Pregnancy complications associated with placental development affect approximately one third of all human pregnancies. Genome health is essential for placental and fetal development, as DNA damage can lead to pregnancy loss and developmental defects. During this developmental phase rapid DNA replication provides an increased opportunity for genome and epigenome damage to occur[1]. Maternal nutrition is one of the principal environmental factors supporting the high rate of cell proliferation and differentiation. Folate functions in one-carbon metabolism and regulates DNA synthesis, DNA repair and gene expression[1]. Deficiencies or defects in gene-nutrient interactions associated with one-carbon metabolism can lead to inhibition of cell division, cell cycle delay and an excessive apoptotic or necrotic cell death rate [2], which may affect placentation. This study is the first to investigate the association between genomic damage biomarkers in late pregnancy complications associated with uteroplacental insufficiency (UPI) including preeclampsia and intrauterine growth restriction (IUGR). The results indicate that genome damage in the form of micronucleated cells in peripheral blood lymphocytes at 20 weeks gestation is significantly increased in women at risk of developing an adverse pregnancy outcome. The observed OR for the high micronuclei frequency may be the highest observed for any biomarker selected in relation to risk of pregnancy complications to date (15.6 – 33.0). In addition, reduced apoptosis was observed in association with increased micronuclei, suggesting that the cells may have escaped specific cell-cycle checkpoints allowing a cell with DNA damage to proceed through mitosis. This study demonstrated that an increase in plasma homocysteine concentration at 20 weeks gestation is associated prospectively with the subsequent development of UPI, indicating a causal relationship. The MTR 2756 GG genotype was significantly associated with increased plasma homocysteine concentration and UPI. Furthermore, the MTHFD1 1958 single nucleotide polymorphism was associated with increased risk for IUGR. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1309296 / Thesis (Ph.D.) -- School of Paediatrics and Reproductive Health, 2007

Pregnancy complications associated with placental development affect approximately one third of all human pregnancies. Genome health is essential for placental and fetal development, as DNA damage can lead to pregnancy loss and developmental defects. During this developmental phase rapid DNA replication provides an increased opportunity for genome and epigenome damage to occur[1]. Maternal nutrition is one of the principal environmental factors supporting the high rate of cell proliferation and differentiation. Folate functions in one-carbon metabolism and regulates DNA synthesis, DNA repair and gene expression[1]. Deficiencies or defects in gene-nutrient interactions associated with one-carbon metabolism can lead to inhibition of cell division, cell cycle delay and an excessive apoptotic or necrotic cell death rate [2], which may affect placentation. This study is the first to investigate the association between genomic damage biomarkers in late pregnancy complications associated with uteroplacental insufficiency (UPI) including preeclampsia and intrauterine growth restriction (IUGR). The results indicate that genome damage in the form of micronucleated cells in peripheral blood lymphocytes at 20 weeks gestation is significantly increased in women at risk of developing an adverse pregnancy outcome. The observed OR for the high micronuclei frequency may be the highest observed for any biomarker selected in relation to risk of pregnancy complications to date (15.6 – 33.0). In addition, reduced apoptosis was observed in association with increased micronuclei, suggesting that the cells may have escaped specific cell-cycle checkpoints allowing a cell with DNA damage to proceed through mitosis. This study demonstrated that an increase in plasma homocysteine concentration at 20 weeks gestation is associated prospectively with the subsequent development of UPI, indicating a causal relationship. The MTR 2756 GG genotype was significantly associated with increased plasma homocysteine concentration and UPI. Furthermore, the MTHFD1 1958 single nucleotide polymorphism was associated with increased risk for IUGR. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1309296 / Thesis (Ph.D.) -- School of Paediatrics and Reproductive Health, 2007

