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High fetal exposure to serotonin and increasing maternal age both contribute to the risk for neurodevelopmental disorders. While identifying covariates for a study of placental protein expression, we found a significant negative correlation between maternal age and the expression of monoamine oxidase A (MAOA), and a significant positive correlation between maternal age and the expression of the serotonin transporter SERT. MAOA and SERT play key roles in placental serotonin metabolism relevant to fetal neurodevelopment. These preliminary findings suggest that the effect of increasing maternal age on neurodevelopmental risk may be mediated in part by changes in placental protein expression relevant to fetal serotonin metabolism.

Introduction Hypoxia-inducible f actor 1A (HIF1A) stability is tightly regulated by hydroxylation and ubiquitination. Emerging evidence indicates that HIF1A is also controlled by the interplay between SUMO-specific ligases, which execute protein SUMOylation, and Sentrin/SUMO-specific proteases that de-SUMOylate target proteins. Given the significance of HIF1A in the human placenta, we investigated whether placental HIF1A is subject to SUMOylation in physiological and pathological conditions. Methods Placentae were obtained from normal and pregnancies complicated by preeclampsia. Human choriocarcinoma JEG3 cells were maintained at either 21% or 3% oxygen or exposed to sodium nitroprusside (SNP). Cells were transfected with SUMO2/3 constructs with and without Mg132, a proteasome inhibitor. Expression, distribution and associations of SUMO/SENPs and HIF1A were evaluated by Western blotting, immunohistochemistry and co-immunoprecipitation. Results HIF1A-SUMO2/3 associations peaked at 9–10 weeks, while its deSUMOylation by SENP3 was greatest at 10–12 weeks. In E-PE, HIF1A deSUMOylation by SENP3 was significantly elevated, while HIF1A-SUMO2/3 associations remained constant. In vitro , overexpression of SUMO2/3 de-stabilized HIF1A in hypoxia, and abrogated HIF1A expression following Mg132 treatment in normoxia. Hypoxia and SNP treatments promoted SENP3 nuclear redistribution from nucleoli to the nucleoplasm where it associates with HIF1A. Conclusion During early placental development, SUMOylation events control HIF1A stability in an oxygen-dependent manner. In E-PE, enhanced deSUMOylation of HIF1A by SENP3 may in part contribute to increased HIF1A activity and stability found in this pathology.

During early pregnancy, placentation occurs in a relatively hypoxic environment which is essential for appropriate embryonic development. Intervillous blood flow increases at around 10-12 weeks of gestation and results in exposure of the trophoblast to increased oxygen tension (PO2). Prior to this time, low oxygen appears to prevent trophoblast differentiation towards an invasive phenotype. In other mammalian systems, oxygen tension effects are mediated by hypoxia inducible factor-1 (HIF-1). We found that the ontogeny of HIF-1alpha subunit expression during the first trimester of gestation parallels that of transforming growth factor-beta3 (TGFbeta3), an inhibitor of early trophoblast differentiation. Expression of both molecules is high in early pregnancy and falls at around 10 weeks of gestation when placental PO2 levels are believed to increase. Antisense-induced inhibition of HIF-1alpha inhibited the expression of TGFbeta3, and stimulated extravillous trophoblast (EVT) outgrowth and invasion. Of clinical significance we found that TGFbeta3 expression was increased in pre-eclamptic placentae when compared to age-matched controls. Significantly, inhibition of TGFbeta3 by antisense oligonucleotides or antibodies restored the invasive capability to the trophoblast cells in pre-eclamptic explants. We speculate that if oxygen tension fails to increase, or trophoblasts do not detect this increase, HIF-1alpha and TGFbeta3 expression remain high, resulting in shallow trophoblast invasion and predisposing the pregnancy to pre-eclampsia. Effective fetal-maternal