Abstracts / Placenta 35 (2014) A1eA112 A7

tissues, ex vivo explants and trophoblast cell culture systems, ADAM12wasimmunolocalized to the distal ends of EVT columns and to highly-invasivematrix-degrading EVTs. Notably, a specific role for the secreted isoform,ADAM12S, in promoting trophoblast invasion, as well as EVT columnoutgrowth was demonstrated. ADAM12 gene expression and promoteractivity were shown to be in part regulated by cyclic adenosine 30-50-monophosphate (cAMP), and this effect was independent of PKA activity.We provide evidence that cAMP-directed induction of ADAM12 iscontrolled by the guanine nucleotide exchange factor Epac1, where inhi-bition of Epac1/Rap1a blocks EVT invasion. Our findings demonstrate theimportance of ADAM12 in directing EVT column formation and tropho-blast cell invasion, and highlight novel upstream factors controllingADAM12 expression in the early placenta.

SIFPAA.EX VIVO DUAL PERFUSION OF A HUMAN PLACENTAL COTELYDON e

MODIFICATIONS OF ACCESS TO THE INTERVILLOUS SPACE

Henning Schneider Department of Obstetrics and Gynecology, InselspitaleUniversity of Berne, Berne, Switzerland

Ex vivo dual perfusion of a human placental cotelydon was first describedby M. Panigel in Paris in 1967. After cannulation of a chorionic arterial andvenous branch the corresponding segment of the fetal vasculature wasperfused. A remnant of a spiral artery of the same cotyledon was cathe-terized. Imaging by cineradioangiography showed, that the flow picture ofthe fetal and maternal circuit for that one cotyledon came very close to thein vivo situation, which had been shown in Rhesus monkeys. In an attemptto simplify access to the IVS an adaptationwith penetration of the decidualplate with 3 to 5 cannulae was introduced. Energy dependant functionssuch as active transport of aminoacids from thematernal to the fetal circuitwere still intact inspite of a delay of 20 to 30 min. needed to set up thepreparation. This unexpected tolerance of prolonged ischemia may beexplained by down regulation of metabolism as had been described for thetransitional stage preceding torpor in hibernating animals. With mediumwith physically dissolved oxygen the supply of oxygen is only a fraction ofthe in vivo estimate of requirement. The special tolerance of chronichypoxia of the perfused tissue can partially be explained by metabolicreprogramming with reduction in mitochondrial oxygen consumption andan increase in anaerobic glycolysis. Improving oxygen supply remains amajor challenge for ex vivo dual perfusion. Different modes of increasingthe oxygen carrying capacity of the medium such as equilibrationwith 95%of oxygen or use of erythrocytes or synthetic oxygen carriers have beenunsatisfactory. Recent studies with direct measurement of oxygen contentat different sites inside the IVS have shown that by increasing the numberof maternal cannulae to 22 a considerable improvement in distribution ofperfusate inside the IVS can be achieved. A systematic study to determinethe optimal number of cannulae for the maternal perfusion circuit is inprogress.

THAN.IDENTIFICATION OF PLACENTAL FAILURE e THE KEY TO SAVINGBABIES’ LIVES?

Alexander Heazell a,b aMaternal and Fetal Health Research Centre,University of Manchester, Manchester, UK; b St Mary's Hospital, ManchesterAcademic Health Science Centre, Manchester, UK

In high-income countries approximately 1 in 200 babies are stillborn.Stillbirth is associated with placental disease in up to 65% of cases.Detailed examination of the placenta offers the opportunity to describeprocesses leading to stillbirth, so that deaths might be prevented. Ourresearch programme focuses on understanding placental abnormalities instillbirth and applying this knowledge to identify pregnancies at greatestrisk.