La placentation humaine est de type hémomonochoriale, le sang maternel est directement en contact avec le syncytiotrophoblaste. Les flux sanguins maternels, dans la chambre intervilleuse, exercent des forces mécaniques de cisaillement (shear stress) sur la surface microvillositaire du syncytiotrophoblaste. Les effets physiologiques du shear stress exercé par les flux sanguins sur l’endothélium vasculaire artériel et veineux ont fait l’objet de nombreux travaux scientifiques. En revanche, les effets biologiques du shear stress sur le syncytiotrophoblaste humain n’ont jamais été explorés. L’objectif de ce travail était premièrement d’évaluer les valeurs du shear stress exercé in vivo sur le syncytiotrophoblaste humain au cours des grossesses normales, puis de mettre au point un modèle de culture primaire dynamique afin de reproduire les conditions physiologique et d’étudier in vitro la réponse biologique du syncytiotrophoblaste au shear stress. En dépit d’un débit sanguin maternel intraplacentaire important, estimé entre 400 et 600 mL.min-1, le shear stress moyen exercée par le syncytiotrophoblaste est estimée entre 0.5±0.2 et 2.3±1.1 dyn.cm-2. Nos résultats montrent cependant que l’intensité du shear stress est très hétérogène tant à l’échelle de la chambre intervilleuse que de la villosité terminale. Nous avons développé un modèle de culture cellulaire dynamique en condition de flux adapté au syncytiotrophoblaste humain. Ce modèle permet d’appliquer un shear stress égal et constant sur toutes les cellules cultivées et reproductible à chaque culture primaire. Aux gammes de shear stress étudiées (1 dyn.cm-2), nous n’avons pas mis en évidence de diminution de la viabilité cellulaire ni de déclenchement des processus précoces d’apoptose en conditions dynamiques comparativement aux conditions statiques. Deux types de chambre de perfusion permettent d’étudier des réponses cellulaires au shear stress à court et long terme selon des temps d’exposition allant de 5 minutes à 24 heures. Ce modèle expérimental a permis de montrer que le syncytiotrophoblaste humain en culture primaire est mécanosensible. La réponse cellulaire à des niveaux de shear stress de 1 dyn.cm-2 est multiple selon les temps d’exposition et le niveau d’intégration étudié. Après 45 minutes de shear stress les taux d’AMP cyclique intracellulaires sont augmentés ce qui a pour effet d’activer la voie de signalisation intracellulaire PKA-CREB. Cette augmentation d’AMP cyclique est secondaire à la synthèse et la libération de prostaglandine E2 qui, par une boucle de régulation autocrine stimule l’adenylate cyclase. L’augmentation de la synthèse/libération de PGE2 est dépendante de l’augmentation rapide du calcium intracellulaire sous shear stress. L’exposition au shear stress de 24 heures stimule l’expression et la sécrétion du PlGF, un facteur de croissance indispensable à l’angiogenèse placentaire et pour l’adaptation maternelle à la grossesse sur le plan vasculaire. Nos travaux montrent que l’augmentation de l’AMPc intracellulaire et l’activation de la PKA contribuent à la phosphorylation de CREB, facteur de transcription régulant l’expression du PlGF. / Human placentation is hemomonochorial, maternal blood circulates in direct contact with the syncytiotrophoblast. In the intervillous space, the maternal blood exerts frictional mechanical forces (shear stress) on the microvillous surface of the syncytiotrophoblast. Flowing blood constantly exerts a shear stress, on the endothelial cells lining blood vessel walls, and the endothelial cells respond to shear stress by changing their morphology, function, and gene expression. The effects of shear stress on the human syncytiotrophoblast and its biological functions have never been studied. The objectives of this study were (1) to determine in silico the physiological values of shear stress exerted on human syncytiotrophoblast during normal pregnancies, (2) to develop a model reproducing in vitro the shear stress on human syncytiotrophoblast and (3) to study in vitro the biological response of human syncytiotrophoblast to shear stress. The 2D numerical simulations showed that the shear stress applied to the syncytiotrophoblast is highly heterogeneous in the intervillous space. In spite of high intraplacental maternal blood flow rates (400-600mL.min-1), the estimated average values of shear stress are relatively low (0.5±0.2 to 2.3±1.1 dyn.cm-2). To study the shear stress-induced cellular responses during exposure times ranging from 5 minutes to 24 hours we have developed two dynamic cell culture models adapted to the human syncytiotrophoblast. We found no evidence of decreased cell viability or early processes of apoptosis in dynamic conditions (1 dyn.cm-2, 24h) compared to static conditions. Shear stress (1 dyn.cm-2) triggers intracellular calcium flux, which increases the synthesis and release of PGE2. The enhanced intracellular cAMP in FSS conditions was blocked by COX1/COX2 inhibitors, suggesting that the increase in PGE2 production could activate the cAMP/PKA pathway in an autocrine/paracrine fashion. FSS activates the cAMP/PKA pathway leading to upregulation of PlGF in human STB. Shear stress-induced phosphorylation of CREB and upregulation of PlGF were prevented by inhibition of PKA with H89 (3 μM). The syncytiotrophoblast of the human placenta is a mechanosenstive tissue.