Naila Abid, Joan Embola and Zoe Tryfonos contributed equally to this study.Abbreviations: 8 Br-cAMP, 8-Bromo adenosine-3', 5'-cyclic monophosphate; BSA, bovine serum albumin; cAMP, adenosine-3', 5'-cyclic monophosphate; DAB, 3,3′-diaminobenzidine; DP44mT, di-2-pyridylketone-4,4,-dimethyl-3-thiosemicarbazone; Epac, exchange proteins directly activated by cAMP; FCS, fetal calf serum; GSK-3β, glycogen synthase kinase-3β; hCG, human chorionic gonadotrophin; HIF, hypoxia-Inducible factor; HRE, hypoxia response element; IGF, insulin-like growth factor; IGFBP4, insulin-like growth factor binding protein; mTOR, mammalian target of rapamycin; mTORC, mammalian target of rapamycin complex; NDRG-1, N-myc downstream-regulated gene 1; NHS, National Health Service; PAPP-A, pregnancy-associated plasma protein-A; PBS, phosphate-buffered saline; PBST, phosphate-buffered saline with tween; PI 3 -Kinase, phosphatidylinositol-3 kinase; PKA, protein kinase A; SGK-1, serum and glucocorticoid-induced kinase-1; STC-1, stanniocalcin-1; TBS, tris-buffered saline; TBST, tris-buffered saline with tween. AbstractStanniocalcin-1 (STC-1) is a multi-functional glycosylated peptide present in the plasma of healthy women postpartum and increased further in pregnancies complicated by preeclampsia. Although the STC-1 gene is expressed by the placenta what regulates its secretion and from which cells at the feto-maternal interface is unknown.Here, we demonstrate for the first time that the syncytiotrophoblast and cytotrophoblast are a major site of STC-1 protein expression in first trimester placental tissue. Further, in response to low oxygen, first trimester chorionic villous tissue from pregnancies at increased risk of developing preeclampsia secreted significantly more STC-1 than normal tissue under the same conditions. Using the human trophoblast cell line BeWo we have shown that low oxygen increased the secretion of STC-1 but it required co-stimulation with the Adenosine-3', 5'-cyclic monophosphate (cAMP) analogue, 8-Bromo adenosine-3', 5'-cyclic monophosphate cAMP to reach significance. Inhibition of Hypoxia inducible factor 2α (HIF-2α) and the Phosphatidylinositol-3 kinase (PI 3 -Kinase)/AKT/Serum and glucocorticoid-induced kinase-1(SGK-1) pathway resulted in significant inhibition of STC-1 secretion. As both low oxygen and cAMP are known to play a central role in placental function, their regulation of STC-1 points to a potentially important role in the maintenance of a normal healthy pregnancy and we would hypothesize that it may act to protect against prolonged placental hypoxia seen in preeclampsia. K E Y W O R D Sfirst trimester, hypoxia, placenta, stanniocalcin-1, trophoblasts

Failure of the placental capillary network to develop normally is associated with early onset fetal growth restriction (FGR) and pre-eclampsia (PE). Although the symptoms are observed at term, the problem begins in the first trimester. However, investigations at this clinically relevant time are hindered by difficulties in identifying earlystage pregnancies that are at risk of developing FGR/PE. Using uterine artery Doppler ultrasound in the first trimester as a proxy measure of poor placentation, we have identified pregnancies at increased risk of developing early onset FGR/PE. Placental endothelial cells (PEC) isolated from pregnancies at increased risk of developing FGR/PE grew more slowly and their basal rate of apoptosis was significantly higher than that seen in the normal group. The pro-apoptotic stimulus, TNFα, induced apoptosis in cells from both groups but this was significantly greater in the high risk group. TNF receptor expression was unaffected. Inhibition of nitric oxide (NO) production significantly increased the sensitivity of cells from the normal pregnancies to TNFα but not in the high risk group establishing a functional role for NO in this system. In conclusion, first trimester PEC from pregnancies at increased risk of developing early onset FGR/PE were inherently more sensitive to apoptotic stimuli and this was functionally linked to the synthesis of NO. This may contribute to the poor placental vascular development seen in on going pregnancies.