Our systematic review verified that stillbirth is associated with a variety ofplacental abnormalities ranging from small placental size to specific his-topathological entities. However, interpretation of these findings is

hampered by variation in classification of “placental causes” of stillbirthand description of lesions. Our morphometric analyses demonstrated thatstillbirths associated with fetal growth restriction (FGR) have a specificplacental phenotype that is not associated with artefacts of storage orabsent fetal circulation. This phenotype is more severe than in live bornFGR infants and is evident in some stillbirths currently classified as “un-explained”. A similar analytical approach identified abnormalities ofplacental structure and dysfunction in groups at increased risk of stillbirth,including women reporting reduced fetal movements (RFM) or advancedmaternal age. Thus, some placental causes of stillbirth display a recog-nisable phenotype, opening the possibility that this dysfunction could bedetected antenatally.We therefore tested the hypothesis that adverse pregnancy outcome (APO)can be identified using markers of fetal wellbeing and placental function.In RFM, APO was associated with abnormal fetal heart rate, a small forgestational age fetus and low levels of progesterone, hCG and hPL; low hPLwas associated with a 7-fold increase in APO. A pilot randomisedcontrolled trial comparing standard management vs. intervention guidedby ultrasound + hPL measurement found a statistically significant reduc-tion in APO, with no increase in maternal anxiety. Thustargeted inter-vention in women with evidence of placental dysfunction may reducestillbirths.

NIH.HUMAN STEM CELLS FROM SINGLE BLASTOMERES REVEAL PATHWAYSOF EMBRYONIC OR TROPHOBLAST FATE SPECIFICATION

Susan Fisher University of California, San Francisco, San Francisco, CA, USA

There are major mechanistic differences among species in how initial cellfate decisions are made in embryos. To gain insights into lineage allocationin humans, we derived ten human embryonic stem cell lines from singleblastomeres of four 8-cell embryos and one 12-cell embryo from a singlecouple (UCSFB1-10). Compared to a large panel of lines from blastocysts,they exhibited unique patterns of gene expression and DNA methylationthat were highly indicative of trophoblastic competence. At a transcrip-tional level, UCSFB lines from different embryos were often more closelyrelated than those from the same embryo. As predicted by the tran-scriptomic data, immunolocalization of eomesodermin and brachyuryshowed differential expression among blastomeres of 8-12-cell humanembryos. The UCSFB lines formed derivatives of the three germ layers.Thus, out data suggested heterogeneity among early-stage blastomeresand that the UCSFB lines had unique properties, suggesting a moreimmature state than lines derived from blastocysts.

Genes controlling extraembryonic or trophoblast development werehypomethylated in the UCSFB lines. Therefore, we investigated theirtrophoblast potential by forming embryonic bodies. Immunoanalyses ofthe outgrowths showed that the cultures contained cells with themorphology and antigenic profile of trophoblasts. Next, we asked whetherwe could derive human trophoblast stem cells from one of the lines. At day3, trophoblast-like cells were manually dissected from the outgrowths andcultured using ourmethod for establishing lines of trophoblast progenitorsfrom human placentas. The resulting cells could be passaged indefinitely.They immunostained, in a nuclear pattern, for transcription factors that arerequired for generation of the trophoblast lineage. Wewere also interestedin their capacity in terms of forming the mature trophoblast cell types ofthe human placenta. In this regard, they form invasive cytotrophoblastsand multinucleated syncytiotrophoblasts. Thus, we succeeded in derivinghuman trophoblast stem cells.

NI.1.EXPRESSION OF THE b-ISOFORM OF THE THROMBOXANE A2 RECEPTORREGULATES MATERNAL AND FETAL DERIVED CHARACTERISTICS OFPRE-ECLAMPSIA

Katie L. Powell a,b, Veronica Stevens a, Sharon McCracken a, VitomirTasevski a,b, Jonathan M. Morris a, Anthony W. Ashton a aDivision ofPerinatal Research, Kolling Institute of Medical Research, St Leonards NSW

Abstracts / Placenta 35 (2014) A1eA112A8

2065, Australia; b Pathology North, Royal North Shore Hospital, St LeonardsNSW 2065, Australia

Objectives: Pre-eclampsia (PE) is the leading cause of maternal morbidityand mortality with the only reliable intervention being delivery of a pre-term baby. The production of isoprostanes and thromboxane (TXA2) areelevated in women with PE and these molecules act via the thromboxanereceptor (TP). We have identified a potentially novel link betweenabnormal placentation associated with PE and the maternal syndrome viachanges in TP isoform expression. The objectives of the study were tocharacterize the maternal and fetal outcomes from a transgenic mousemodel expressing the human specific TPb isoform (TPb-Tg) and identifychanges in cellular function in TPa/TPb expressing trophoblasts.

