- Email: [email protected]
PlGF) as an aid in differential diagnosis of pregnancy-related hypertensive disorders
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Poster Presentations / Pregnancy Hypertension: An International Journal of Women’s Cardiovascular Health 1 (2011) 273–299
normal controls. Real-time PCR and immunofluorescence analysis of EGFL7 expression in normal and preeclamptic human placental tissue were performed. Results: Real-time PCR analysis revealed that EGFL7 is strongly down regulated in the villi of PE placentas when compared to normal samples. In agreement with published data, VEGF expression was also decreased in PE placentas, although to a lesser extent compared to EGFL7. At variance, expression of the pan-endothelial marker CD31 was not significantly altered in PE as compared to normal placentas. These results were confirmed by immunofluorescence analysis. Conclusion: Our results suggest a role for Egfl7 in the pathogenesis of PE. We hypothesize that in PE placental villi the total number of endothelial cells is not significantly decreased (as indicated by the unchanged expression of CD31), but that their spatial distribution and function is severely affected, possibly due to the decrease in EGFL7 and VEGF protein. These preliminary results implicate a role for Egfl7 in PE human placentas: our preliminary studies also show that it interacts with the Notch pathway. The Notch signaling pathway appears to be crucial for both placental implantation and development and targeted mutations on several pathway genes result in defective growth of vascular vessels. To test our hypothesis, further studies are necessary in order to assess the importance of Egfl7 in human placental development and preeclampsia and to examine if Egfl7 function in human placenta is mediated through its interactions with Notch. First Author < 35 years Yes. doi:10.1016/j.preghy.2011.08.068
P8. Circulating ficolins in normal pregnancy and preeclampsia A. Molvarec, A. Szarka, B. Stenczer, J. Szijártó, J. Rigó Jr. (First Department of Obstetrics and Gynecology, Semmelweis University, Budapest, Hungary) Objective: Ficolins are soluble molecules of the innate immune system that recognize carbohydrate molecules on pathogens, apoptotic and necrotic cells. They act through two distinct routes: initiate the lectin pathway of complement activation and mediate a primitive opsonophagocytosis. The purpose of this study was to determine whether circulating levels of ficolins are altered in normal pregnancy and preeclampsia and related to the clinical features and laboratory parameters of the patients, including angiogenic factors (soluble fms-like tyrosine kinase-1, placental growth factor) and markers of endothelial activation (von Willebrand factor antigen) and injury (fibronectin). Methods: Sixty preeclamptic patients, 60 healthy pregnant women and 59 healthy non-pregnant women were involved in this case-control study. Levels of H-ficolin and Lficolin in maternal plasma were measured by enzyme-linked immunosorbent assay. Circulating levels of soluble fms-like tyrosine kinase-1 (sFlt-1), placental growth factor (PlGF), von Willebrand factor antigen (VWF:Ag) and fibronectin
were also determined. For statistical analyses, non-parametric methods were applied. Results: Plasma levels of L-ficolin were significantly lower in healthy pregnant than in healthy non-pregnant women, while H-ficolin levels did not differ significantly between the two groups. Furthermore, preeclamptic patients had significantly lower H-ficolin and L-ficolin concentrations than healthy non-pregnant and pregnant women. In the preeclamptic group, plasma L-ficolin levels showed a significant positive correlation with serum PlGF concentrations and significant inverse correlations with serum levels of sFlt-1, blood urea nitrogen and creatinine, serum lactate dehydrogenase activities, as well as with plasma VWF:Ag and fibronectin concentrations. Conclusions: Circulating levels of L-ficolin are decreased in the third trimester of normal pregnancy. There is a further decrease in plasma L-ficolin concentrations in preeclampsia, which might contribute to the development of the maternal syndrome of the disease through impaired removal of the aponecrotic trophoblast debris released into the maternal circulation by the ischemic and oxidatively stressed preeclamptic placenta. First Author < 35 years: No. doi:10.1016/j.preghy.2011.08.069
