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Aim The aim of research was to evaluate the maternal serum concentration of selected endogenous steroid hormones during spontaneous parturition at term and to determinate their association with the need for oxytocin augmentation. Methods Blood of 108 healthy pregnant women whose parturition started with the regular spontaneous uterine contractions was drawn at the beginning of the labor. Liquid chromatography coupled to the tandem mass spectrometry device was utilized for measurement of sex hormone binding globulin, aldosterone, androstenedione, cortisol, cortisone, corticosterone, dehydroepiandrosterone, dehydroepiandrosteron sulphate, 17-hydroxyprogesterone, progesterone, and testosterone. Mann-Whitney U test, chi-square test, univariate and multivariate logistic regression, and receiver operating characteristic (ROC) analysis were used for the data analysis. Results Reference ranges of the selected hormones assessed by liquid chromatography coupled to the tandem mass spectrometry in maternal serum were established. Statistically significant differences in the serum concentration of corticosterone, dehydroepiandrosterone, and androstenedione between mothers requiring oxytocin augmentation and the rest of women having spontaneous parturition were found (p = 0.002, p = 0.008, and p = 0.04, respectively). Concentrations were lower in the group of mothers who required oxytocin infusion for progression of labor. ROC analysis showed that all the mothers with dehydroepiandrosterone serum concentration above 21.6 nmol/L have lower chance to use oxytocin infusion for the labor progression ( area under the curve (AUC) = 0.649, sensitivity = 71.7%, specificity = 59.6%, p = 0.006). Conclusion This research provided reference ranges for the selected maternal serum steroid hormones at the beginning of parturition. Association of corticosterone, dehydroepiandrosterone, and androstenedione with the need for the oxytocin infusion usage has been established. Dehydroepiandrosterone could be potential predictor of oxytocin infusion augmentation for progression of the parturition.

Aim To evaluate the roles of four selected genetic variations in fetal and maternal progesterone receptor gene (PGR) and to identify women who may have higher or lower odds for spontaneous premature birth compared to the general population. Methods A preliminary case–control study with two groups of pregnant women (with term and premature delivery, 218 in total) and two groups of newborns (term and preterm, 218 in total) was performed. Four single nucleotide polymorphisms (SNPs) of the progesterone receptor gene (rs1042838, rs1042839, rs10895068, and rs1942836) were genotyped. Results There was statistically significant difference between cases and controls in the distribution of newborns' allele frequency of minor C allele of the PGR SNP rs1942836 (p = 0.03, Fishers' exact test) in favor of premature birth. A statistically significant difference between the frequency of the mothers' minor T allele of rs1042838 (p = 0.005 ; chi-squared test) and the mothers' minor T allele of rs1042839 (p = 0.005 ; chi-squared test) in favor of extremely premature birth has been found. There was a statistically significant difference between the frequency of the newborns' minor C allele of rs1942836 (p = 0.03 ; chi-squared test) and newborns' heterozygotes CT genotype of rs1942836 (p = 0.03 ; Fishers' exact test) when comparing the group of term births and the group of early premature birth. Conclusion Our study suggests that patients with selected genetic variants of the progesterone receptor gene could have greater odds for premature birth compared to term birth. Replication studies with a larger population and different ethnicity are needed in order to confirm these findings.

