermore maternal asthma is an increased risk factor for the development of atopy or asthma in the offspring. There is evidence that this is associated with differential placental gene expression. We have demonstrated that maternal asthma is associated with increased basal cytokine expression in the placentae of female neonates compared to controls. Such placental inammatory responses may program the development of the fetal immune system. The aim of current study was to assess the cytokine and chemokine prole in cord blood from pregnancies complicated by maternal asthma. Methods: Following delivery, cord blood was collected from control pregnancies (n7), pregnancies complicated by untreated asthma (n14) and those treated with inhaled glucocorticoids (n17). Plasma was assayed for 19 cytokines and chemokines with a Lincoplex assay kit on a Luminex multi-measurement platform. Results: Asthma was associated with an increased cord blood concentration of granulocyte colony stimulating factor (G-CSF) compared to controls (p0.003). In cord blood from females, increased concentrations of interleukin (IL)-10 (p0.038) and IL-12 (p0.025) were also demonstrated. In cord blood from males, cytokine and chemokine concentrations did not change with asthma. None of the measured cytokines or chemokines were affected by inhaled glucocorticoids for asthma treatment. Conclusion: Increased cytokine concentrations were observed in cord blood from females following maternal asthma. This data suggests that the developing immune system may be programmed differentially in females and males, given that G-CSF stimulates the production of haematopoietic stem cells, and both IL-10 and IL-12 are involved in cell differentiation. These ndings may help elucidate mechanisms contributing to increased rates of allergy and atopy currently observed in female children. Keywords: cord blood, cytokine, allergy, programming
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[P16.06]. EFFECTS OF MATERNAL MOOD AND ANTIDEPRESSANT USE ON PLACENTAL GENE EXPRESSION KL Ponder*1,2, AL Salisbury1,2, BG McGonnigal1,2, AM LaLiberte2, JF Padbury1,2. 1Warren Alpert Medical School of Brown University, United States, 2 Department of Pediatrics, Women & Infants Hospital, United States Introduction: Previous work has shown that stress, drug exposure and growth restriction modify placental neuroendocrine gene expression. It is not known whether maternal mood disorders (depression/anxiety) and antidepressant treatment have similar effects and inuence fetal programming. We sought to determine how maternal depression, anxiety, and treatment with Selective Serotonin Reuptake Inhibitors (SSRIs) affect placental human serotonin transporter (hSERT), norepinephrine transporter (hNET), and 11beta-hydroxysteroid dehydrogenase type 2 (11betaHSD-2) expression. Methods: We collected placental samples from 52 controls, 56 women with depression and/or anxiety symptoms during pregnancy, and 27 women who used SSRIs in at least the third trimester. A subset of 25 women who consented to structured interview was also included. Inventory of Depressive Symptomatology and Hamilton anxiety scores were determined. RNA expression was determined by real-time PCR. ANOVA was used to determine statistical signicance between control, depression/anxiety, and SSRI groups. Results: Exclusions included other psychotropic medications, nicotine or illicit drug use, diabetes, and gestational age <37 weeks. There was a signicant overall effect of mood and SSRIs on expression of SERT, NET, and 11beta-HSD-2. 11beta-HSD-2 expression was signicantly increased in the depression/anxiety group, but not the SSRI group, compared to controls (p<.05). NET was positively correlated with 11beta-HSD-2 and SERT expression (p<0.001). Discussion: Maternal mood has a signicant effect on placental 11betaHSD-2 gene expression. 11beta-HSD-2 is induced by cortisol and converts cortisol to its inactive form. Placental 11beta-HSD-2 therefore serves as a barrier to maternal cortisol passage to the fetus. Depression is associated with increased blood cortisol levels. The increase in 11beta-HSD-2 expression in the depression/anxiety group could represent a compensatory response to increased maternal cortisol levels. This nding is consistent with previous in vitro studies in human trophoblast cells. These observations suggest possible mechanisms for long term effects of maternal mood and antidepressant treatment on fetal/neonatal neurodevelopmental programming. Keywords: gene expression, depression, antidepressants, fetal programming
[P16.07]. ADVANCED PATERNAL AGE AFFECTS BIRTH WEIGHT AND PLACENTAL THICKNESS OF FEMALE BUT NOT MALE INFANTS: AN ANALYSIS OF THE COLLABORATIVE PERINATAL PROJECT (CPP) CM Salaa*1,2, DP Misra3, AK Charles4, RK Miller5. 1Placental Analytics LLC, United States, 2Institute for Basic Research, United States, 3Wayne State University, United States, 4Princess Margaret Hospital, Australia, 5University of Rochester, United States Background: Advanced paternal age has recently been identied as a risk factor in female offspring for neurodevelopmental and neuropsychiatric outcomes such as autism and schizophrenia. The mechanism has been speculated to involve age-pathology of the paternally-derived X chromosome. We tested the hypothesis that advanced paternal age would affect the placenta (in which paternal genes are preferentially expressed) of female but not male infants, and that those placentas would yield a smaller birthweight than placentas from pregnancies with a younger father. Methods: 11,120 male and 10,542 female newborns had complete data for chorionic larger and smaller diameters, disk thickness, distance from cord insertion to the disk edge, cord length, placental weight, and categorization of placental shape as round/oval versus other, and parental ages. Univariate and multivariate linear regressions were performed. Results: In bivariate regression, advanced paternal age was associated with greater birth weight in males (p<0.001) and females (p<0.0001). In male infants, paternal age effects disappeared after adjustment for maternal age, while in female infants, such adjustment led to a signicant negative effect of paternal age (p<0.0001). Paternal age also showed an inverse relationship to disk thickness in female (p<0.0001) but not in male infants (p0.62). Paternal age was not associated with placental weight or chorionic disk area (calculated from the larger and smaller placental diameters) in either sex. Conclusion: Advanced paternal age is a risk factor for autism and schizophrenia, in females. Autism and schizophrenia are also disorders of abnormal neuronal connectivity. One speculation has been that the paternally derived X chromosome carries altered/mutated genes and/or abnormal/altered epigenetic programming. We propose that the observed effect of thinner (less well arborized) placentas of girlswith older fathers mirror fetal branching in other organs, including neurons. Study of the placenta may therefore be a proxy for analysis of the brain proper. Keywords: paternal age, placental growth, birth weight, fetal programming