P9. The elecsys assay for PlGF, sFlt1 and their ratio (sFlt1/ PlGF) as an aid in differential diagnosis of pregnancyrelated hypertensive disorders T. Engels, J. Pape, K. Schoofs, B. Denk, E. Beinder, S. Verlohren) Objectives: We have shown previously that automated measurement of sFlt1 and PlGF is a reliable tool in the assessment of preeclampsia (PE). The aim of this study was first to test if the sFlt1/PlGF ratio (sFlt1/PlGF) is superior to the single measurement of sFlt1 or PlGF in PE diagnosis. Secondly, we wanted to characterize sFlt1/PlGF in different types of hypertensive pregnancy disorders. Patients and methods: In a single center case control trial we investigated 64 patients with PE/HELLP, 18 with pregnancy induced hypertension (PIH), 2 with chronic hypertension (cHT), 22 with gestational proteinuria (GP) and 232 controls. For further distinction the PE/HELLP group was divided into early onset PE (n = 24), late onset PE (n = 40), mild PE (n = 31), severe PE (n = 20), superimposed PE (n = 7) and HELLP (n = 6), respectively. sFlt1 and PlGF were measured in serum samples on an automated Elecsys platform, sFlt1/ PlGF was calculated (mean ± SEM). Receiver operated curves (ROC) and their areas under the curve (AUC) were calculated. Results: PE patients showed a higher sFlt1 and sFlt1/PlGF and a lower PlGF, respectively, as compared to controls (10,888 ± 878 vs. 2456 ± 116; 268 ± 39 vs. 16 ± 2 and 68 ± 6 vs. 439 ± 37, each p < 0.001). ROC curves and their AUC showed best performance for sFlt1/PlGF (ratio 96.4 % vs. sFlt1 92.8% and PlGF 92.4%). Early and late onset PE patients both showed higher ratios than their gestational age
Poster Presentations / Pregnancy Hypertension: An International Journal of Women’s Cardiovascular Health 1 (2011) 273–299
matched controls, values of early onset PE being significantly higher than in late onset PE (497 ± 81 vs. 13 ± 4, p < 0,001 and 131 ± 15 vs. 29 ± 5, p < 0.001; p for early vs. late PE <0.001). Again ROC curves and their AUC showed best performance for the sFlt1/PlGF ratio, being 98.6% (vs. 97.6% for sFlt1 and 97.4% for PlGF) in early onset PE vs. controls and 90.8% (vs. 82.1% for sFlt1 and 88.4% for PlGF) in late onset PE. Patients with superimposed, mild or severe PE and HELLP each showed higher ratios than controls (202 ± 110; 137 ± 27; 497 ± 91 and 254 ± 72 vs. 16 ± 2, each p < 0.001); in calculated ROC curves, their AUC were 93.6%, 94.8%, 99.4% and 98.6%, respectively. Whereas sFlt1/PlGF values compared to controls were significantly higher in patients with PIH and GP, no significant difference was observed in patients with cHT (PIH 65 ± 18 vs. 16 ± 2, p < 0.001; GP 62 ± 25 vs. 16 ± 2, p = 0.01 and cHT 13 ± 8 vs. 16 ± 2, p = 0.6). Ratio values of patients with each PIH and GP were significantly lower than in patients with mild PE (p < 0.007), severe PE (p < 0.001) and HELLP (p < 0.003). Compared to patients with superimposed PE a significant difference was observed only vs. GP (p = 0.015) whereas vs. PIH no significant difference was observed (p = 0.178). Conclusion: The automated measurement of sFlt1/PlGF is a reliable tool to discriminate between different types of pregnancy-related hypertensive disorders, particularly in early onset PE, severe PE and HELLP, being the most severe variations of PE. Thus, sFlt1/PlGF gives additional valuable information and can be used as an aid in diagnosis and therefore adapt clinical management. First Author < 35 years: Yes.
