Elevated hsa-miR-99a levels in maternal plasma may indicate congenital heart defects.

The current standard for prenatal screening is mostly based on biochemical marker tests and the use of ultrasonography. There is no secure stand-alone screening marker for congenital heart defects (CHDs). MicroRNAs (miRNAs) that are associated with cardiogenesis enter the maternal peripheral bloodstream during pregnancy and allow non-invasive prenatal testing (NIPT). The present study investigated the plasma expression profile of fetal hsa-miR-99a in maternal blood. Peripheral blood samples were collected from 39 pregnant patients, comprising 22 with CHD-positive fetuses and 17 with CHD-free controls. miRNAs were isolated from the maternal serum and reverse transcription-quantitative polymerase chain reaction was carried out to determine the expression of hsa-miR-99a. While the miRNA concentrations were almost identical among the affected and control groups (5.54 vs. 6.40 ng/µl), significantly upregulated hsa-miR-99a levels were identified in the affected group (1.78×10-2±3.53×10-2 vs. 1.09×10-3±3.55×10-3 ng/µl, P=0.038). In conclusion, according to the present study, hsa-miR-99a is involved in cardiac malformation and may serve as a biomarker during fetal development, and therefore presents as a candidate for monitoring cardiomyogenesis and potential use as a NIPT-biomarker for fetal CHD.

Detection of sex chromosome aneuploidies using quantitative fluorescent PCR in the Hungarian population.

BACKGROUND: Aneuploidies are the most frequent chromosomal abnormalities at birth. Autosomal aneuploidies cause serious malformations like trisomy 21, trisomy 18 and trisomy 13. However sex chromosome aneuploidies are causing less severe syndromes. For the detection of these aneuploidies, the "gold standard" method is the cytogenetic analysis of fetal cells, karyograms show all numerical and structural abnormalities, but it takes 2-4 weeks to get the reports. Molecular biological methods were developed to overcome the long culture time, thus, FISH and quantitative fluorescent PCR were introduced. In this work we show our experience with a commercial kit for the detection of sex chromosome aneuploidies. METHODS: We analyzed 20.173 amniotic fluid samples for the period of 2006-2013 in our department. A conventional cytogenetic analysis was performed on the samples. We checked the reliability of quantitative fluorescent PCR and DNA fragment analysis on those samples where sex chromosomal aneuploidy was diagnosed. RESULTS: From the 20.173 amniotic fluid samples we found 50 samples with sex chromosome aneuploidy. There were 19 samples showing 46, XO, 17 samples with 46, XXY, 9 samples with 47, XXX and 5 samples with 47, XYY karyotypes. The applied quantitative fluorescent PCR and DNA fragment analyses method are suitable to detect all abnormal sex chromosome aneuploidies. CONCLUSIONS: Quantitative fluorescent PCR is a fast and reliable method for detection of sex chromosome aneuploidies.

PP028. Serum cytokine profile in relation to the clinical features and laboratory parameters in women with preeclampsia.

INTRODUCTION: Preeclampsia is characterized by an excessive maternal systemic inflammatory response. OBJECTIVES: To determine circulating levels of cytokines, chemokines and adhesion molecules in normal pregnancy and preeclampsia, and to investigate their relationship to the clinical features and laboratory parameters of the patients. METHODS: Serum levels of IL-1beta, IL-1ra, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p40, IL-12p70, IL-18, IFN-gamma, TNF-alpha, TGF-beta1, IP-10, MCP-1, ICAM-1 and VCAM-1 were measured in 60 preeclamptic, 60 healthy pregnant and 59 healthy non-pregnant women. RESULTS: In addition to a shift towards Th1-type immunity (increased IL-2/IL-4 and IFN-gamma/IL-4 ratios), 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, 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. CONCLUSION: 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 preeclampsia.

PP088. The role of microRNA in pathogenesis of preeclampsia-miRNA network analysis.