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[P16.09]. BIRTHWEIGHTS SMALLER OR LARGER THAN THE PLACENTA WOULD PREDICT IS ASSOCIATED WITH DIASTOLIC BLOOD PRESSURE AT AGE 7 YEARS: AN ANALYSIS OF THE COLLABORATIVE PERINATAL PROJECT (CPP) CM Salaa*1,2, DP Misra3, AK Charles4, RK Miller5. 1Placental Analytics LLC, United States, 2Institute for Basic Research, United States, 3Wayne State University, United States, 4Princess Margaret Hospital, Australia, 5University of Rochester, United States Background: Fetal growth is a complex process depending on parental genetics, maternal /uterine environment and placental function. Placental growth measures collected in the CPP (disk shape, larger and smaller diameters, disk thickness, site of cord insertion and cord length) account for w40% of birthweight variance and have been shown to have independent effects on birth weight. (Salaa CM et al. Birth Defects Res A Clin Mol Teratol. 2007;79(4):281-8.) We hypothesized that altered placental proportions (and different vascular structure) affect the fetal cardiovascular system, and be reected in variation in childhood blood pressure. Methods: Using linear regression, we generated a birth weight predicted by the available placental measures (disk shape, smaller and larger diameters, disk thickness, site of cord insertion, and cord length). The ratio of the actual birth weight to that predicted by placental parameters (observed/expected ratio, OER) was used as the independent variable in analyses of age 7 year diastolic and systolic blood pressures in the 15,902 singleton liveborns delivered between 34-42 weeks with complete. Results: After adjustment for precise ages at blood pressure measurement, the OER had a signicant independent effect on diastolic blood pressure (b3.4.63, p<0.001) at 7 years of age. This effect persisted after further adjustment for gestational age, sex, socioeconomic status, and maternal pre-pregnancy height and weight, and was also independent of BMI, previously shown to be correlated with OER. Conclusion: Placental proportions, proxied here by the observed/expected ratio, reect the vascular architecture of the arborizing placenta; different proportions imply different numbers and distributions of vessels at different levels of the villous tree. We interpret our ndings as consistent with the hypothesis that placentas with different vascular composition affect fetal cardiac work and/or reect differences in intrinsic patterns of the cardiovascular system, and may either mediate or mark differences in childhood diastolic blood pressure. Keywords: blood pressure, placental growth, birth weight, fetal programming
[P17.01]. PRENATAL ETHANOL EXPOSURE IN THE RAT RESULTS IN GROWTH RESTRICTION AND DECREASED AQUAPORIN-1 GENE EXPRESSION IN FEMALE BUT NOT MALE PLACENTAE LB Wilson, DG Simmons, ME Probyn, KM Moritz*. University of Queensland, Australia Background: Exposure of the developing foetus to alcohol (ethanol) can result in fetal growth restriction and impaired development. This may occur in part due to effects of ethanol directly on the placenta. High doses of ethanol during pregnancy in animal models can reduce placental weight and function. In this study the effects of maternal consumption of a low to moderate amount of ethanol throughout pregnancy on placental development was examined in a rat model. Methods: Pregnant rats were fed a control diet or a diet containing 6% (vol/ vol) ethanol (ETOH). At day 20 of pregnancy, dams were killed and foetuses and placentae collected and weighed. RNA was extracted from the labyrinth and gene expression of Glut-1, aquaporin-1 (AQP-1) and alcohol dehydrogenase (ADH) examined using real-time PCR. Placentae from male and female foetuses were examined separately. Results: ETOH exposed females, but not males, were lighter than control foetuses (P<0.05). The placentae from all offspring (male, female, control, ethanol) were of a similar weight. ETOH resulted in decreased Aqp1expression in the placenta of female but not male foetuses and tended to decrease ADH expression in both sexes. Gene expression is shown in the table below (*P<0.05):
Gene Control (n7-10 samples from 5 litters) Male Female Male Female Male Female 1.020.19 1.240.18 1.080.23 1.360.23 1.130.24 1.000.19 Ethanol (n7-10 samples from 4 litters) 1.180.2 1.250.18 0.680.25 0.600.21* 0.750.43 0.430.16
Glut1 Aqp1 ADH
Discussion: Prenatal ethanol exposure has caused sex specic decreases in placental Aqp1 gene expression and fetal growth. Aqp1 is expressed in vascular endothelium and thus decreased expression may reect decreased development of the vasculature in the placenta of female foetuses thereby contributing to the observed growth restriction. Keywords: alcohol, aquaporin 1, sex
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[P17.02]. SEX SPECIFIC DIFFERENCES IN HUMAN PLACENTAL MICRO RNA EXPRESSION A Osei-Kumah*, N Hodyl, W Kong, J Owens, V Clifton. University of Adelaide, Robinson Institute, Australia Introduction: We have previously identied sex specic differences in placental gene expression associated with strategies for optimal growth and fetal survival. Specically, we have shown differences in placental cytokine mRNA and glucocorticoid receptor expression. We have also identied sex specic differences in global placental gene expression. Micro RNAs (miRs) are non-coding small RNAs and act as important post-transcriptional regulators of gene expression by altering the abundance or translational efciency of mRNAs. The current study aims to examine miR expression in the placenta, to determine if they are involved in the regulation of differential gene expression observed in male and female placentae. Hypothesis: We hypothesise that post-transcriptional regulation of genes by miRs play an important role in conferring sexual dimorphism in placental gene expression during development. Methods: RNA was extracted from male (n6) and female (n6) placenta from normal pregnancies and miR analysis was conducted using exiqon arrays. Target prediction algorithm database was used to identify predicted targets for identied miRs. Ingenuity Pathways Analysis (IPA) software was used to identify functional networks of differentially expressed miRs. Results: One hundred and six miRs were differentially expressed between male and female placentae based on a P-value of less than 0.05. Sixty miRs were up-regulated and 46 were down-regulated in female placentae compared with males. Most of the differentially expressed miRS were clustered on chromosomes 13, 14, 16,17, 19, 20 and X. Pathway analysis identied networks involved in innate immune activation, cytokine signalling, glucocorticoid receptor signalling, cellular growth and proliferation. Conclusions: There are differentially expressed miRs in male and female placenta which target genes involved in cytokine gene expression and other immune pathways in the placenta. This may be related to the differential gene regulatory mechanisms initiated by males and females in-utero for growth and survival. Keywords: micro RNA, placenta, gene expression, cytokine