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and gestation matched normal control pregnancies (n = 9) and snap frozen within 30 min of delivery of the placenta. Biopsies were stored at 80 °C until RNA extraction. cDNA was synthesised and Real Time PCR was carried out for relative quantification of specific genes. Total protein was extracted and proteins were measured using ELISA. Statistical analysis was performed using non parametric Mann–Whitney U test. Correlation between parameters was assessed using the Spearman rank test. Values of p..0.05 were considered significant. The preliminary results of the study show that tissue factor (TF) mRNA levels are reduced (p < 0.001) while TFPI-2 is increased (p < 0.05) in PET compared to normal pregnancy. PAI-1 and PAI-2 are also increased in PET versus normal pregnancy (p < 0.01). TFPI-1, VEGF A, PlGF and sFLT-1 were not significantly altered in term PET. Protein results show a similar trend to that of RNA for all molecules. Correlation analysis has shown a significant relationship between TF and VEGF-A (.. = 0.750, p < 0.02), TFPI-1 and PlGF (.. = 0.667, p < 0.05) in PET placenta, no correlation was found in control samples. This study shows the disturbances in the regulation of some haemostatic genes in term PET placenta compared to controls. The interplay between haemostasis and the angiogenic pathway is crucial in pregnancy and the possible dysregulation shown here may contribute to the development of preeclampsia. However, larger sample numbers are required to confirm these observations. First Author < 35 years. doi:10.1016/j.preghy.2011.08.071
doi:10.1016/j.preghy.2011.08.070
P10. Placental expression of haemostatic and angiogenic markers in preeclampsia Lynne Kelly a, Shanthi Muttukrishna a, Lucy Norris b, John Higgins a (a Anu Research Centre, Department of Obstetrics and Gynaecology, University College Cork, Ireland, b Department of Obstetrics and Gynaecology, Trinity College Dublin, Ireland) Pre-eclampsia (PET), affecting between 3% and 5% of pregnant women, is a major contributor to perinatal and maternal morbidity and mortality worldwide. In normal pregnancy, an activation of haemostasis occurs to prevent blood loss during childbirth. Previous work has suggested that placental expression of some haemostatic proteins is altered in PET and that this may contribute to the pathogenesis of the disease. Studies have also shown an upregulation in the expression of some angiogenic factors in PET compared to control pregnancies. A small number of studies have previously reported conflicting results and further investigations are necessary to define inter-relationship between regulation of haemostasis and angiogenesis at a molecular level in the placenta. The objective of this ongoing study is to investigate placental expression of the haemostatic and angiogenic genes and proteins and to evaluate their relationship in PET. Placental tissue was collected at term (..37 weeks of gestation) from PET patients (n = 9)
P11. Differentially expressed micro-RNAs in microparticles from haemoglobin perfused placentas T. Rasmussen a, K. May b, M. Familari c, S. Guller d, H. Schneider e, B. Åkerström f, S.R. Hansson a (a Division of Obstetrics and Gynecology, Department of Clinical Sciences, Lund University Hospital, Lund University, Lund, Sweden, b Department of Clinical Pharmacology, Ernst Moritz Arndt University of Greifswald, Germany, c Department of Zoology, University of Melbourne, Parkville, Victoria, Australia, d Department of Obstetrics, Gynecology, and Reproductive Sciences, Reproductive Immunology Unit, School of Medicine, Yale University, New Haven, CT, USA, e Department of Obstetrics and Gynecology, Inselspital, University of Bern, Switzerland, f Division of Infection Medicine, Department of Clinical Sciences, Lund University, Lund, Sweden) Objectives: We have previously shown that free fetal haemoglobin (HbF) may play a role in the patophysiology of preeclampsia (PE). HbF causes functional and structural damage to the placenta and we hypothesize that this also leads to an altered micro-RNA (miRNA) profile. The levels of microparticles (MPs) are elevated in PE and as carriers of miRNAs they may play a role in the pathophysiology. We therefore examined the miRNA profile in MPs from placentas perfused with free HbF. Methods: Human term placentas, obtained from uncomplicated singleton pregnancies delivered by Caesarean sec-