The purpose of this study was to evaluate the relationships between interleukin 10 (IL10) (rs1800896) and interleukin 1B (IL1B) (rs16944) genetic polymorphisms and the risk for cervical cancer in a cohort of women from Croatia.A case-control study of 81 patients with cervical cancer and 80 age-matched healthy controls was performed. We collected peripheral blood samples, extracted deoxiribonucleic acid (DNA), and analyzed two single-nucleotide polymorphisms (SNPs) rs1800896 and rs16944 using TaqMan assays (Fa. Thermo Fisher Scientific, Waltham, MA, USA) and real-time polymerase chain reaction (PCR). We investigated a possible association between two cytokine genetic polymorphisms and the occurrence of cervical cancer.Our results showed no significant difference in the frequency of IL10 (rs1800896) and IL1B (rs16944) genotypes between the patients and the controls (χ2 test, P 0.05).In this study, no association was found between IL10 rs1800896 and IL1B rs16944 polymorphisms and cervical cancer development.GRUNDLAGEN: Das Ziel der vorliegenden Studie war, die Beziehung zwischen den genetischen Polymorphismen IL10 (rs1800896) und IL1B (rs16944) und dem Risiko für Gebärmutterhalskrebs in einer Kohorte von Frauen aus Kroatien zu untersuchen.Es wurde eine Fall-Kontroll-Studie mit 81 Frauen mit invasivem Gebärmutterhalskrebs und 80 altersentsprechenden gesunden Kontrollen durchgeführt. Dazu wurden periphere Blutproben gesammelt, DNA extrahiert und 2 Einzelnukleotidpolymorphismen (SNP; rs1800896 und rs16944) unter Verwendung von TaqMan-Assays (Fa. Thermo Fisher Scientific, Waltham, MA, USA) und der Echtzeit-Polymerasekettenreaktion (PCR) analysiert. Die Autor*innen untersuchten einen möglichen Zusammenhang zwischen dem genetischen Polymorphismus zweier Zytokine und dem Auftreten des Zervixkarzinoms.Dabei zeigte sich kein signifikanter Unterschied in der Häufigkeit der Genotypen IL10 (rs1800896) und IL1B (rs16944) zwischen den Patienten und gesunden Kontrollen (χIn dieser Studie wurde kein Zusammenhang zwischen den Polymorphismen IL10 rs1800896 und IL1B rs16944 und der Entwicklung von Gebärmutterhalskrebs festgestellt.

Women’s metabolism during pregnancy undergoes numerous changes that can lead to gestational diabetes mellitus (GDM). The cause and pathogenesis of GDM, a heterogeneous disease, are not completely clear, but GDM is increasing in prevalence and is associated with the modern lifestyle. Most diagnoses of GDM are made via the guidelines from the International Association of Diabetes and Pregnancy Study Groups (IADSPG), which involve an oral glucose tolerance test (OGTT) between 24 and 28 weeks of pregnancy. Diagnosis in this stage of pregnancy can lead to short- and long-term implications for the mother and child. Therefore, there is an urgent need for earlier GDM markers in order to enable prevention and earlier treatment. Routine GDM biomarkers (plasma glucose, insulin, C-peptide, homeostatic model assessment of insulin resistance, and sex hormone-binding globulin) can differentiate between healthy pregnant women and those with GDM but are not suitable for early GDM diagnosis. In this article, we present an overview of the potential early biomarkers for GDM that have been investigated recently. We also present our view of future developments in the laboratory diagnosis of GDM.

Cardiovascular diseases are the world’s leading cause of death. Human physiologic activities and state during illness are under the control of circadian rhythm. The aim of the study was to determine the potential association of chronotype and daytime sleepiness with susceptibility to myocardial infarction. We conducted a case-control study on 200 patients hospitalized due to myocardial infarction and 200 healthy controls. Systematic information on the past and present medical history was obtained from all participants. Chronotype was assessed using the Morningness-Eveningness Questionnaire (MEQ), and daytime sleepiness was assessed by the Epworth Sleepiness Scale (ESS). The mean age of the study population was 64±13 years, and 54.5% were male. There was no significant difference in MEQ (58.88±6.52 vs. 58.46±7.78, p=0.601) or ESS (5 (interquartile range, IQR 4-7.5) vs. 6 (IQR 3-8), p=0.912) score between patients and controls. Nevertheless, we found statistically significant differences related to risk factors for cardiovascular diseases, such as hypertension, dyslipidemia, and diabetes mellitus. However, there was no association of MEQ and ESS score with myocardial infarction in the study population., Kardiovaskularne bolesti su vodeći uzrok smrtnosti u svijetu. Pod kontrolom cirkadijalnog sata su humana fiziologija i fiziološka stanja tijekom bolesti. Cilj ovoga istraživanja bio je utvrditi potencijalnu povezanost kronotipa i dnevne pospanosti s infarktom miokarda. Provedeno je istraživanje slučajeva i kontrola na 200 bolesnika s infarktom miokarda te 200 zdravih kontrolnih ispitanika. Od svih ispitanika prikupljeni su podaci o prošloj i trenutnoj medicinskoj anamnezi. Kronotip je procijenjen pomoću upitnika MEQ (Morningness-Eveningness Questionnaire), a dnevna pospanost pomoću Epworthove ljestvice pospanosti (ESS, Epworth Sleepiness Scale). Prosječna životna dob ispitivane populacije bila je 64±13 godina, a 54,5% ispitanika činili su muškarci. Statistički značajna razlika nije pronađena između bolesnika i kontrola u kronotipu (58,88±6,52 nasuprot 58,46±7,78; p=0,601) i dnevnoj pospanosti (5 (IQR 4-7,5) nasuprot 6 (IQR 3-8); p=0,912). Unatoč tome, pronađena je statistički značajna razlika povezana sa čimbenicima rizika za kardiovaskularne bolesti kao što su hipertenzija, dislipidemija i dijabetes. Međutim, u ovom istraživanju nije utvrđena povezanost kronotipa i dnevne pospanosti s infarktom miokarda.