[P17.03]. EFFECT OF TESTOSTERONE ON PLACENTAL CYTOKINE PRODUCTION AND p38MAPKINASE M Stark*1, N Hodyl1, N Scott2, E Green2, A Osei-Kumah1, V Clifton1. 1The University of Adelaide, Australia, 2The University of Newcastle, Australia Introduction: Testosterone is known to have a suppressive effect on innate immune function that may mediate the differential responses we have previously demonstrated in pro-inammatory cytokine expression between male and female placenta. Activation of p38MAPkinase is central to pro-inammatory cytokine production. We questioned whether placental cytokine production and p38MAPkinase activity differed in a sex-specic manner, and were altered in the presence of testosterone. Hypotheses: We hypothesise that placental TNFa production will be greater in females than males due to increased activation of the p38 MAPkinase signalling pathway. This sexually dimorphic production of TNFa will be decreased in female placentae in the presence of testosterone. Methods: Placentae were collected from women pregnant with a male (n9) or female (n7) infant. Placental p38MAP kinase protein and activity were measured by Western blot and activity assay, respectively. Placental explants were stimulated with LPS (1ng/ml) in the presence and absence of testosterone. Supernatant was collected and tumour necrosis factor (TNF) alpha concentrations were determined (ELISA). Results: Placental p38MAPkinase levels and activity were increased in females compared to males (p<0.05). Following LPS stimulation, increased TNFa concentrations were observed in placentae from females compared to males (p<0.05). This was signicantly reduced by co-treatment of explants with testosterone (p<0.05), to a level equivalent to males. Conclusions: This data suggests that the sex-specic levels and activation of p38MAPkinase results in increased TNFa production in female compared to male placentae. Testosterone appears to play a key regulatory role in this pathway, as the sex-specic differences in cytokine production are abolished in its presence. This may be due to testosterone mediated increases in p38 MAP kinase phosphorylation that has been previously reported in the adult human literature. Whether a similar mechanism exists in the human placenta is a focus of our ongoing research. Keywords: cytokine, testosterone, p38MAP kinase, sex differences
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[P18.01]. IL-6 PROMOTES SHEDDING OF NECROTIC TROPHOBLASTS FROM PLACENTAL EXPLANTS LM Chen*1,2, B Liu2, HB Zhao1, P Stone2, L Chamley2, Q Chen1,2. 1The Hospital of Obstetrics & Gynaecology, Fudan University, China, 2Department of Obstetrics & Gynaecology, The University of Auckland, New Zealand Preeclampsia (PE) is characterised by elevated maternal blood pressure, preceded by endothelial cell dysfunction. Trophoblasts shed from the placenta may be one of the factors that trigger PE. Women with PE frequently have elevated serum levels of inammatory markers such as, IL6 and TNF alpha but their functional signicance is unclear. In this study we investigated whether these or other cytokines can alter trophoblast shedding. Placental explants were treated with 9 different cytokines for 72 hours. Shed trophoblasts then were harvested using our published method. The numbers of trophoblasts shed were quantied by automated cell counter. Expression of active of caspases 3&7 by the shed trophoblasts was determined using a FLICA kit and confocal microscopy. The trophoblasts shed from cytokine-treated or control explants were exposed to endothelial cell monolayers and endothelial activation determined ELISA for cell surface ICAM-1. Treatment of explants with IL-6 caused a 50% increase (p0.001), while TNF alpha and TGFbeta1, caused smaller but statistically signicant increases in the numbers of trophoblasts shed from explants. Trophoblasts shed from explants treated with IL-6, TGF beta1, or TGFbeta3 expressed signicantly less active caspases 3&7 than controls or trophoblasts shed from explants treated with other cytokines. Exposing trophoblasts shed from IL-6- or TGFbeta1-treated explants to endothelial cells caused a signicant (P<0.001) increase in endothelial cell-surface ICAM-1 compared to controls. Normally trophoblasts shed from the placenta die by an apoptosis-like process and their phagocytosis is silent but a shift to shedding of necrotic trophoblasts can lead to endothelial cell activation. It remains unclear what might trigger a shift from apoptotic to necrotic trophoblast death. This study suggests that IL-6 and possibly other cytokines can alter both the number and the nature of shed trophoblasts such that, the trophoblasts are more necrotic and their phagocytosis by maternal endothelial cells could contribute to the pathogenesis of preeclampsia. Keywords: cytokines, trophoblast deportation, endothelium
[P18.02]. MEGALIN, A LDL RECEPTOR, MEDIATES INTERNALISATION ANTIPHOSPHOLIPID ANTIBODIES INTO SYNCYTIOTROPHOBLAST
OF
Q Chen*1,2, B Liu1, LM Chen2, P Stone1, LW Chamley1. 1Department of Obstetrics & Gynaecology, The University of Auckland, New Zealand, 2The Hospital of Obstetrics & Gynaecology, Fudan University, China Antiphospholipid autoantibodies (aPL) increase the risk of a women developing preeclampsia nine fold. Previously we have shown that adding aPL to placental explants resulted in the internalisation of the aPL into the syncytiotrophoblast and subsequently shed syncytial knots. The aPL also increased the number of necrotic syncytial knots shed from the explants. These necrotic syncytial knots in turn activated endothelial cells. The mechanism by which aPL were internalised into the syncytiotrophoblasts is unclear but aPL bind to the plasma protein b2 glycoprotein I which is transported into epithelial cells via megalin, an endocytic LDL receptor. Here we investigated whether aPL are internalised into the syncytiotrophoblast via megalin. Placental explants were treated with monoclonal aPL (ID2 or IIC5) or a control antibody (CD45) in the presence or absence of the megalin inhibitors, receptor associated protein (RAP), or 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB). Internalization of the antibodies was monitored by confocal microscopy. Effects on endothelial cell activation were determined by adding trophoblasts shed from explants treated with aPL, or a control antibody, +/- megalin inhibitors, to endothelial cell monolayers and monitoring the expression of cell surface ICAM-1 by ELISA. Confocal microscopy conrmed that aPL, but not control antibodies, were internalised into the trophoblasts and that both megalin inhibitors blocked this internalisation. Furthermore, trophoblasts shed from explants treated with aPL, but not control antibodies, activated endothelial cell monolayers while trophoblasts shed from explants treated with aPL plus megalin inhibitors did not activate endothelial cells. These results suggest that megalin is involved in the selective transport of aPL in the syncytiotrophoblast and that once internalised, via megalin, aPL lead to aberrant shedding of trophoblasts. Since both altered trophoblast shedding and endothelial cell activation are thought to be involved in the pathogenesis of preeclampsia this might explain why aPL predispose women to developing preeclampsia. Keywords: antiphospholipid antibodies, megalin, internalisation, endothelial cell activation
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[P18.03]. THE ABILITY OF TROPHOBLASTS TO INTERNALIZE ANTIPHOSPHOLIPID ANTIBODIES CORRELATES WITH MEGALIN EXPRESSION C Viall*, Q Chen, S Lau, P Stone, L Chamley. Department of Obstetrics & Gynaecology, The University of Auckland, New Zealand Antiphospholipid antibodies (aPL) are autoantibodies that, by an unknown mechanism, increase the risk of developing preeclampsia nine fold. We have recently shown that aPL cause an increase in the number of syncytial knots shed from placental explants and a change in the trophoblast death process towards a more necrosis-like death, which might contribute to the pathogenesis of preeclampsia. Prior to inducing changes in trophoblasts aPL, but not control antibodies, are internalised into the syncytiotrophoblast of explants suggesting a specic mechanism for internalisation of the aPL. Internalisation of aPL into the syncytiotrophoblast appears to be required for aPL to affect trophoblast shedding and we have preliminary evidence that aPL-internalisation is mediated by the endocytic receptor, megalin. Monoclonal aPL IIC5 (13mg/ml)or ID2 (6mg/ml), were incubated with monolayers of the choriocarcinoma cell lines, Jar, Jeg-3, BeWo, or rst trimester placental explants for 24 hours. Internalisation of aPL into trophoblasts was visualised following uorescent immuno-staining. The expression of megalin by the cell lines and explants was examined by immuno-staining using an afnity-puried rabbit anti-megalin (Sigma). Experiments were repeated at least three times. Confocal microscopy demonstrated that BeWo cells, but not Jar or Jeg3 cells, were able to internalise the aPL. Despite treating explants and BeWos with the same amount of aPL, the level of antibody internalised within BeWos was less than internalised into the syncytiotrophoblast of explants. Megalin was expressed by BeWos but not Jars or Jeg3 cells. However, BeWos expressed substantially less megalin than the syncytiotrophoblast of placental explants. The internalization of aPL into syncytiotrophoblasts may play an important role in regulating the trophoblast death process that leads to the shedding of syncytial knots, this study adds to our previous data indicating that megalin is likely to be the receptor that mediates aPL internalization into trophoblasts making this pathway a potential therapeutic target. Keywords: antiphospholipid, megalin, internalisation
[P18.04]. ANTIPHOSPHOLIPID ANTIBODIES INDUCE DEATH AND EXCESS SHEDDING OF SYNCYTIAL KNOTS BY DISRUPTING MITOCHONDRIAL FUNCTION Q Chen*1,2, Y Kang1, B Liu2, P Stone2, L Chamley2. 1The hospital of Obstetrics & Gynaecology, Fudan University, China, 2Department of Obstetrics & Gynaecology, The University of Auckland, New Zealand Excess trophoblast shedding and deportation is known to be associated with preeclampsia a hypertensive disease of pregnancy whose symptoms are preceded by endothelial cell activation. Antiphospholipid antibodies (aPL) are autoantibodies that increase the risk of developing preeclampsia nine fold. We have recently found that aPL are internalised into the syncytiotrophoblast of placental explants where they subsequently induce increased shedding of necrotic or aponecrotic trophoblasts that are characterised by signicantly reduced levels of active caspases 3&7. These more-necrotic trophoblasts can induce the activation of endothelial cells. Here we examine whether aPL alter the trophoblast death process via an effect on the mitochondria. Placental explants were treated with the monoclonal aPL IIC5 (13ug/ml) or ID2 (6ug/ml), or control antibodies, for 2 or 24 hours. Trophoblasts shed from the treated explants were collected and contaminating leucocytes removed. Mitochondrial membrane potential was examined using DiOC2 and confocal microscopy. Cytochrome C levels in shed trophoblasts were quantied by ELISA (R&D). Confocal microscopy showed that mitochondrial function was compromised in syncytial knots shed from aPL-treated explants after 2 hours and did not recover 24 hours post treatment. Signicantly higher levels of cytochrome C (P0.001) were released from the mitochondria of syncytial knots that were shed from aPL-treated explants than controls. Our current data, coupled with our previous data showing aPL reduce the levels of active caspases 3&7 in syncytial knots shed from aPL-treated explants, suggest that aPL, once internalised into the syncytiotrophoblast, disrupt normal mitochondrial function in this cell layer which then results in aberrant death of portions of the syncytiotrophoblast. The release of cytochrome C suggests apoptotic death, but the syncytial knots shed form aPLtreated explants are clearly more necrotic than apoptotic as demonstrated by their ability to activate endothelial cells leading us to conclude that aPL induce aponecrotic death in the syncytiotrophoblast. Keywords: antiphospholipid antibodies, mitochondrial, internalisation, cyto chrome C