Poster Presentations / Pregnancy Hypertension: An International Journal of Women’s Cardiovascular Health 1 (2011) 273–299
normal controls. Real-time PCR and immunofluorescence analysis of EGFL7 expression in normal and preeclamptic human placental tissue were performed. Results: Real-time PCR analysis revealed that EGFL7 is strongly down regulated in the villi of PE placentas when compared to normal samples. In agreement with published data, VEGF expression was also decreased in PE placentas, although to a lesser extent compared to EGFL7. At variance, expression of the pan-endothelial marker CD31 was not significantly altered in PE as compared to normal placentas. These results were confirmed by immunofluorescence analysis. Conclusion: Our results suggest a role for Egfl7 in the pathogenesis of PE. We hypothesize that in PE placental villi the total number of endothelial cells is not significantly decreased (as indicated by the unchanged expression of CD31), but that their spatial distribution and function is severely affected, possibly due to the decrease in EGFL7 and VEGF protein. These preliminary results implicate a role for Egfl7 in PE human placentas: our preliminary studies also show that it interacts with the Notch pathway. The Notch signaling pathway appears to be crucial for both placental implantation and development and targeted mutations on several pathway genes result in defective growth of vascular vessels. To test our hypothesis, further studies are necessary in order to assess the importance of Egfl7 in human placental development and preeclampsia and to examine if Egfl7 function in human placenta is mediated through its interactions with Notch. First Author < 35 years Yes. doi:10.1016/j.preghy.2011.08.068
P8. Circulating ficolins in normal pregnancy and preeclampsia A. Molvarec, A. Szarka, B. Stenczer, J. Szijártó, J. Rigó Jr. (First Department of Obstetrics and Gynecology, Semmelweis University, Budapest, Hungary) Objective: Ficolins are soluble molecules of the innate immune system that recognize carbohydrate molecules on pathogens, apoptotic and necrotic cells. They act through two distinct routes: initiate the lectin pathway of complement activation and mediate a primitive opsonophagocytosis. The purpose of this study was to determine whether circulating levels of ficolins are altered in normal pregnancy and preeclampsia and related to the clinical features and laboratory parameters of the patients, including angiogenic factors (soluble fms-like tyrosine kinase-1, placental growth factor) and markers of endothelial activation (von Willebrand factor antigen) and injury (fibronectin). Methods: Sixty preeclamptic patients, 60 healthy pregnant women and 59 healthy non-pregnant women were involved in this case-control study. Levels of H-ficolin and Lficolin in maternal plasma were measured by enzyme-linked immunosorbent assay. Circulating levels of soluble fms-like tyrosine kinase-1 (sFlt-1), placental growth factor (PlGF), von Willebrand factor antigen (VWF:Ag) and fibronectin
were also determined. For statistical analyses, non-parametric methods were applied. Results: Plasma levels of L-ficolin were significantly lower in healthy pregnant than in healthy non-pregnant women, while H-ficolin levels did not differ significantly between the two groups. Furthermore, preeclamptic patients had significantly lower H-ficolin and L-ficolin concentrations than healthy non-pregnant and pregnant women. In the preeclamptic group, plasma L-ficolin levels showed a significant positive correlation with serum PlGF concentrations and significant inverse correlations with serum levels of sFlt-1, blood urea nitrogen and creatinine, serum lactate dehydrogenase activities, as well as with plasma VWF:Ag and fibronectin concentrations. Conclusions: Circulating levels of L-ficolin are decreased in the third trimester of normal pregnancy. There is a further decrease in plasma L-ficolin concentrations in preeclampsia, which might contribute to the development of the maternal syndrome of the disease through impaired removal of the aponecrotic trophoblast debris released into the maternal circulation by the ischemic and oxidatively stressed preeclamptic placenta. First Author < 35 years: No. doi:10.1016/j.preghy.2011.08.069