Cervical cancer is the most common gynaecological cancer in women. Cell mediated immunity plays a significant role in the progression or regression of neoplastic cervical lesions caused by human papilloma virus infection. Engagement of antigen-specific T cell receptors is a prerequisite for T cell activation. The initial events of T cell activation involve the movement of the T cell receptor into specialised microdomains known as lipid rafts. Gangliosides play an active role in the formation, stabilisation and biological functions of lipid rafts. This study aims to determine whether polymorphisms in the genes involved in the biosynthesis of gangliosides represent risk a factor for cervical cancer. Taqman methods for single nucleotide polymorphism genotyping was used. All subjects carried the homozygous wild-type genotypes for all analysed genes (CC for gene B4GALT5, AA for gene ST3GAL5, AA for gene ST8SIA1 and CC for gene B4GALNT1). A χ2 test showed significant differences in genotype failure for B4GALT5 rs138960078 (χ2 = 32.02, df = 1, p = .001) and genotype failure for B4GALNT1 rs144643461 (χ2 = 41.03, df = 1, p = .001) between cervical cancer group and control group. Genotype failures were significantly more frequent in the cervical cancer group. Unknown adjacent SNPs to rs138960078 in gene B4GALT5 and rs144643461 in gene B4GALNT1 could be associated with cervical cancer development.

Introduction/Background Cervical cancer is worldwide the second most common cancer in women. Immune response appears to be a key determinant in controlling human papilloma virus (HPV) infection. Cell mediated immunity plays a significiant role in progression or regression of neoplastic cervical lesions. Lipid rafts have essential role in T lymphocite activation. Gangliosides play an important role in the formation and stabilization of specific cell lipid membrane domains. This study aims to determine whether ganglioside GM1 expressions in T cell membrane represent risk factor for cervical cancer. Methodology In total, 20 women participated in our study. Peripheral blood samples were obtained from 10 cervical cancer patients and 10 healthy controls at University Hospital for Tumors, Zagreb, Croatia. A 3 mL sample of venous blood was collected from each subject into a test tube containing EDTA as anticoagulant. For T cell subtypes isolation we used Anti CD4 Phycoerythrin antibody and Anti CD8 Peridinin Chlorophyll Protein Complex antibody. Ganglioside GM1 expression in T lymphocytes membranes was analyzed using double labeling antibodies on ganglioside GM1. Results We have not found difference in ganglioside GM1 expression in T lymphocytes membranes between the two groups. Conclusion The present study did not show association of ganglioside GM1 expression in T lymphocytes membranes with cervical cancer development. Disclosure Nothing to disclose.

Single nucleotide polymorphism (SNP) in genes encoding drug-metabolizing enzymes (DME) could have a critical role in individual responses to anastrozole. Frequency of CYP3A4*1B, CYP3A5*3 and UGT1A4*2 SNPs in 126 Croatian breast cancer (BC) patients and possible association with anastrozole-induced undesirable side effects were analyzed. Eighty-two postmenopausal patients with estrogen receptor (ER)-positive BC treated with anastrozole and 44 postmenopausal ER-positive BC patients before hormonal adjuvant therapy were included in the study. Genomic DNA was genotyped by TaqMan Real-Time PCR. Bone mineral density (BMD) was measured by dual-energy X-ray absorptiometry. The homozygotes for the variant G allele of CYP3A5*3 were predominant (88%), and the homozygotes for the reference A allele were not detected. While homozygotes for the variant G allele of CYP3A4*1B were not detected, predominantly wild type homozygotes for A allele (94%) were present. CYP3A4*1B and CYP3A5*3 SNPs were in 84.3% linkage disequilibrium (D&rsquo, = 0.843) and 95.1% (D&rsquo, = 0.951) in group treated with anastrozole and w/o treatment, respectively. Homozygotes for the A allele of UGT1A4*2 were not detected in our study groups. Although the variant CYP3A5*3 allele, which might result in poor metabolizer phenotype and more pronounced side effects, was predominant, significant association with BMD changes induced by anastrozole were not confirmed.