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[P18.05]. EFFECT OF siRNA-MEDIATED KNOCKDOWN OF CAVEOLIN-1 ON OXIDATIVE STRESS-INDUCED TROPHOBLAST CELL DEATH GP Collett*, EA Linton, R Dragovic, CWG Redman, IL Sargent. University of Oxford, United Kingdom Objectives: Pre-eclampsia is associated with increased shedding of proinammatory placental debris which may occur as a result of exacerbated oxidative stress-induced cell death in the syncytiotrophoblast. Caveolin-1 (cav-1) is expressed in human trophoblast and has been shown to both promote and protect from oxidative stress-induced death in other cell types. Furthermore, in the BeWo human trophoblast cell line hydrogen peroxide (H2O2) treatment is associated with translocation of cav-1 from the plasma membrane to the mitochondria, thus potentially affecting the response to oxidative stress. In the present study we examined the effect of cav-1 knockdown on oxidative stress-induced BeWo cell death. Methods: BeWo cells were transfected with a cav-1 siRNA duplex (50nM) or a scrambled, non-silencing control siRNA. After 48h, when cav-1 knockdown was conrmed by western blotting, the transfection medium was removed and the cells were treated with hydrogen peroxide (0-5mM). Total cell death was assessed by measuring lactate dehydrogenase release into the culture medium, apoptosis was determined by assessing poly (ADP-ribosyl) polymerase cleavage, and necrotic cell death was measured by monitoring the release of high mobility group box-1 protein (HMGB1) into the culture medium. Results: H2O2 treatment resulted in a dose-dependent increase in BeWo cell death. The mode of cell death switched from predominantly apoptotic at low (<1mM) concentrations of H2O2 to predominantly necrotic at higher concentrations. Cav-1 knockdown had no effect on either the extent or mode of cell death at any of the H2O2 concentrations used, compared with non-silencing controls. Furthermore, intracellular levels of ATP, which fell rapidly upon H2O2 treatment, were unaffected by cav-1 knockdown. Conclusion: Our results suggest that cav-1 has no effect on H2O2-induced trophoblast cell death, and therefore may not play a role in the shedding of trophoblast debris as a result of exacerbated oxidative stress. Keywords: caveolin-1, trophoblast, oxidative stress
[P18.06]. NEW DIMENSIONS IN THE QUANTIFICATION AND CHARACTERISATION OF PLACENTAL AND OTHER CELLULAR MICROPARTICLES AND NANOPARTICLES IN NORMAL PREGNANCY AND PRE-ECLAMPSIA RA Dragovic*1, DS Tannetta1, P Harrison2, CWG Redman1, IL Sargent1. Nufeld Department of Obstetrics & Gynaecology, John Radcliffe Hospital, University of Oxford, United Kingdom, 2Oxford Haemophilia & Thrombosis Centre, Churchill Hospital, Oxford, United Kingdom
1
Introduction: Pre-eclampsia is associated with altered levels of circulating particles derived from the placenta, platelets, immune cells and endothelium. These particles are comprised of microparticles (MPs; 100 nm 1 mM) and nanoparticles (exosomes 30 nm 100 nm). We have previously measured total particles in plasma ultracentrifuge pellets using an ELISA, which does not distinguish between the two populations. We have therefore investigated the use of a new technology, Nanosight Tracking Analysis NTA (which can detect particles in the range of 30 nm - 1 mM) in parallel with ow cytometry to quantify and characterise MP subpopulations in pregnancy and pre-eclampsia. Methods: Control syncytiotrophoblast particles (STBMs) were prepared from normal caesarean section placentas. Particles were measured in both platelet free plasma (PFP) and PFP ultracentrifuge pellets from non-pregnant (nonP), normal-pregnant (normP) and pre-eclamptic (PET) women, using ELISA, NTA and ow cytometry (BD LSRII digital ow cytometer). Particle count and phenotype was measured by labelling with a novel combination of cellular particle markers (Lactadherin or Bodipy-maleimide) and antibodies to platelet (CD61) and placental particles (NDOG2). Results: Flow cytometric analysis resulted in >90% of STBMs labelling positive for NDOG2 (detection range 290nm - 1 mM). NanoSight analysis showed a highly polydisperse distribution (mean size w190 nm). In comparison to ow cytometry w350 fold more particles were detected and 85% were <290 nm. Total cellular marker positive particles and cellular marker/CD61 positive particles were signicantly increased in both PFP and the resuspended pellets from nonP vs normP and nonP vs PET samples. No NDOG2 particles from normP or PET samples were detected by ow cytometry, although NDOG2 positive particles were detected by ELISA and were signicantly elevated in PET vs normP samples. Discussion: NTA provides a powerful tool for cellular particle analysis and suggests the majority of these particles are <290 nm and are therefore too small to be detected by ow cytometry. Keywords: Microparticles, Nanoparticles, Pre-eclampsia
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[P18.07]. DEPTH OF TROPHOBLAST INVASION AND VASCULAR REMODELLING ON DAY 18 AND 21 OF A TRANSGENIC RAT MODEL FOR PRE-ECLAMPSIA Nele Geusens1, Catherine Luyten1, Lisbeth Vercruysse1, Myriam Hanssens*1, Robert Pijnenborg1, Ralf Dechend2. 1Dept Obstet & Gynaecol K.U.Leuven, Belgium, 2Dept Cardiology Charite University Medicine Berlin, Germany Background & Objectives: We studied trophoblast invasion and associated uterine spiral artery (SA) remodeling in a transgenic rat model with symptoms comparable to human pre-eclampsia (PE). Design & Methods: Transgenic Sprague Dawley (SD) rats bearing the human angiotensinogen gene and human renin gene were crossed to obtain PE (hAogen \ x hRen _), reversely mated (hAogen _ x hRen \) and a control group consisted ofnormal SP rats . Rats were sacriced on day 18 or day 21 of pregnancy and implantation sites were collected for histology and immuno stained for cytokeratin (MNF), smooth muscle cell a-actin, the endothelial marker CD31, and brinoid (PAS). The mesometrial triangle (MT) is divided into 3 equal parallel layers to evaluate depth of invasion. The KS-400 image analysis system is used to quantify endovascular trophoblast invasion and associated vascular changes of SA. Statistics are performed with the Mann-Whitney test. Results: Overall there is, in the entire MT, signicantly (P<0.001) more endovascular trophoblast overlying the SA lumen contours in the PE group compared to that observed in the control and RM group. This is particularly true in the deeper layers. Yet, control rats seem to catch up with the PE rats by day 21. Inspite of the deeper endovascular trophoblast invasion in the PE rats, vascular smooth muscle cell remodelling was less pronounced in that group as compared to that observed in the reversely mated and the control rats both at 18 and 21 days of pregnancy. Conclusion: The ndings of more endovascular trophoblast with less pronounced smooth muscle cell remodelling in the SA of the PE group are quite different from observations in human PE where less endovascular trophoblast and almost absent remodelling of the myometrial segment of the SA is seen. Keywords: trophoblast invasion, remodellling spiral arteries, transgenic rat model, pre-eclampsia
