Levente Lázár

Circulating cytokines, chemokines and adhesion molecules in normal pregnancy and preeclampsia determined by multiplex suspension array

BackgroundPreeclampsia is a severe complication of pregnancy characterized by an excessive maternal systemic inflammatory response with activation of both the innate and adaptive arms of the immune system. Cytokines, chemokines and adhesion molecules are central to innate and adaptive immune processes. The purpose of this study was to determine circulating levels of cytokines, chemokines and adhesion molecules in normal pregnancy and preeclampsia in a comprehensive manner, and to investigate their relationship to the clinical features and laboratory parameters of the study participants, including markers of overall inflammation (C-reactive protein), endothelial activation (von Willebrand factor antigen) and endothelial injury (fibronectin), oxidative stress (malondialdehyde) and trophoblast debris (cell-free fetal DNA).ResultsSerum levels of interleukin (IL)-1beta, IL-1 receptor antagonist (IL-1ra), IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p40, IL-12p70, IL-18, interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha, transforming growth factor (TGF)-beta1, interferon-gamma-inducible protein (IP)-10, monocyte chemotactic protein (MCP)-1, intercellular adhesion molecule (ICAM)-1 and vascular cell adhesion molecule (VCAM)-1 were measured in 60 preeclamptic patients, 60 healthy pregnant women and 59 healthy non-pregnant women by multiplex suspension array and ELISA. In normal pregnancy, the relative abundance of circulating IL-18 over IL-12p70 and the relative deficiency of the bioactive IL-12p70 in relation to IL-12p40 might favour Th2-type immunity. Although decreased IL-1ra, TNF-alpha and MCP-1 concentrations of healthy pregnant relative to non-pregnant women reflect anti-inflammatory changes in circulating cytokine profile, their decreased serum IL-10 and increased IP-10 levels might drive pro-inflammatory responses. In addition to a shift towards Th1-type immunity (expressed by the increased IL-2/IL-4 and IFN-gamma/IL-4 ratios), circulating levels of the pro-inflammatory cytokines IL-6 and TNF-alpha, the chemokines IL-8, IP-10 and MCP-1, as well as the adhesion molecules ICAM-1 and VCAM-1, were raised in preeclampsia compared with healthy pregnancy, resulting in an overall pro-inflammatory systemic environment. Increased IP-10, MCP-1, ICAM-1 and VCAM-1 concentrations of preeclamptic patients showed significant correlations with blood pressure values, renal and liver function parameters, as well as with CRP, malondialdehyde, von Willebrand factor antigen and fibronectin levels.ConclusionsAccording to our findings, preeclampsia was associated with an overall pro-inflammatory systemic environment. Elevated amounts of pro-inflammatory cytokines, chemokines and adhesion molecules in the maternal circulation might play a central role in the excessive systemic inflammatory response, as well as in the generalized endothelial dysfunction characteristics of the maternal syndrome of preeclampsia.

Increased serum heat-shock protein 70 levels reflect systemic inflammation, oxidative stress and hepatocellular injury in preeclampsia

It has been previously reported that serum levels of 70-kDa heat-shock protein (Hsp70) are elevated in preeclampsia. The aim of the present study was to examine whether increased serum Hsp70 levels are related to clinical characteristics and standard laboratory parameters of preeclamptic patients, as well as to markers of inflammation (C-reactive protein), endothelial activation (von Willebrand factor antigen) or endothelial injury (fibronectin), trophoblast debris (cell-free fetal DNA) and oxidative stress (malondialdehyde). Sixty-seven preeclamptic patients and 70 normotensive, healthy pregnant women were involved in this case-control study. Serum Hsp70 levels were measured with enzyme-linked immunosorbent assay (ELISA). Standard laboratory parameters (clinical chemistry) and C-reactive protein (CRP) levels were determined by an autoanalyzer using the manufacturer’s kits. Plasma von Willebrand factor antigen (VWF:Ag) levels were quantified by ELISA, and plasma fibronectin concentration by nephelometry. The amount of cell-free fetal DNA in maternal plasma was determined by quantitative real-time polymerase chain reaction analysis of the sex-determining region Y gene. Plasma malondialdehyde levels were measured by the thiobarbituric acid-based colorimetric assay. Serum Hsp70 levels were increased in preeclampsia. Furthermore, serum levels of blood urea nitrogen, creatinine, bilirubin and CRP, serum alanine aminotransferase and lactate dehydrogenase (LDH) activities, as well as plasma levels of VWF:Ag, fibronectin, cell-free fetal DNA and malondialdehyde were also significantly higher in preeclamptic patients than in normotensive, healthy pregnant women. In preeclamptic patients, serum Hsp70 levels showed significant correlations with serum CRP levels (Spearman R = 0.32, p = 0.010), serum aspartate aminotransferase (R = 0.32, p = 0.008) and LDH activities (R = 0.50, p < 0.001), as well as with plasma malondialdehyde levels (R = 0.25, p = 0.043). However, there was no other relationship between serum Hsp70 levels and clinical characteristics (age, parity, body mass index, blood pressure, gestational age, fetal birth weight) and laboratory parameters of preeclamptic patients, including markers of endothelial activation or injury and trophoblast debris. In conclusion, increased serum Hsp70 levels seem to reflect systemic inflammation, oxidative stress and hepatocellular injury in preeclampsia. Nevertheless, further studies are required to determine whether circulating Hsp70 plays a causative role in the pathogenesis of the disease.