P9. The elecsys assay for PlGF, sFlt1 and their ratio (sFlt1/ PlGF) as an aid in differential diagnosis of pregnancyrelated hypertensive disorders T. Engels, J. Pape, K. Schoofs, B. Denk, E. Beinder, S. Verlohren) Objectives: We have shown previously that automated measurement of sFlt1 and PlGF is a reliable tool in the assessment of preeclampsia (PE). The aim of this study was first to test if the sFlt1/PlGF ratio (sFlt1/PlGF) is superior to the single measurement of sFlt1 or PlGF in PE diagnosis. Secondly, we wanted to characterize sFlt1/PlGF in different types of hypertensive pregnancy disorders. Patients and methods: In a single center case control trial we investigated 64 patients with PE/HELLP, 18 with pregnancy induced hypertension (PIH), 2 with chronic hypertension (cHT), 22 with gestational proteinuria (GP) and 232 controls. For further distinction the PE/HELLP group was divided into early onset PE (n = 24), late onset PE (n = 40), mild PE (n = 31), severe PE (n = 20), superimposed PE (n = 7) and HELLP (n = 6), respectively. sFlt1 and PlGF were measured in serum samples on an automated Elecsys platform, sFlt1/ PlGF was calculated (mean ± SEM). Receiver operated curves (ROC) and their areas under the curve (AUC) were calculated. Results: PE patients showed a higher sFlt1 and sFlt1/PlGF and a lower PlGF, respectively, as compared to controls (10,888 ± 878 vs. 2456 ± 116; 268 ± 39 vs. 16 ± 2 and 68 ± 6 vs. 439 ± 37, each p < 0.001). ROC curves and their AUC showed best performance for sFlt1/PlGF (ratio 96.4 % vs. sFlt1 92.8% and PlGF 92.4%). Early and late onset PE patients both showed higher ratios than their gestational age
Poster Presentations / Pregnancy Hypertension: An International Journal of Women’s Cardiovascular Health 1 (2011) 273–299
matched controls, values of early onset PE being significantly higher than in late onset PE (497 ± 81 vs. 13 ± 4, p < 0,001 and 131 ± 15 vs. 29 ± 5, p < 0.001; p for early vs. late PE <0.001). Again ROC curves and their AUC showed best performance for the sFlt1/PlGF ratio, being 98.6% (vs. 97.6% for sFlt1 and 97.4% for PlGF) in early onset PE vs. controls and 90.8% (vs. 82.1% for sFlt1 and 88.4% for PlGF) in late onset PE. Patients with superimposed, mild or severe PE and HELLP each showed higher ratios than controls (202 ± 110; 137 ± 27; 497 ± 91 and 254 ± 72 vs. 16 ± 2, each p < 0.001); in calculated ROC curves, their AUC were 93.6%, 94.8%, 99.4% and 98.6%, respectively. Whereas sFlt1/PlGF values compared to controls were significantly higher in patients with PIH and GP, no significant difference was observed in patients with cHT (PIH 65 ± 18 vs. 16 ± 2, p < 0.001; GP 62 ± 25 vs. 16 ± 2, p = 0.01 and cHT 13 ± 8 vs. 16 ± 2, p = 0.6). Ratio values of patients with each PIH and GP were significantly lower than in patients with mild PE (p < 0.007), severe PE (p < 0.001) and HELLP (p < 0.003). Compared to patients with superimposed PE a significant difference was observed only vs. GP (p = 0.015) whereas vs. PIH no significant difference was observed (p = 0.178). Conclusion: The automated measurement of sFlt1/PlGF is a reliable tool to discriminate between different types of pregnancy-related hypertensive disorders, particularly in early onset PE, severe PE and HELLP, being the most severe variations of PE. Thus, sFlt1/PlGF gives additional valuable information and can be used as an aid in diagnosis and therefore adapt clinical management. First Author < 35 years: Yes.