INTRODUCTION: Preeclampsia (PE) is a leading cause of maternal and fetal mortality and morbidity, affecting least 5-8% of all pregnancies worldwide. Several theories (e.g., immunological, placental ischemia, and genetic) have been described to explain pathogenesis of PE. The analysis of different contributing factors (proteins, mRNA, miRNA) are in the higlight of preeclampsia research, leading to an increasing pool of data. Recently, microRNA-s seems to provide feasible biochemical mechanism playing key role in protein translation regulation. MATERIALS AND METHODS: The aim of our strudy was to collect datas from literature regarding to different subset of miRNA-s connected to pathways playing role in pathogenesis of preeclampsia. miRNA expression datas were analysied by biostatistical methods, connections in miRNA gene network and graphical representation of connections and crosspoints of supposed pathways were performed. CONCLUSIONS: In more than a hundered miRNA-s analysed in different studies most of the reserches concentrate on angiogenesis, trophoblast cell invasion, vascular developement, oxiadtive stress and blood presure regulation. This condition resulting as major crosspoints miRNA-s involved in this pathways: miR-29b, miR-155, miR-195, miR-16, miR-20a, miR-20b. Complex network analysis and graphical presentation in dynamic manner of miRNA pathways and possible consecquences of aletartion in miRNA expression could help in setting of major directions in PE research.

[Cell-free nucleic acid-based non-invasive prenatal diagnosis of fetal aneuploidies]. / Magzati aneuploidiák szabadnukleinsav-alapú, nem invazív diagnosztikája.

Prenatal detection of fetal aneuploidies is one of the main goals of the prenatal diagnostic approach. As a benefit of the development of advanced ultrasound equipment and advances in molecular biology in the last decade, there is a significant progress in screening methods for fetal aneuploidies, although invasive methods remain the gold standard for aneuploidy detection. Non-invasive prenatal diagnosis has substantial medical impact as it targets the development of safer and more effective methods to avoid the risk of fetal loss associated with currently used invasive methods. Identification of fetal-specific messenger ribonucleic acids, digital polymerase chain reaction and next-generation sequencing give the real chance for non-invasive prenatal diagnosis of fetal aneuploidies. Although all these methods have both advantages and limitations, some of them are moving closer to clinical implementation. In this review the authors highlight the most recent advances in methods for non-invasive prenatal diagnosis of aneuploidies.

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-miR­325 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-miR­325 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.

Early prenatal detection of a fast-growing fetal epignathus.

Epignathus is a rare congenital orofacial teratoma. We present a case of a fast-growing tumor, where early prenatal diagnosis was made and where fetopathological examination revealed the reason of the remarkable ultrasonographic signs and underlined the expected poor prognosis. Ultrasonographic examination at 18 weeks' gestation showed that there was a growing tumor protruding from the fetus's mouth. The fetal stomach could not be seen and extreme polyhydramnios was also detected. After counseling, the couple opted for a termination of pregnancy. Fetopathological examination showed that the tumorosus mass was not only protruding from the mouth, but also inexplicably grew downwards, was connected to the hard palate and the periosteum of the vertebral corpus, making an airway and esophageal obstruction, causing the ultrasonographic findings. Postnatal treatment and surgical removal of this tumor seemed to be impossible. In case of an early detection of a fast-growing fetal epignathus, pregnancy termination should be considered.

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

BACKGROUND: Preeclampsia 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). RESULTS: Serum 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. CONCLUSIONS: According 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.

[Quantity of total cell free and cell free fetal DNA in pregnancies with no complications and with preeclampsia]. / A vérplazma összes szabad DNS, valamint szabad magzati DNS mennyisége praeeclampsiával szövôdött és szövôdménymentes terhességek esetében.

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.

Leptin receptor (LEPR) SNP polymorphisms in HELLP syndrome patients determined by quantitative real-time PCR and melting curve analysis.