interactions during early placentation are critical for a successful pregnancy. Optimal placental perfusion requires the controlled invasion of trophoblast cells deep into the decidua to the spiral arteries. Trophoblast stem cells, also referred to as cytotrophoblast cells, reside in chorionic villi of two types, floating and anchoring villi. Floating villi, which represent the vast majority of chorionic villi, are bathed in maternal blood and primarily perform gas and nutrient exchange for the developing embryo. During early placentation, cytotrophoblast cells in the floating villi proliferate and differentiate by fusing to form the multinucleate syncytiotrophoblast layer. Cytotrophoblast cells in anchoring villi either fuse to form the syncytiotrophoblast layer, or break through the syncytium at selected sites and form multilayered columns of non-polarized extravillous trophoblast cells, which physically connect the embryo to the uterine wall (Figure 1). The extravillous trophoblast cells invade into the uterine wall as far as the first third of the myometrium and its associated spiral arteries, where they disrupt the endothelium and the smooth muscle layer and replace the vascular wall. This results in the conversion of the narrow calibre arteries into distended uteroplacental arteries, thereby increasing blood flow to the placenta and allowing an adequate supply of oxygen and nutrients to the growing fetus. The invasive activity of the extravillous trophoblast cells is at a maximum during the first trimester of gestation, peaking at around 10-12 weeks and declining thereafter. Insufficient invasion contributes to the development of pre-eclampsia, which often results in fetal intrauterine growth restriction, maternal hypertension and proteinuria. In contrast, unrestricted invasion is associated with premalignant conditions, such as invasive mole, and with malignant choriocarcinoma. Invading trophoblast cells undergo striking and rapid changes in cellular functions that are temporally and spatially regulated along the invasive pathway (Figure 1) (Cross, Werb and Fisher, 1994. The formation of the anchoring villi is accompanied by changes in synthesis and degradation of extracellular matrix proteins and their receptors, and changes in the spatial distribution of extracellular matrix proteins, as well as changes in the expression of adhesion molecules (Damsky, Fitzgerald and

Members of the TGF-beta family have been shown to play an important role in numerous tissues during development. In the present study we have investigated the spatial and temporal expression of TGF-beta 3, in human umbilical cord development. Total TGF-beta 3 protein content, assessed by immunoblotting, increased with advancing gestation as did immunostaining and mRNA in Wharton's jelly fibroblasts. Immunohistochemical analysis revealed that TGF-beta 3 was present in all cell types. Temporal changes in TGF-beta 3 expression were observed in the vascular smooth muscle cells, such that with advancing gestation TGF-beta 3 protein expression and became mostly restricted to the extracellular compartment of the vascular media. This was associated with a decrease in TGF-beta 3 mRNA expression in umbilical vascular smooth muscle cells. Of clinical significance, umbilical cords from pregnancies complicated by pre-eclampsia, showed a significant reduction in total TGF-beta 3 protein expression when compared to those of age-matched patients. Both TGF-beta 3 mRNA and protein expression were downregulated in the endothelium and smooth muscle layers of the umbilical arteries, as well as in the Wharton jelly fibroblasts. Our data demonstrate that during umbilical cord development TGF-beta 3 expression is spatially and temporally regulated and that TGF-beta 3 expression is altered in umbilical cords of pregnancies complicated by pre-eclampsia. We speculate that the downregulation of TGF-beta 3 expression found in pre-eclamptic umbilical cord may contribute to the abnormal structure and mechanical properties seen in these pathological umbilical cords.