Methods: An in vivo TPb-Tg mouse model and in vitro trophoblast cell linesexpressing TPa/TPb were developed to assess their role in the develop-ment of PE. Blood pressure was monitored throughout pregnancy and PEhallmarks determined by ELISA and IHC. Western blotting and functionalcell assays were used to characterize altered trophoblast function.Results: During pregnancy TPb-Tg mice spontaneously develop a PE-likecondition and express molecular markers of the human disease. TPb-Tgmice developed hypertension at D14.5 of an 18-19 day gestation (p<0.05).TPb-Tg pups had a lower birth weight consistent with an IUGR phenotype,but higher placental weight (p<0.05). TPb-Tg mice at D18.5 of gestationhad elevated circulating levels of sFlt-1 and enhanced placental MMP14expression. In vitro, we identified that amino acids 347-366 and 385-407(full length TPb) contributed to diminished syncytialisation and migration,whereas 328-347 and 385-407 contributed to a reduction in cell prolifer-ation in TPb expressing trophoblasts.Conclusion: The data characterizing the TPb-Tg mouse model stronglysupport its use as a model of PE. Therefore this model is ideal for testingnovel therapies including ones designed to target specific regions withinthe TPb receptor tail responsible for impaired trophoblast function asso-ciated with PE.

NI.2.IMPACT OF THE TRANSCRIPTION FACTOR NRF2 ON MURINE PLACENTALDEVELOPMENT.

Nisreen Kweider a, Jessica Lambertz a, Thomas Pufe a, ChristophWruck a, Werner Rath b aDepartment of Anatomy and Cell Biology;Medical Faculty; RWTH Aachen University, Aachen, Germany; bObstetricsand Gynecology; Medical Faculty, University Hospital of the RWTH Aachen,Aachen, Germany

Objectives: The placenta is the key organ for successful and healthypregnancy. Increased oxidative stress during early human placentation isknown to induce multiple pregnancy-related disorders such as pre-eclampsia and intra uterine growth restriction (IUGR). The transcription ofmany antioxidative-genes is mediated mainly through the transcriptionfactor Nrf2. We have previously reported a link between Nrf2 and vascu-logenesis which is implicated in the pathogenesis of preeclampsia.

Herein, we investigated the placental phenotype, placental and fetalweight, and the vascular function of the placentas from Nrf2 knockoutmice (Nrf2-/-) at different stages of pregnancy.Methods: Placentae (n¼6-7) from Nrf2 -/- (n¼6) and wild type Nrf2 +/+

(n¼7) mice were collected on days 8.5, 11.5, 15.5 and 18.5 post coitum.Markers for oxidative stress, VEGF, VEGFR1 and 2, Nrf2 and HO-1 weremeasured by immunohistochemical Methods: Sterological analysis hasbeen applied to assess the volume of the labyrinth zone.Results: At 18.5 days post coitum the fetal weight was reduced (P<0.05) inNrf2 -/- (0.8391± 0.01569) versus Nrf2+/+ (0.9715± 0.01897) indicating adecrease in placental efficiency.In the placentas of Nrf2-/- there was a significant decrease in HO-1, animportant endogenous antioxidant enzyme and a key regulator oftrophoblast invasion and placental function. Furthermore, Nrf2-/- labyrinthcells showed increased levels of the lipid peroxidation product 4-hydroxynonenal (4-HNE). Further analysis showed that the labyrinth zoneof Nrf2-/- placenta was smaller when compared to the wild type one.