[P18.08]. P53 MEDIATED APOPTOSIS IN A MODEL OF HUMAN PLACENTAL VILLOUS FUNCTION A.N. Sharp, A.E.P. Heazell, P.N. Baker, I.P. Crocker*. University of Manchester, United Kingdom Introduction: The placental conditions of pre-eclampsia and intrauterine growth restriction are characterised by altered villous cell turnover and exaggerated expression of the cell regulator p53, implying a role in trophoblast apoptosis. In BeWo cells stimulation of the p53 pathway can be achieved with the small molecule activator, Nutlin-3, whilst p53 activity can be inhibited with a second compound, Pithrin-a, which may correct these aberrant apoptotic events. The effects of these compounds were tested in vitro on placental villous fragments. Methods: Third trimester placental explants from uncomplicated pregnancies were cultured at 6%O2 in the presence of Nutlin-3 alone or in combination with Pithrin-a. Culture medium was collected for assessment of lactate dehydrogenase (necrosis) and human chorionic gonadotrophin (hCG) secretion (trophoblast differentiation). Tissue was formalin xed and wax embedded for immunohistochemistry and quantitative PCR performed for mRNA expression. Trophoblast apoptosis was assessed by M30 staining and reported in proportion to villous area. Results: Nutlin-3 increased trophoblast apoptosis in cultured explants (control vs. 40mM Nutlin, 0.750.11 median (SD) vs. 3.520.38, p<0.01, Kruskal-Wallis, n5) with no effect on LDH or hCG release. Nutlin-3 treatment increased p21 gene expression (29.435.7 vs. 4.662.49, p<0.01, Mann-Whitney Test), but not p53 or Mdm2. Dual treatment with Nutlin-3 and Pithrin-a (5mM) returned apoptotic levels to control values (0.840.40 vs. 1.480.50, ns, Kruskal-Wallis, n5), suppressing Nutlin-3 induced apoptosis of trophoblast by 58%. Discussion: Nutlin-3 modulates p53 in third trimester placental trophoblasts, stimulating apoptosis rather than necrosis. Pithrin-a can be used to restrict this pathway, potentially offering a way of regulating excessive placental apoptosis. Further experiments will establish a role for these compounds in complicated pregnancies in vivo. Keywords: pre-eclampsia, p53, apoptosis, trophoblast
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[P19.01]. EXPRESSION OF ANGIOGENIC FACTORS IN PLACENTA OF STRESSED RATS Isis Paloppi Correa1, Rodrigo Ruano1, Nilton Hideto Takiuti1, Salet Terezinha de Souza1, Estela Bevilacqua2, Marcelo Zugaib*1. 1Obstetrics Department, Faculty of Medicine, University of Sao Paulo, Brazil, 2Institute of Biomedical Sciences, University of Sao Paulo, Brazil The aim of present study was to analyze the inuence of the stress in pregnant rats on gestational parameters, on the placental morphology and on the placental gene expression of VEGF, PlGF and, its receptors. These parameters were evaluated on gestation day 20 after chronic and acute stress protocols during gestation. Fetal and placental weights were statistically smaller in the stressed animals comparing to controls. Morphologically the placentas exhibited reduction in the junctional zone and degenerative cells. Although VEGF, PlGF, VEGFR1 and VEGFR2 were expressed in all placentas, stress condition signicantly changed the placental gene expression in comparison to control group, increasing VEGFA (p<0.05) and, reducing PlGF, VEGFR-1 and VEGFR-2 expression (p>0.05). In conclusion, the present study showed that in the stress pregnant group, placental and fetal growth restriction is observed, suggesting abnormalities in the gestational physiology. These animals showed persistent hypertension which suggests a hypotensive response such as those induced by VEGF; consistent with that, placental VEGF was increased. However, because at the same sites PlGF and receptors VEGFR1/ R2 expressions were relevantly reduced, it is possible that the VEGF action on stress-induced hypertension has been limited. This model of investigation shows at least one mechanism by which stress can act on the placental physiology and, may further help us understand stress-induced gestational disorders. Financial support: FAPESP and CNPq. Keywords: Stress, VEGF, PlGF, VEGFR
[P19.02]. MATRIX METALLOPROTEINASE-12, AN IMPORTANT MEDIATOR OF ELASTOLYSIS IN REMODELLING HUMAN SPIRAL ARTERIES? LK Harris*1, PN Baker1, JE Cartwright2, GS Whitley2, V Dive3, A Yiotakis4. University of Manchester, United Kingdom, 2St Georges University of London, United Kingdom, 3Institut de Biologie et de Technologies de Salcay, France, 4University of Athens, Greece
Introduction: Remodelling of the uterine spiral arteries involves degradation of elastin bres within the arterial media to facilitate a permanent increase in vessel diameter; however, the mediator(s) of elastolysis are currently unknown. We have previously shown that rst trimester cytotrophoblasts (CTB) and vascular smooth muscle cells (SMC) utilize matrix metalloproteinases (MMP) to degrade elastin in vitro. As SMC and CTB express high levels of the elastolytic enzyme MMP-12, we hypothesized that these cells employ MMP-12 to catabolise elastin. Methods: CTB were isolated from rst trimester placenta by trypsin digestion and density gradient centrifugation. Triton-X100 extracts of CTB and SMC were incubated with the elastase substrate N-succinyl-(Lalanine)3-p-nitroanilide, in the presence of a broad spectrum MMP inhibitor or a specic inhibitor of MMP-12. Elastase activity was calculated from a standard curve prepared using porcine pancreatic elastase. CTB cultured with elastin in the presence or absence of MMP inhibitors were stained with an antibody to cytokeratin-7, and the number of cells engulng elastin bres was quantied. Results: Triton-X100 extracts of SMC and CTB exhibited MMP-dependent elastase activity, consistent with the presence of membrane-associated elastases. A specic inhibitor of MMP-12 reduced elastolysis to 23.38.7% of control activity in SMC and 31.710.9% in CTB, compared to a broad spectrum MMP inhibitor which reduced activity to 2.652.7% in SMC and 0.130.1% in CTB. Inhibition of MMP activity did not reduce the percent of cells containing intracellular elastin fragments. Discussion: These data implicate MMP-12 as a primary mediator of elastolysis in rst trimester CTB and SMC, but demonstrate that MMP-12 is not required for elastin engulfment. As we have previously shown that MMP12 expression is induced in the media of excised spiral arteries cultured with CTB-conditioned medium, we hypothesise that the coordinated actions of invasive trophoblast and spiral artery SMC mediate local elastin catabolism during vessel transformation. Keywords: trophoblast invasion, MMP-12, elastin, arterial remodelling
