Sonia T. Chelbi

Serum profile in preeclampsia and intra-uterine growth restriction revealed by iTRAQ technology.

In order to identify new protein markers modified in placental diseases, high-throughput analysis of proteins in the plasma of pregnant women was carried out for normal and pathological pregnancies (Preeclampsia and/or Intra-Uterine Growth Restriction) using iTRAQ technology. We could identify 166 proteins that were modified (p<0.05) and the technique used allowed the detection of previously undetected factors, such as various members of the SERPINA clade. The modifications of two proteins (C reactive protein and antichymotrypsin, SERPINA3) were validated on individual samples. Complement and coagulation cascades proteins were significantly enriched among modified protein clusters in the case of intra-uterine growth restriction (p<2.6.10(-11)). Several proteins were specifically enriched in isolated preeclampsia and depleted when preeclampsia was complicated by intra-uterine growth restriction. These findings suggest that the growth restricted foeto-placental unit is able to moderate some changes in maternal plasma composition. Overall, the use of iTRAQ technology, for the first time on this subject, enabled us to provide a new list of proteins modified in placental diseases, among which proteins expressed at a low level that were not accessible by other methods.

STOX1 Overexpression in Choriocarcinoma Cells Mimics Transcriptional Alterations Observed in Preeclamptic Placentas

Background Mutations in STOX1 were proposed to be causal for predisposing to preeclampsia, a hypertensive disorder originating from placental defects, affecting up to 10% of human pregnancies. However, after the first study published in 2005 three other groups have dismissed the polymorphism described in the first paper as a causal mutation. Methodology and Principal Findings In the present study, we have produced a choriocarcinoma cell line overexpressing STOX1. This overexpression results in transcriptional modification of 12.5% of the genes, some of them being direct targets as shown by chromatin immunoprecipitation. STOX1 overexpression correlates strongly and specifically with transcriptomic alterations in preeclamptic placentas (r = 0.30, p = 9.10−7). Numerous known key modulators of preeclampsia (such as Endoglin, Syncytin, human chorionic gonadotrophin -hCG-, and Glial Cell Missing Homolog -GCM1-) were modified in these transformed choriocarcinoma cells. Conclusions Our results contribute to reconcile contradictory data concerning the involvement of STOX1 in preeclampsia. In addition, they strongly suggest that anomalies in STOX1 expression are associated with the onset of preeclampsia, thus indicating that this gene should be the target of future studies. Our cellular model could constitute an invaluable resource for studying specific aspects of this human disease.

Hypoxia-activated genes from early placenta are elevated in Preeclampsia, but not in Intra-Uterine Growth Retardation

BackgroundAs a first step to explore the possible relationships existing between the effects of low oxygen pressure in the first trimester placenta and placental pathologies developing from mid-gestation, two subtracted libraries totaling 2304 cDNA clones were constructed. For achieving this, two reciprocal suppressive/subtractive hybridization procedures (SSH) were applied to early (11 weeks) human placental villi after incubation either in normoxic or in hypoxic conditions. The clones from both libraries (1440 hypoxia-specific and 864 normoxia-specific) were spotted on nylon macroarrays. Complex cDNAs probes prepared from placental villi (either from early pregnancy, after hypoxic or normoxic culture conditions, or near term for controls or pathological placentas) were hybridized to the membranes.ResultsThree hundred and fifty nine clones presenting a hybridization signal above the background were sequenced and shown to correspond to 276 different genes. Nine of these genes are mitochondrial, while 267 are nuclear. Specific expression profiles characteristic of preeclampsia (PE) could be identified, as well as profiles specific of intra-uterine growth retardation (IUGR).Focusing on the chromosomal distribution of the fraction of genes that responded in at least one hybridization experiment, we could observe a highly significant chromosomal clustering of 54 genes into 8 chromosomal regions, four of which containing imprinted genes. Comparative mapping data indicate that these imprinted clusters are maintained in synteny in mice, and apparently in cattle and pigs, suggesting that the maintenance of such syntenies is requested for achieving a normal placental physiology in eutherian mammals.ConclusionWe could demonstrate that genes induced in PE were also genes highly expressed under hypoxic conditions (P = 5.10-5), which was not the case for isolated IUGR. Highly expressed placental genes may be in syntenies conserved interspecifically, suggesting that the maintenance of such clusters is requested for achieving a normal placental physiology in eutherian mammals.