BACKGROUND: Several studies have shown overexpression of leptin in microarray experiments in pre-eclampsia (PE) and in hemolysis, elevated liver enzymes, low platelets (HELLP) syndrome. We decided to study four leptin receptor (LEPR) SNP polymorphisms in HELLP syndrome patients by using quantitative real-time PCR and melting curve analysis. METHODS: DNA was isolated from blood samples from 83 normotensive pregnant women and 75 HELLP syndrome patients. Four SNPs, LEPR c.326A>G (K109), LEPR c.668A>G (Q223R), LEPR c.1968G>C (K656N) and LEPR c.3024A>G (S1008) were determined by quantitative real-time PCR and melting curve analysis. Investigators were blinded to clinical outcomes. RESULTS: LEPR c.326A>G, LEPR c.668A>G, LEPR c.1968G>C and LEPR c.3024A>G allele, genotype and haplotype polymorphisms were not different in HELLP syndrome patients and normotensive healthy pregnants. There were strong linkage disequilibrium (LD) between loci c.326A>G and c.6687A>G (D' = 0.974), and c.668A>G and c.1968G>C (D' = 0.934), and c.326A>G and c.1968G>C (D' = 0.885), and c.1968G>C and c.3024A>G (D' = 1.0). However, linkages of c.3024A>G with c.668A>G (D' = 0.111) and c.326A>G (D' = 0.398) were weak. The Hardy-Weinberg equilibrium was observed for all polymorphisms. However the LEPR c.326A>G AG genotype was twice more frequent and the (AG AG GG AG) haplotype was three times more frequent in HELLP syndrome patients. The introduced quantitative real-time PCR combined with melting curve analysis is a fast and reliable method for the determination of LEPR SNPs. CONCLUSION: Although certain LEPR haplotypes are more frequent in HELLP syndrome, we conclude that there is no compelling evidence that the four studied LEPR SNP polymorphisms associated with the development of HELLP syndrome.

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

UNLABELLED: 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 manufacturer's 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 (beta)=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.

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

BACKGROUND: The 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. METHODS: Circulating 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 CONCLUSION: Circulating 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/microl; 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.

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

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 (> or =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.

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.

Factors affecting reproductive outcome following abdominal myomectomy.

PURPOSE: Fibroids 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 METHODS: Two 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. RESULTS: There 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. CONCLUSION: Opening the uterine cavity does not impair postoperative pregnancy rates. Preoperative location, size and number of fibroids do not influence postoperative reproductive results.

[Detection of Toxoplasma gondii in amniotic fluid using quantitative real-time PCR method]. / Toxoplasma gondii kimutatása magzatvízbol kvantitatív valós ideju PCR-módszerrel.

INTRODUCTION: The infection caused by parasite Toxoplasma gondii is often asymptomatic or a mild clinical disease. Congenital toxoplasmosis is the result of transplacental transmission of Toxoplasma gondii from an acute infected mother. Toxoplasmosis can cause several fetal symptoms. Early diagnosis of the infection can enhance the success of the medical treatment. Congenital toxoplasmosis can be detected by serological or PCR amplification methods. AIMS: The authors decided to develop a quantitative real-time PCR technique for detection of T. gondii in amniotic fluid samples. METHODS: DNA was isolated using silica adsorption method. Quantitative real-time PCR method was used to detect T. gondii infection in the samples. RESULTS: From the studied 74 samples in 6 cases T. gondii was detected. CONCLUSION: The introduced quantitative real-time PCR method is a fast and sensitive PCR based method and makes possible the quantification of the protozoa number in the sample.

[Non invasive detection of fetal Rh using real-time PCR method]. / Noninvazív magzati Rh-meghatározás real-time PCR-módszerrel.

INTRODUCTION: In the last ten years the detection of fetal origin cells and cell free fetal DNA in maternal circulation opened new horizons in non-invasive prenatal diagnosis. The diagnostic possibilities are based on the differences between the maternal and fetal origin DNA. One of the differences could be the Rh blood group and the genetical background. The Rh incompatibility is the most frequent blood group incompatibilities in the clinical practice, which can cause fetal anemia, hydrops and even fetal death. AIMS: The aim of this study was to detect the fetal DNA in maternal circulation, to determine the Rh status of the fetus, and to compare the reliability of the method with the data found in other studies. METHODS: Blood samples and amnionic fluid samples were collected from 30 pregnant women, with Rh negative status, between 11-22 week of gestation presented for genetic amniocentesis at the 1st. Department of Obstetrics and Gynecology, Semmelweis University. After DNA isolation real-time PCR was performed in order to detect the exon 7 of the RhD gene located on the first chromosome (1p36.11.). RESULTS: In 24 cases the PCR reaction gave same result in case of the DNA isolated from plasma and amniotic fluid, but in six cases there was no PCR product of plasma samples and the product was detectable in amniotic fluid samples. The exon 7 was detectable in 25 cases, and there was no product in 5 cases. CONCLUSIONS: The real-time PCR method seems to be an easy and reliable method to determine the fetal Rh blood group. The sensitivity and specificity of the method in this study is in concordance with international data. The use of more than one probe could increase the sensitivity of the method.