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[P19.03]. LEUKOCYTE MEDIATED MECHANISMS OF VASCULAR REMODELING IN VITRO A D Hazan*1,2, S D Smith3,4, R L Jones3,4, S J Lye1,2, C E Dunk2. 1University of Toronto, Canada, 2Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Canada, 3University of Manchester, United Kingdom, 4St. Marys Hospital, United Kingdom Introduction: While there is an abundant population of immune cells in the decidua, their functional role in vascular transformation has yet to be fully established. In this study we investigate the association of uterine Natural Killer cells (uNK) and macrophages with vascular remodeling during early gestation and the potential mechanisms by which they regulate these processes. Methods: Patient-matched rst trimester (6-9 weeks) placental and decidual tissues are collected. Decidual explants are cultured alone (n13) or with placenta (n13) over 6 days. Placenta-induced vascular transformation and leukocyte localization in these decidua are assessed by immunohistochemistry and quantitative image analysis at increasing depths from the site of placental contact (0-500, 500-1000, & 10002000mm) and 15mm radiating distances from vessel lumens. Results: Vascular transformation is indicated by a decrease (p<0.05) in smooth muscle actin/vessel lumen area in the proximal 1000mm of cocultures compared to decidual controls. In actively remodeling vessels at day 3 there is an increase (p<0.0003) in the number of uNK and macrophages within 15mm of the vessel lumen. Farther from the vessel lumen (15-30mm) there is an increase in macrophages (p<0.01). Concurrently there is disruption of the vascular smooth muscle cell (vsmc) layers, separation and migration of individual vsmc, and gradual loss of smooth muscle actin staining, suggesting dedifferentiation of vsmc. We also observe matrix metalloproteinase(MMP)-9 expression by uNKs and vascular cells. Inhibition of MMP-2/9 results in similar smooth muscle/ lumen ratios between co-cultures and controls. TUNEL staining demonstrates that vsmc and endothelium are apoptotic in remodeling vessels and dual immunouorescence using lysozyme muramidase indicates macrophage-mediated phagocytosis. Discussion: These experiments identify, for the rst time, potential mechanisms involved in decidual vascular remodeling induced by the presence of placenta. Temporal differences in macrophage and uNK association with vessels during remodeling indicate distinct functional roles for these cells in a tightly regulated process. Keywords: leukocytes, decidua, trophoblast, vascular remodeling
[P19.04]. NEW ROUTES OF TROPHOBLAST INVASION? INVESTIGATIONS WITH A CO-CULTURE CONFRONTATION MODEL SYSTEM G Moser*, K Orendi, M Gauster, M Siwetz, N Flieser, B Huppertz. Medical University Graz, Austria Objectives: Secretory products delivered by the uterine glands into the intervillous space prior to the establishment of maternal blood ow into the placenta are considered to play an important role in the nutrition of the embryo (Burton et al. 2002). However, these authors did not provide any idea of how such secretory products may reach the intervillous space. Therefore, we tested putative new routes of trophoblast invasion with double tissue co-culture in-vitro confrontation models for trophoblast invasion. Methods: Pieces of decidua parietalis (6-10 weeks gestation) were confronted directly with villous explants from the same pregnancy (direct confrontation without epithelium) or after separate preculture for 72h (indirect confrontation with epithelium). For indirect confrontation decidua pieces re-epithelialized during preculture under constant agitation. All confrontations were harvested after 72h, cryosectioned and processed for immunohistochemistry and/or double labeling immunouorescence using antibodies against cytokeratin 7, HLA-G and entactin. Results: In both models (direct and indirect confrontation) confrontation of villous and decidual tissues resulted in formation of trophoblastic cell columns and their adhesion to decidual tissues. Immunohistochemical staining with antibodies against HLA-G showed invasion of extravillous trophoblast (EVT) into decidual tissues. Immunouorescent double labeling against either HLA-G/entactin or cytokeratin 7/entactin showed that EVT invaded through the decidual stroma and along the basal lamina of decidual epithelium and glands. Thus, during invasion along the basal lamina of the epithelium EVT reached glands and single trophoblasts were also found in the lumen of glands. Conclusions: Enders et al. (1983) showed that in the rhesus monkey extravillous trophoblast extended along the basal lamina of the uterine epithelium and into the neck of adjacent uterine glands. Our data show that extravillous trophoblast in the human may follow similar routes, so far not described yet. They may reach and open uterine glands to facilitate histiotrophic embryo nutrition. Keywords: uterine glands, confrontation model, extravillous trophoblasts, histiotrophic nutrition
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[P19.05]. MECHANISM OF PROPROTEIN CONVERTASE 6 ACTION DURING DECIDUALIZATION: REGULATION OF BONE MORPHOGENETIC PROTEIN 2 ACTIVATION S Heng, B Hardman, S Paule, H Singh, L Salamonsen, G Nie*. Prince Henrys Institute of Medical Research, Melbourne, Australia Introduction: Proprotein convertase 5/6 (PC6), a member of the proprotein convertase (PC) family, is a critical endometrial factor for implantation. PC6 is up-regulated in the endometrium specically at implantation in association with epithelial differentiation (in human and monkey) and stromal cell decidualization (in the mouse, human and monkey). Knockdown of PC6 inhibits decidualization. PCs function by converting a range of important precursor proteins into their bioactive forms. One group of such proteins are the transforming growth factor beta (TGF-beta) superfamily proteins. They are rst synthesized as larger biologically inactive precursors, and then are processed by PCs into their active forms. Bone morphogenetic protein 2 (BMP2) is a TGF-beta superfamily member and demonstrated to be essential for decidualization. We hypothesized that BMP2 is one of the proteins that PC6 activates during decidualization. Methods: Freshly isolated stromal cells from human endometrium were decidualized in culture with and without inhibition of PC6 activity. The full-length (precursor, non-active) and processed (activated) forms of BMP2 were determined in cellular lysates and media. The proteolytic processing of BMP2 by PC6 was conrmed in vitro. To further conrm the role of PC6 in activating BMP2 in decidualization, active BMP2 was added into cells to rescue decidualization arrest caused by PC6 inhibition. Results: PC6 was the only PC member that was signicantly up-regulated during decidualization. The precursor form of BMP2 was reduced whereas the active form was increased during decidualization. Inhibition of PC6 activity inhibited decidualization, and this inhibition was accompanied by a total inhibition of the production of active BMP2. Addition of recombinant active BMP2 partially rescued the decidualization arrest caused by PC6 inhibition. Discussion: Both PC6 and BMP2 are known to be critical for decidualization. This study demonstrated that PC6 regulates decidualization by activating molecules such as BMP2 that are essential for decidualization. Keywords: PC6, BMP2, Decidualization, Activation