miR-34a expression, epigenetic regulation, and function in human placental diseases

Preeclampsia (PE) is the major pregnancy-induced hypertensive disorder responsible for maternal and fetal morbidity and mortality that can be associated with intrauterine growth restriction (IUGR). PE and IUGR are thought to be due to a placental defect, occurring early during pregnancy. Several placental microRNAs (miRNAs) have been shown to be deregulated in the context of placental diseases and could thus play a role in the pathophysiology of PE. Here, we show that pri-miR-34a is overexpressed in preeclamptic placentas and that its placental expression is much higher during the first trimester of pregnancy than at term, suggesting a possible developmental role. We explored pri-miR-34a regulation and showed that P53, a known activator of miR-34a, is reduced in all pathological placentas and that hypoxia can induce pri-miR-34a expression in JEG-3 cells. We also studied the methylation status of the miR-34a promoter and revealed hypomethylation in all preeclamptic placentas (associated or not with IUGR), whereas hypoxia induced a hypermethylation in JEG-3 cells at 72 h. Despite the overexpression of pri-miR-34a in preeclampsia, there was a striking decrease of the mature miR-34a in this condition, suggesting preeclampsia-driven alteration of pri-miR-34a maturation. SERPINA3, a protease inhibitor involved in placental diseases, is elevated in IUGR and PE. We show here that miR-34a overexpression in JEG-3 downregulates SERPINA3. The low level of mature miR-34a could thus be an important mechanism contributing to SERPINA3 upregulation in placental diseases. Overall, our results support a role for miR-34a in the pathophysiology of preeclampsia, through deregulation of the pri-miRNA expression and its altered maturation.

Combination of promoter hypomethylation and PDX1 overexpression leads to TBX15 decrease in vascular IUGR placentas.

Preeclampsia (PE) and vascular intra-uterine growth restriction (vIUGR) are two pathological obstetrical conditions originating from placental dysfunction. Recently, methylation changes at the placental level have been shown to be indicative of these diseases. The alteration of such epigenetic marks is therefore a novel pathway that might be critical for these pathologies. Here, we identified a region located in the distal promoter of the T-box-containing transcription factor TBX15 that is differentially methylated in pathological placentas. The level of methylation correlated significantly with the weight and stature of the newborn. The promoter was found to be hypomethylated in vIUGR coinciding with the down-regulation of its expression. PDX1, a transcription factor important for the regulation of insulin metabolism regulation was able to repress the TBX15 promoter in a methylation-dependent manner, which might, at least partially, explain the specific mRNA decrease of TBX15 observed in vIUGR placentas. Overall, the data presented herein suggest that TBX15 might be involved in the pathophysiology of placental diseases.

Re-evaluation of the role of STOX1 transcription factor in placental development and preeclampsia

Preeclampsia is a common disease of pregnancy, characterized by high blood pressure and proteinuria appearing from the second trimester of gestation. Preeclampsia has been shown to have a strong genetic component. In 2005 a positional cloning project led to the discovery of the STOX1 transcription factor, and mutations of this gene were proposed as causal for preeclampsia in Dutch families. Despite the publication of three contradictory studies, we have shown by analyzing the functional effects of STOX1 that its overexpression in choriocarcinoma cells recapitulates several transcriptomic aspects of preeclampsia. In this review, the current literature is analyzed to evaluate the possible involvement of STOX1 in the pathogenesis of this disease. While preeclampsia obviously cannot be considered as a disease caused by mutation in a single gene, we argue that STOX1 may be at the center of common pathways leading to preeclampsia.

Why preeclampsia still exists

Preeclampsia (PE) is a deadly gestational disease affecting up to 10% of women and specific of the human species. Preeclampsia is clearly multifactorial, but the existence of a genetic basis for this disease is now clearly established by the existence of familial cases, epidemiological studies and known predisposing gene polymorphisms. PE is very common despite the fact that Darwinian pressure should have rapidly eliminated or strongly minimized the frequency of predisposing alleles. Consecutive pregnancies with the same partner decrease the risk and severity of PE. Here, we show that, due to this peculiar feature, preeclampsia predisposing-alleles can be differentially maintained according to the familial structure. Thus, we suggest that an optimal frequency of PE-predisposing alleles in human populations can be achieved as a result of a trade-off between benefits of exogamy, importance for maintaining genetic diversity and increase of the fitness owing to a stable paternal investment.