Relationship of circulating cell-free DNA levels to cell-free fetal DNA levels, clinical characteristics and laboratory parameters in preeclampsia

BackgroundThe aim of our study was to examine whether increased circulating total cell-free DNA levels are related to the clinical characteristics and standard laboratory parameters of preeclamptic patients, to markers of inflammation, endothelial activation or injury, oxidative stress and to cell-free fetal DNA levels.MethodsCirculating total cell-free DNA was measured by real-time quantitative PCR in plasma samples obtained from 67 preeclamptic and 70 normotensive pregnant women. Standard laboratory parameters, C-reactive protein, plasma von Willebrand factor antigen, plasma fibronectin, plasma malondialdehyde and cell-free fetal DNA levels were also determined.Results and ConclusionCirculating total cell-free and fetal deoxyribonucleic acid levels were significantly elevated in pregnancies complicated by preeclampsia (median: 11.395 vs. 32.460 and 0.001 vs. 0.086 pg/μl; P < .001). The quantity of plasma total cell-free DNA did not correlate with most of the laboratory parameters, except for serum aspartate aminotransferase and alanine aminotransferase activities (correlation coefficient: 0.31; P = 0.012 and 0.46; P < .001). There was no correlation with clinical characteristics, including body mass index. The releases of both free fetal and total cell-free deoxyribonucleic acid were found to be affected in preeclampsia. Hepatocellular necrosis seems to be responsible - at least partly - for increased circulating total DNA levels in preeclampsia, as suggested by the significant correlation with liver enzyme activities.

Incidence of chromosomal abnormalities in the presence of fetal subcutaneous oedema, such as nuchal oedema, cystic hygroma and non-immune hydrops

Introduction: The authors investigated the incidence of chromosomal abnormalities in subcutaneous oedema detected in the fetus by intrauterine ultrasonography. Material and Method: In the 10-year period, intrauterine karyotyping was performed in pregnancies with positive ultrasound findings for subcutaneous oedema, such as nuchal oedema, cystic hygroma and non-immune hydrops. Results: Intrauterine karyotyping in fetal subcutaneous oedema was carried out in 434 cases. The chromosomal investigation was made in nuchal oedema in 374 cases, in 120 patients the chromosomal examination was made in the first trimester because of nuchal translucency, and in 254 cases in the second trimester because of nuchal thickening. Cystic hygroma cases (27 patients), non-immune hydrops cases (20 patients), and combined cases of non-immune hydrops and cystic hygroma (13 patients) were investigated separately. In nuchal oedema, pathological karyotypes were detected in 8.33% in the first trimester and in 5.51% in the second trimester. Chromosomal abnormality was found in 48.15, 20, and 53.8% in cystic hygroma, non-immune hydrops, and combined occurrence of non-immune hydrops and cystic hygroma, respectively. Considering all of the changes accompanied by subcutaneous oedema, 50, 25 and 18.75% of the pathological karyotypes was X-monosomy, trisomy 18 and trisomy 21, respectively. Discussion: It was important to distinguish nuchal oedema and cystic hygroma, and in the case of non-immune hydrops, it was also important to discuss cases with or without cystic hygroma separately. During the investigations, cases of non-immune hydrops with or without cystic hygroma were evaluated as separate categories. Conclusions: The authors emphasize the differentiation of the various types of subcutaneous oedema and the importance of precise information about the risks, provided during genetic counselling.