Conclusion: This is the first data describes the placental phenotype ofNrf2-/- mice. Our results demonstrated that disruption of Nrf2 signallingresults in reduction in the efficiency of the placenta, increases placentaloxidative stress and negatively affects the placental nutrient exchangecapacity. Further studies need to be performed to investigate the mecha-nisms through which Nrf2 interfere with placental function.

NI.3.ALTERED PLACENTAL FUNCTION IN THE TRYPTOPHAN HYDROXYLASE 1KNOCK-OUT MOUSE e FROM FETAL DEVELOPMENT IN UTERO TOERYTHROPOIESIS IN POST-NATAL LIFE

Kathy Deroy a, France Cot�e b, Thomas Sanderson a, CathyVaillancourt a a INRS-Institut Armand Frappier and BioMed ResearchCentre, Laval, Quebec, Canada; b INSERM U1163 / CNRS ERL 8254, InstitutImagine, Hopital Necker, Universit�e Sorbonne Paris Cit�e, Paris, France

Objectives: Peripheral serotonin-deficient TPH1-/- mice (knock-out fortryptophan hydroxylase 1) have developmental problems in post-natal lifesuch as an ineffective erythropoiesis compared to wild-type (WT). As theplacenta is a critical source of hematopoietic stem cells for post-natal life,we asked if placental hematopoiesis, which occurs principally betweenE10.5 and E15.5, is affected in TPH1-/- mice. First, we characterized thedevelopmental effects of reduced placental serotonin levels, finding thatTPH1-/- mice have more than double the fetal mortality rate with 3 inutero deaths per 4 litters (n¼26) comparedwith 1 death per 3 litters inWTmice (n¼24).

Methods and Results: Placental index (fetal weight/placental weight) ofTPH1-/- mice was significantly reduced on E11.5 (by 26%; p¼0.0032) andE15.5 (18%; p˂0.0001), but was 36% greater than that of WT mice on E17.5(p¼0.0079). Second, we characterized the effects of reduced serotoninlevels on placental hematopoiesis, showing that hematopoietic stem cellsare increased by 270% (p˂0.0001) in TPH1-/- placentas compared to WTon E13.5. Also, the proportion of early erythroid progenitor cells wasincreased on E11.5 (410%; p¼0.0021) and E13.5 (260%; p¼0.0386) inplacentas from TPH1-/- compared to WT mice. Moreover, we observed adefinitive erythropoiesis in TPH1-/- placentas with an increased pro-duction of pro-erythroblast cells from E12.5 (p¼0.0263) to E15.5(p˂0.0001) that was not accompanied by an increase in red blood cellproduction. This maturation defect observed during embryonic devel-opment may be due to ineffective erythropoiesis as is the case in adultTPH1-/- mice.Conclusion: These results indicate that reduced peripheral serotoninlevels have adverse effects on vital systems that may impair fetal devel-opment and have possible insidious effects later in life due to altered fetalprogramming.

NI.4.3D SURFACE RECONSTRUCTION OF HUMAN TERMINAL VILLI AND THEFETAL CAPILLARY BED

Romina Plitman Mayo, Steve Charnock-Jones, Graham Burton, MichelleOyen University of Cambridge, Cambridge, UK

Objectives: Placental transport is the main factor affecting the health anddevelopment of both the fetus and the placenta. The surface and geometryof terminal villi and their capillary beds are directly related to placentalefficiency, and therefore their spatial arrangements are of great interest.Histomorphological changes are being widely investigated. However, thepublished data are sparse and inconsistent mainly due to the differentinvestigation criteria.

Methods: 3D surface reconstructions of 2D stacks of images have provento provide significant insights in many fields of bioengineering and havethe potential of becoming a powerful tool in developmental biology. Thosetechniques are applied herein to reconstruct the geometry and 3Darrangement of terminal villi from fluorescent confocal laser scanningmicroscopic (CLSM) images of perfused placentas (Fig 1). These surfaces