277
and gestation matched normal control pregnancies (n = 9) and snap frozen within 30 min of delivery of the placenta. Biopsies were stored at 80 °C until RNA extraction. cDNA was synthesised and Real Time PCR was carried out for relative quantification of specific genes. Total protein was extracted and proteins were measured using ELISA. Statistical analysis was performed using non parametric Mann–Whitney U test. Correlation between parameters was assessed using the Spearman rank test. Values of p..0.05 were considered significant. The preliminary results of the study show that tissue factor (TF) mRNA levels are reduced (p < 0.001) while TFPI-2 is increased (p < 0.05) in PET compared to normal pregnancy. PAI-1 and PAI-2 are also increased in PET versus normal pregnancy (p < 0.01). TFPI-1, VEGF A, PlGF and sFLT-1 were not significantly altered in term PET. Protein results show a similar trend to that of RNA for all molecules. Correlation analysis has shown a significant relationship between TF and VEGF-A (.. = 0.750, p < 0.02), TFPI-1 and PlGF (.. = 0.667, p < 0.05) in PET placenta, no correlation was found in control samples. This study shows the disturbances in the regulation of some haemostatic genes in term PET placenta compared to controls. The interplay between haemostasis and the angiogenic pathway is crucial in pregnancy and the possible dysregulation shown here may contribute to the development of preeclampsia. However, larger sample numbers are required to confirm these observations. First Author < 35 years. doi:10.1016/j.preghy.2011.08.071
doi:10.1016/j.preghy.2011.08.070
P10. Placental expression of haemostatic and angiogenic markers in preeclampsia Lynne Kelly a, Shanthi Muttukrishna a, Lucy Norris b, John Higgins a (a Anu Research Centre, Department of Obstetrics and Gynaecology, University College Cork, Ireland, b Department of Obstetrics and Gynaecology, Trinity College Dublin, Ireland) Pre-eclampsia (PET), affecting between 3% and 5% of pregnant women, is a major contributor to perinatal and maternal morbidity and mortality worldwide. In normal pregnancy, an activation of haemostasis occurs to prevent blood loss during childbirth. Previous work has suggested that placental expression of some haemostatic proteins is altered in PET and that this may contribute to the pathogenesis of the disease. Studies have also shown an upregulation in the expression of some angiogenic factors in PET compared to control pregnancies. A small number of studies have previously reported conflicting results and further investigations are necessary to define inter-relationship between regulation of haemostasis and angiogenesis at a molecular level in the placenta. The objective of this ongoing study is to investigate placental expression of the haemostatic and angiogenic genes and proteins and to evaluate their relationship in PET. Placental tissue was collected at term (..37 weeks of gestation) from PET patients (n = 9)
P11. Differentially expressed micro-RNAs in microparticles from haemoglobin perfused placentas T. Rasmussen a, K. May b, M. Familari c, S. Guller d, H. Schneider e, B. Åkerström f, S.R. Hansson a (a Division of Obstetrics and Gynecology, Department of Clinical Sciences, Lund University Hospital, Lund University, Lund, Sweden, b Department of Clinical Pharmacology, Ernst Moritz Arndt University of Greifswald, Germany, c Department of Zoology, University of Melbourne, Parkville, Victoria, Australia, d Department of Obstetrics, Gynecology, and Reproductive Sciences, Reproductive Immunology Unit, School of Medicine, Yale University, New Haven, CT, USA, e Department of Obstetrics and Gynecology, Inselspital, University of Bern, Switzerland, f Division of Infection Medicine, Department of Clinical Sciences, Lund University, Lund, Sweden) Objectives: We have previously shown that free fetal haemoglobin (HbF) may play a role in the patophysiology of preeclampsia (PE). HbF causes functional and structural damage to the placenta and we hypothesize that this also leads to an altered micro-RNA (miRNA) profile. The levels of microparticles (MPs) are elevated in PE and as carriers of miRNAs they may play a role in the pathophysiology. We therefore examined the miRNA profile in MPs from placentas perfused with free HbF. Methods: Human term placentas, obtained from uncomplicated singleton pregnancies delivered by Caesarean sec-