Role of hsa-miR-325 in the etiopathology of preeclampsia

Preeclampsia (PE) is a common pregnancy-specific syndrome characterized by hypertension and proteinuria. Evidence has demonstrated that hypertensive disorders in pregnancy are associated with alterations in the expression of different microRNAs (miRNAs). miRNAs are endogenously expressed non-coding RNAs that have significant biological and pathological functions due to their potential mechanisms of regulation of gene expression. The purpose of the present study was to investigate the expression of hsa-miR-325 in placental samples of preeclamptic and uncomplicated pregnancy patients. hsa-miR-325 was isolated from placenta tissue samples obtained from 31 preeclamptic and 28 normotensive pregnant females. Quantitative real-time polymerase chain reaction was used to analyze miRNA expression. The expression of hsa-miR‑325 was elevated in uncomplicated pregnancies compared with preeclamptic patients. ΔCt (mean±SD) values were 0.117±0.07 in PE tissues and 0.135±0.051 in normotensive cases (p<0.05). The expression levels correlated with patient blood pressure (p=0.015, r=-0.23), and tended to correlate with body mass index (p=0.065, r=0.261). The expression of hsa-miR-325 was downregulated in the case of PE. Changes in hsa-miR‑325 expression in the case of pregnancy-related hypertensive disorders might affect the oxidative stress pathways and heat-shock protein production. These factors have a strong correlation with the development of PE. We, therefore, suggest that hsa-miR-325 contributes to the pathogenesis of PE.

Plasma osteopontin concentrations in preeclampsia - Is there an association with endothelial injury?

Abstract Background: It has been previously reported that plasma osteopontin (OPN) concentrations are increased in cardiovascular disorders. The goal of the present study was to determine plasma OPN concentrations in healthy pregnant women and preeclamptic patients, and to investigate their relationship to the clinical characteristics of the study subjects and to markers of inflammation [C-reactive protein (CRP)], endothelial activation [von Willebrand factor antigen (VWF:Ag)] or endothelial injury (fibronectin), oxidative stress [malondialdehyde (MDA)] and trophoblast debris (cell-free fetal DNA). Methods: Forty-four patients with preeclampsia and 44 healthy pregnant women matched for age and gestational age were involved in this case-control study. Plasma OPN concentrations were measured with ELISA. Serum CRP concentrations were determined with an autoanalyzer using the manufacturers reagents. Plasma VWF:Ag was quantified by ELISA, while plasma fibronectin concentrations were measured by nephelometry. Plasma MDA concentrations were estimated by the thiobarbituric acid-based colorimetric assay. The amount of cell-free fetal DNA in maternal plasma was determined by quantitative real-time PCR analysis of the sex-determining region Y (SRY) gene. For statistical analyses, non-parametric methods were applied. Results: Serum levels of CRP, as well as plasma concentrations of VWF:Ag, fibronectin, MDA and cell-free fetal DNA were significantly higher in preeclamptic patients than in healthy pregnant women. There was no significant difference in plasma OPN concentrations between controls and the preeclamptic group. However, preeclamptic patients with plasma fibronectin concentrations in the upper quartile had significantly higher plasma OPN concentrations than those below the 75th percentile, as well as healthy pregnant women [median (interquartile range): 9.38 (8.10–11.99) vs. 7.54 (6.31–9.40) and 7.40 (6.51–8.80) ng/mL, respectively, p<0.05 for both]. Furthermore, in preeclamptic patients, plasma OPN concentrations showed a significant positive linear association with plasma fibronectin (Spearman R=0.38, standardized regression coefficient (β)=0.41, p<0.05 for both). Conclusions: Plasma OPN concentrations are increased in preeclamptic patients with extensive endothelial injury. However, further studies are warranted to explore the relationship between OPN and endothelial damage. Clin Chem Lab Med 2010;48:181–7.