s / Placenta 34 (2013) A1–A99 A10 decreased JunB and increased Cyclin-D1 levels in control villous explants. Normal villous explants treated by PE-PDMSCs CM showed decrease p18 protein levels (7.6 Fold Decrease) relative to normal-PDMSCs CM and untreated control explants. Conclusion: Our data demonstrated that p18 contributes to the aberrant Cyclin-D1 levels described in PE-PDMSCs. While normal PDMSCs CM is able to promote p18 expression in order to counterbalance increased Cyclin-D1 levels in normal villous explants, 72h treatment is not sufficient to restore proper JunB/Cyclin-D1/p18 ratio in PE placentae. http://dx.doi.org/10.1016/j.placenta.2013.06.033 NI.10. MASS SPECTROMETRY IMAGING OF SPHINGOLIPIDS IN PREECLAMPTIC PLACENTAE Leonardo Ermini , Isabella Caniggia , Megan Melland-Smith , Martin Post 1 Hospital for Sick Children Research Institute, Toronto, Canada; 2 Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Canada Objectives: Sphingolipids comprise a class of bioactive lipids that are involved in a variety of patho-physiological processes including cell death and survival. The aim of the present study was to examine the sphingolipid profile (content and tissue location) in preeclampsia. Methods: Sphingolipid analysis of physiological and pathological placentae was performed using tandem mass spectrometry (MS/MS). Although chemical imaging techniques, such as immunohistochemistry and dye staining, are typically used to localize biological compounds in tissues, these techniques are limited in their ability to locate specific lipids in tissues. Recent advancements in imagingmass spectrometry (IMS)has createdanew generation of mass spectrometers suitable for direct analysis of lipids in tissues. In the present study, we used Matrix-Assisted Laser Desorption Ionization (MALDI)-IMS to visualize the spatial distribution of sphingolipids. In MALDI-IMS analyte ions are produced directly from a tissue slice coated withMALDImatrix and sequentialmass spectra are acquiredby rastering the laser beamacross the tissue surface. Images are thenobtainedby plotting the intensity of the analyte of interest as a function of the x-y coordinates. Results: Analysis of sphingolipid levels in placentae from preeclamptic (PE) and normotensive preterm control pregnancies (PTC) revealed significant increases in ceramides and sphingomyelins with various fatty acyl-CoA lengths in PE placentae compared to PTC. Of note, the relative abundance of individual ceramides and sphingomyelins (% of total) remained unchanged. The IMS analysis confirmed the elevated tissue content of ceramides in PE placentae and demonstrated that the increase in ceramides was primarily noted in preeclamptic villi exhibiting the presence of syncytial knots. Conclusion: Taken together, these data are compatible with the idea that, in preeclampsia, elevated pro-apoptotic ceramides contribute to the increased placental cell death and turnover found in this disease. (Supported by CIHR). http://dx.doi.org/10.1016/j.placenta.2013.06.034 NI.11. DECIDUAL MESENCHYMAL STEM CELLS HAVE REDUCED RESISTANCE TO OXIDATIVE STRESS IN PREECLAMPSIA, WHICH IS RESTORED BY THE ALDEHYDE DEHYDROGENASE AGONIST ALDA-1 Gina Kusuma , Mohamed Abumaree , Shaun Brennecke , Bill Kalionis 1 University of Melbourne Department of Obstetrics and Gynaecology and Department of Perinatal Medicine Pregnancy Research Centre, Royal Women's Hospital, Australia; King Abdullah International Medical Research Center, King Saud Bin Abdulaziz University for Health Sciences, Riyadh, Saudi Arabia Objectives: Decidua basalis contains mesenchymal stem cells (DMSCs) but their role in pregnancy disorders is unknown. During the latter stages of preeclampsia (PE), the decidua is a source of circulating reactive oxygen species that cause widespread maternal vascular endothelium damage. Stem cells resist oxidative stress through elevated expression of aldehyde dehydrogenase (ALDH) enzymes, which detoxify harmful aldehydes. The study aims were (1) to quantify ALDH expression in PE and normotensive DMSCs and (2) to inactivate ALDH1A1, the principal gene responsible for aldehyde detoxification in stem cells, and determine whether resistance to oxidative stress is restored by the ALDH agonist Alda-1. Methods: Informed consent was obtained from PE-affected and normotensive patients (n1⁄410 each). DMSCs were isolated from decidua attached to the placenta using published methods. The flow cytometry-based Aldefluor assay was employed to quantify cells with high level expression of ALDH (i.e. ALDHbr cells) in PE-DMSCs and DMSCs. ALDH1A1 was inactivated by siRNA transfection in the DMSC-derived cell line (DMSC23). DMSC23 cells were subjected to H2O2-induced oxidative stress and their response was measured by cytotoxicity assays. The ALDH agonist Alda-1 was added following ALDH1A1-siRNA transfection and the percentage of ALDHbr cells was determined by Aldefluor assay. Results: The percentage of ALDHbr cells was significantly reduced (p-value1⁄40.0011) in PE-DMSCs (6.318 0.831%) compared with normotensiveDMSCs (23.16 4.338%). siRNA methodology substantially reduced ALDH1A1 expression in DMSC23 as verified by real-time PCR. The knockdown showed reduced percentage of ALDHbr cells (si7: 6.3 1.071% vs. NC: 15.18 1.272%, p-value