[P19.07]. IDENTIFICATION OF CHEMOKINES INVOLVED IN RECRUITMENT OF DECIDUAL LEUKOCYTES TO REMODELLING SPIRAL ARTERIES SD Smith*1, JD Aplin1, LK Harris1, CE Dunk1,2, RL Jones1. 1University of Manchester, United Kingdom, 2University of Toronto, Canada Introduction: Remodelling of uterine spiral arteries in the rst 20 weeks of gestation is essential for a healthy pregnancy. We have recently shown that macrophages and uNK cells inltrate the wall of remodelling decidual spiral arteries prior to extravillous trophoblast (EVT) colonisation, providing evidence for a role for decidual leukocytes in the early stages of remodelling. However, leukocytes do not inltrate the wall of arteries distant from the implantation site (decidua parietalis) suggesting a local trigger for leukocyte recruitment. We hypothesized that trophoblastsecreted factors activate spiral arteries to produce chemokines, which chemoattract leukocytes into remodelling vessels. Methods: Decidual endothelial cells were isolated from rst trimester decidua (8-12 weeks gestation). Vascular smooth muscle cells (VSMC, derived from human aorta) and decidual endothelial cells were treated with EVT-conditioned medium generated from villous tip outgrowths. Chemokine expression by treated and untreated VSMC and endothelial cells was analysed using an unbiased PCR genearray approach, to examine expression of 80 chemokines/cytokines/receptors. Immunohistochemistry for selected chemokines in decidua basalis (8-12 weeks gestation) was performed to validate expression patterns in vivo. Results: 40 genes were differentially regulated in both VSMC and decidual endothelial cells in response to EVT conditioned medium. Of particular interest were macrophage chemoattractants: CCL5, CCL7, CCL3 and CCL4, that were upregulated between 2-36 fold in treated compared to control in both cell types. Furthermore, uNK chemoattractants CXCL12 and CX3CL1 were upregulated in VSMC and endothelial cells respectively. Immunohistochemistry of decidua basalis conrmed CXCL12, CCL4 and CX3CL1 localisation to spiral artery VSMC and endothelial cells. Discussion: These data suggest that factors secreted by EVT promote chemokine expression by vascular cells. We have identied chemoattractants released by vascular cells that may recruit macrophages (CCL5, CCL7, CCL3 and CCL4) and uNK cells (CXCL12 and CX3CL1) to spiral arteries, where they participate in the early stages of remodelling. Keywords: Decidua, Leukocytes, Trophoblast, Chemokines
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[P19.08]. PRODUCTION OF ANGIOPOIETIN-2 BY DECIDUAL ENDOTHELIAL CELLS IN HYPOXIA, AND IN FIRST VERSUS THIRD TRIMESTER MATERNAL SERUM CA Woolnough*1,2, Y Wang2, V Tasevski1,2, E Gallery2, J Morris2. 1 Fetal Maternal Medicine (PaLMS), Royal North Shore Hospital, Australia, 2 Perinatal Research, Kolling Institute, Royal North Shore Hospital, Australia Introduction: During pregnancy the placenta, fetus and uterus undergo angiogenesis which is accompanied by a rise in Angiopoietin-2 (Ang-2) levels in maternal serum peaking in the rst trimester and gradually declining throughout gestation. The rst objective of this study was to determine whether decidual endothelial cells (DECS) may be the source of the Ang-2 observed in maternal serum. The second objective was to investigate potential factors which may regulate Ang-2 production by DECS (1) rst compared to third trimester maternal serum and (2) hypoxia. Methods: DECS (n8) were isolated from maternal decidua obtained at caesarean section clinically following normal pregnancies, and cultured until passage ve. DECS were exposed to hypoxia (1% and 3%) and normoxia. In each oxygen condition, DECS were cultured in rst trimester and third trimester serum. DEC supernatants were collected and assayed for Ang-2 using ELISA, and Ang-2 in cellular lysates was measured by western blotting. Cellular RNA was extracted for reverse transcriptase PCR and Ang-2 semi-quantication. Results: Ang-2 was detected in DEC supernatant, cellular protein and cellular RNA. DECS released statistically signicantly more Ang-2 when cultured in rst compared to third trimester serum, and when cultured in 1% compared with 3% hypoxia and normoxia (Figure 1). Cellular Ang-2
levels followed similar patterns however Ang-2 RNA levels were not signicantly different across the culturing conditions.
Figure 1. Ang-2 released by DECS under different culturing conditions *p<0.05 Ang-2 levels are higher in rst compared to third trimester serum, D p<0.05 Ang-2 levels are higher in 1% hypoxia compared to normoxia.
Discussion: DECS release substantial amounts of Ang-2 indicating that these cells are a source of the Ang-2 seen in maternal serum. Protein levels of Ang-2 are higher when DECS are cultured in rst trimester serum and in 1% hypoxia indicating that serum factors in addition to a hypoxic environment may trigger DECS to produce more Ang-2 in the rst trimester. There were no signicant differences in Ang-2 RNA levels across the different treatments indicating that DEC Ang-2 is controlled at translation. Keywords: Angiopoietin-2, Hypoxia, Decidua, Endothelial cells

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Placenta 30 (2009) A.1A.

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38.0 2.0 52.1 1.7* 143.4 7.1 139.1 6.3

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N10. THE REGULATORY ROLE TROPHOBLAST INVASION

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N12. GENOMIC INVESTIGATION NETWORK

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[P02.03]. HEME OXYGENASE-1 GENE PLACENTA OF PREECLAMPSIA

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Table 1: miRNAs that changed in expression with syncytialisation.

Keywords: microRNA, syncytialisation, BeWo cells, microarray

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Keywords: glucocorticoids, mRNA, hypoxia, microarray

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[P04.14]. CD11c IDENTIFIES MACROPHAGES

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[P05.06]. BOVINE TROPHOBLAST EMBEDDED SPHEROIDS

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Keywords: Ectopic pregnancy, Epidermal growth factor, Methotrexate, Therapy

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[P06.30]. DETERMINATION EMBRYOGENESIS

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