Prenatal sonographic findings in 207 fetuses with trisomy 21.

OBJECTIVE: The objective was to evaluate the contribution of second trimester ultrasound examination to the prenatal diagnosis of trisomy 21 in 207 fetuses with this aneuploidy. The type and frequency of abnormal sonographic findings were determined. Possible multiple malformation patterns, characteristic of trisomy 21 were sought. STUDY DESIGN: Singleton fetuses that had prenatal sonography during the second trimester, then underwent cytogenetic evaluation in our institution, made up the study population. The sonographic findings of 207 fetuses with trisomy 21 were analyzed. RESULTS: Between 1990 and 2004, fetal karyotyping was performed in 22,150 patients for different indications. An abnormal karyotype was diagnosed in 514 cases (2.3%); among them 207 fetuses with trisomy 21 were detected (40.3%). Abnormal sonography was seen in 63.8% of the cases. Structural anomalies were detected in 28.5% of the trisomy 21 fetuses, among them cardiac defects (15.9%), central nervous system anomalies (14.5%), and cystic hygromas (6.8%) were the most common. Of the minor markers, increased nuchal translucency (28%), pyelectasis (20.3%), and shorter extremities (8.7%) were common findings. CONCLUSIONS: Appropriate diagnosis of structural anomalies, looking for relatively easily detectable minor markers and incorporating fetal echocardiography into the second trimester sonographic protocol, may increase the contribution of mid-trimester ultrasound examination to diagnosing trisomy 21.

Detection of DeltaF508del using quantitative real-time PCR, comparison of the results obtained by fluorescent PCR.

OBJECTIVE: Cystic fibrosis (CF) is the most common autosomal recessive genetic disorder in the Caucasian population. The molecular diagnosis is difficult since there are about 1,000 mutations in the CF transmembrane regulator gene. The DeltaF508del is the cause of the CF in 64% of the cases in Hungary. Our aim was to compare two polymerase chain reaction (PCR)-based method for the detection of DeltaF508del. METHODS: One hundred and sixteen DNA samples isolated from different tissues (84 blood samples, 18 chorionic villus samples and 14 amniotic fluid samples) were involved in the study. Fluorescent PCR with DNA fragment analysis and quantitative real-time PCR with melting curve analysis were performed on the DNA samples for the detection of DeltaF508del. RESULTS: Sixty-five healthy normal samples, 43 heterozygous samples, 6 DeltaF508del homozygous samples and 2 DeltaF508C homozygous samples were detected by using quantitative real-time PCR combined with melting curve analysis. The fluorescent PCR method did not detect the DeltaF508C mutation and these two samples were diagnosed as normal healthy ones. CONCLUSIONS: The quantitative real-time PCR with melting curve analysis is a reliable and fast method for the detection of DeltaF508del. The results are ready in 1 h following the DNA isolation. The applied primer-probe set with melting curve analysis gives additional information for the presence of other mutations in the DeltaF508del region.

[Detection of deltaF508 deletion causing cystic fibrosis, using quantitative real-time PCR]. / A cystis fibrosist okozó F508 deléció kimutatása kvantitatív real-time PCR-módszerrel.

INTRODUCTION: Cystic fibrosis is the most common autosomal recessive lethal genetic disorder in the Caucasian population. There are about 1400 mutation in the cystic fibrosis transmembrane regulator gene, which makes the molecular diagnosis difficult, while luckily in Hungary the cause is the deltaF508del in almost 60% of the cases. METHOD: The authors introduced the quantitative real-time PCR and melting curve analysis method for the detection of deltaF508del. They studied 94 samples (70 blood, 16 chorionic villi, 8 amniotic fluids). RESULTS: They found 52 healthy normal, 36 heterozygotic and 5 homozygotic samples and one deltaF508C homozygotic sample. DISCUSSION: The quantitative real-time PCR and melting curve analysis is a reliable and fast method for detection of deltaF508del. The results are available in one hour following the DNA isolation. The primer-probe set makes available the deltaF508Cdel detection too.