Leptin gene (TTTC)n microsatellite polymorphism in pre-eclampsia and HELLP syndrome

Abstract Background: Leptin plays an important role in energy homeostasis. There is polymorphism on the leptin (LEP) gene. Our aim was to compare the tetranucleotide repeat (TTTC)n polymorphism in the 3′-flanking region in the LEP gene on DNA samples from patients with pre-eclampsia (PE), hemolysis, elevated liver enzymes, and low platelet (HELLP) syndrome and healthy pregnant controls. Methods: Blood samples were collected from healthy pregnant women (n=88), patients with PE (n=79) and HELLP (n=77) syndrome. Fluorescent PCR and DNA fragment analysis was performed from the isolated DNA for the detection of (TTTC) repeats. The electrophoretograms were evaluated and patients were assigned to two groups; class I low (<190 bp) or class II high (≥190 bp) PCR fragments. Results: We observed similar distributions of the class I and class II (TTTC) alleles in the groups studied (class I allele: healthy pregnant 58.5%; severe pre-eclamptic 58.3%; HELLP syndrome 52.6%). We detected a higher frequency of the II/II genotype in HELLP syndrome patients (32.4%) compared to healthy controls (22.7%). However, the difference was not statistically significant. Conclusions: In an ethnically homogenous population, the LEP gene (TTTC) microsatellite polymorphism in the 3′-flanking region does not show a significant difference in the allele and genotype distribution in healthy pregnant, pre-eclamptic and HELLP syndrome patients. Furthermore, we recommend a new classification of the class I and class II alleles based on the distribution of the (TTTC) microsatellites. Clin Chem Lab Med 2009;47:1033–7.

Factors affecting reproductive outcome following abdominal myomectomy

PurposeFibroids may cause infertility and recurrent pregnancy loss. Studies have analysed the reproductive results after myomectomy according to the size, location and number of fibroids removed, but data are insufficient about comparison of opening the uterine cavity or not during surgery.Materials and methodsTwo hundred twenty-nine abdominal myomectomies with the indication of infertility and/or recurrent pregnancy loss were analysed retrospectively. The main purpose was to compare postoperative pregnancy, delivery and miscarriage rates according to either the uterine cavity was opened or not during the surgery. As a secondary outcome postoperative pregnancy rates were assessed by location, size and number of fibroids.ResultsThere was no significant difference in reproductive results according to either the uterine cavity was opened or remained closed. Preoperative location, size and number of fibroids did not influence significantly the postoperative pregnancy rates.ConclusionOpening the uterine cavity does not impair postoperative pregnancy rates. Preoperative location, size and number of fibroids do not influence postoperative reproductive results.

Quantity of total cell free and cell free fetal DNA in pregnancies with no complications and with preeclampsia

UNLABELLED Several researches focused on the factors which could influence the quantity of cell free DNA in case of normal and pathological pregnancies. The aim of our study was to evaluate the quantity of total cell free and cell free fetal DNA in case of normal pregnancies and preeclampsia. STUDY DESIGN Plasma samples were obtained from 67 preeclamptic and 70 normotensive pregnant women. The quantity of total cell free DNA and cell free fetal DNA was measured using real-time polymerase chain reaction. RESULTS We confirmed that circulating total free and fetal DNA levels are significantly elevated in pregnancies complicated by preeclampsia (median: 0.0114 vs. 0.0325 and 0.001E-3 vs. 0.086E-3 ng/microl; P < 0.001). The quantity of total plasma-free DNA did not correlate with the body mass index. CONCLUSION The releases of both free fetal and maternal DNA were found to be affected in preeclampsia. Hepatocellular necrosis seems to be responsible - at least partly - for increased circulating total DNA levels in preeclampsia, and the abnormal trophoblast invasion could be responsible for increased trophoblast destruction and elevation of cell free fetal DNA level.

The Correlation of Circulating Cell-Free DNA, Cell-Free Fetal DNA and MicroRNA 325 Levels to Clinical Characteristics and Laboratory Parameters in Pre-eclampsia

Objective: Elevated amounts of circulating DNA in maternal plasma have been detected in pregnancies complicated by preeclampsia. In order to confirm this, we simultaneously examined whether increased circulating cell-free DNA and microRNA levels are related to the clinical and laboratory parameters of preeclamptic patients. The quantity of total plasma-free DNA did not correlate with most of the laboratory parameters except for serum aspartate aminotransferase and alanine aminotransferase activities. MicroRNA expression was significantly lower in preeclamptic patients than in normotensive cases. There was no correlation with clinical characteristics, including body mass index. The quantity of markers of inflammation, endothelial activation/injury and oxidative stress did not show any correlation with cf DNA levels, and neither did cff DNA levels. Hepatocellular necrosis seems to be responsible – at least partly – for the increased circulating total DNA levels in preeclampsia, as suggested by the significant correlation with liver enzyme activities. MiRNA 325 also seems to play role in the developement of preeclampsia.