Downregulation of notch signaling pathway in late preterm and term placentas from pregnancies complicated by preeclampsia.

Preeclampsia (PE) is a major cause of maternal mortality and morbidity, affecting 3-5% of all pregnancies. The Notch signaling pathway plays an important role during placental development, activating several target genes. Defects in the Notch pathway have adverse effect on placentation. The aim of this study was to investigate the expression of receptors NOTCH1,-2,-3,-4, ligands DLL1,-3,-4, JAG1,-2 and target genes HEY1,-2 in placental tissue samples from 20 late preterm or term pregnancies complicated by PE versus 20 normal pregnancies. mRNA levels of the studied molecules were measured by quantitative Real-Time PCR (qRT-PCR), while the protein expression of the intracellular domain of NOTCH2 (NICD2) and NOTCH3 (NICD3) was measured by Western Blot (WB). qRT-PCR analysis revealed that NOTCH1, NOTCH4 and DLL1 were not expressed in the placenta. On the contrary, NOTCH2, NOTCH3, DLL3, DLL4, JAG1, JAG2, HEY1 and HEY2 mRNA levels were downregulated in PE samples vs. controls (p<0.01). WB confirmed that NICD2 (p = 0.014) and NICD3 (p<0.001) protein levels were also lower in PE specimens. Statistical analysis revealed several significant associations: of NOTCH3 mRNA expression with smoking during pregnancy (p = 0.029), of NICD3 protein levels (p = 0.028) and DLL3 mRNA levels (p = 0.041) with birth weight centile, and of HEY2 transcript levels with parity (p = 0.034) and mode of delivery (p = 0.028). Our results suggest that Notch pathway downregulation is associated with PE. Further studies are required in order to determine the role of these molecules in PE pathogenesis and to evaluate their potential use for the early detection and treatment of PE

Expression analysis of Notch signaling pathway between normal and PE placentas.

<p><b>P-value (c):</b> crude p-value; <b>P-value (a):</b> P-value adjusted for gestation period, mode of delivery and smoking during pregnancy. All values are presented as mean ± SEM.</p><p>Expression analysis of Notch signaling pathway between normal and PE placentas.</p

Notch signaling pathway pair-wise mRNA co-expression analysis in normal placentas.

<p><b>CC</b>: Correlation coefficient.</p><p>Notch signaling pathway pair-wise mRNA co-expression analysis in normal placentas.</p

Box and whisker plots depicting statistically significant associations in preeclampsia-complicated late preterm and term placentas.

<p>(A) <i>NOTCH3</i> was not expressed in preeclamptic (PE) women that smoked during their pregnancy versus women with PE that did not smoke (0.00±0.00 vs. 0.29±0.09, p = 0.029). (B) <i>DLL3</i> mRNA expression was higher in babies born from PE pregnancies with Birth Weight Centile (BWC) <5 compared with babies born from PE pregnancies with BWC >5 (0.91±0.30 vs. 0.42±0.19, p = 0.041). (C) <i>HEY2</i> transcript levels were increased in women with PE who were on their fist parity versus women with PE that gave birth to their 2^nd or 3^rd child (0.44±0.13 vs. 0.05±0.02, p = 0.034). (D) <i>HEY2</i> mRNA expression was higher in women with PE who gave birth with a Caesarian Section compared with women with PE who gave birth naturally (0.63±0.17 vs. 0.15±0.05, p = 0.028). (E) NOTCH3 intracellular domain (NICD3) protein levels were higher in babies born from women with pregnancies complicated by PE with BWC <5 versus babies born from women with pregnancies complicated by PE with BWC >5 (1.15±0.24 vs. 0.16±0.07, p = 0.028). The thick line near the center of each rectangular box represents the median value, the bottom and top edges of the box indicate the 1^st (Q₁) and 3^rd (Q₃) quartiles, and the ends of the whiskers depict the 10^th (P₁₀) and 90^th (P₉₀) percentiles.</p

Notch signaling pathway pair-wise mRNA co-expression analysis in preeclamptic placentas.

<p><b>CC</b>: Correlation coefficient.</p><p>Notch signaling pathway pair-wise mRNA co-expression analysis in preeclamptic placentas.</p

Clinical characteristics of the study groups.

<p>^a Student’s T test</p><p>^b Mann-Whitney U test</p><p>^c Chi-square test</p><p>^d Fisher’s exact test</p><p>All tests were 2-tailed.</p><p>Clinical characteristics of the study groups.</p

Primer sequences, annealing temperatures and amplicon sizes for the qRT-PCR analysis of the studied genes.

<p>Primer sequences, annealing temperatures and amplicon sizes for the qRT-PCR analysis of the studied genes.</p

Schematic representation of Notch pathway molecules mRNA expression profile in our series of preeclampsia-complicated late preterm and term placentas.

<p>Red: Downregulation; White: Normal expression; Sky Blue: Overexpression.</p

Schematic depiction of the studied Notch signaling pathway molecules in late preterm and term placentas.

<p>Schematic depiction of the studied Notch signaling pathway molecules in late preterm and term placentas.</p