The objectives of this study were to validate the direct triplet-primed PCR method (dTP-PCR) for determination of dynamic mutations in the

Hereditary hemochromatosis (HH) is a disorder of iron accumulation in tissues, which is related to coronary heart diseases. Free radicals and reactive oxygen species, created because of iron deposition, promote oxidation of LDL cholesterol and could lead to the development of atherosclerosis. Studies have shown that HFE gene mutation carriers might be at higher risk of developing cardiovascular diseases compared with non-carriers. This study aimed to determine the frequency of HFE gene mutations in patients with myocardial infarction compared to a healthy group in eastern Slavonia. A retrospective case-control study was carried out on a population of 400 participants. In the first group there were 200 patients (114 males and 86 females) with myocardial infarction. The second group consisted of 200 controls (103 males and 97 females) without a history of cardiovascular diseases. All patients were genotyped for the three most common mutations of the HH in the HFE gene: C282Y, H63D, and S65C, by real-time PCR. The difference in the frequency of carriers of these mutations between the patients and the controls was not significant (C282Y: 4.5 vs. 8.1%; H63D: 19 vs. 24.5%; S65C: 3.5 versus 4%), and neither was the frequency and distribution of possible HFE gene genotypes and compound heterozygotes. There were no statistically significant associations of cardiovascular risk factors and HFE gene mutations in patients with myocardial infarction. In this study, no association was found between the HFE gene mutation for HH and myocardial infarction in the population of eastern Slavonia.

Disruption of circadian clock may trigger the onset of diabetes mellitus and myocardial infarction. Type 2 diabetes mellitus (T2DM) is well-known risk factors for cardiovascular diseases and myocardial infarction. We performed a case-control study, where we explored the possible association between single nucleotide polymorphisms in three circadian rhythm genes (ARNTL, CLOCK, and PER2) and myocardial infarction in 657 patients with T2DM. The study group consisted of 231 patients with myocardial infarction and T2DM and a control group of 426 T2DM patients. We hypothesized that variations in the circadian rhythm genes in patients with T2DM could be an additional risk factor for myocardial infarction. The statistically significant difference was found in allelic (p = 1.1 × 10–5) and genotype distribution (p = 1.42 × 10–4) between two groups of the rs12363415 at the ARNTL gene locus. We provide evidence that genetic variability in the ARNTL gene might be associated with myocardial infarction in patients with T2DM.

Disruption of endogenous circadian rhythms has been shown to increase the risk of developing myocardial infarction (MI), suggesting that circadian genes might play a role in determining disease susceptibility. We conducted a case-control study on 200 patients hospitalized due to MI and 200 healthy controls, investigating the association between MI and single nucleotide polymorphisms (SNPs) in four circadian genes (ARNTL, CLOCK, CRY2, and PER2). The variants of all four genes were chosen based on their previously reported association with cardiovascular risk factors, which have a major influence on the occurrence of myocardial infarction. Statistically significant differences, assessed through Chi-square analysis, were found in genotype distribution between cases and controls of the PER2 gene rs35333999 (p=0.024) and the CRY2 gene rs2292912 (p=0.028); the corresponding unadjusted odds ratios, also significant, were respectively OR=0.49 (95% CI 0.26-0.91) and OR=0.32 (95% CI 0.11-0.89). Our data suggest that genetic variability in the CRY2 and PER2 genes might be associated with myocardial infarction.

Purpose Human physiological activities and diseases are under the control of the circadian rhythm. There are strong epidemiological associations between disrupted circadian rhythms, sleep duration and diseases. Sleep disorders are associated with vascular outcomes, such as myocardial infarction (MI). Methods We conducted an association study of genotype-phenotype interaction, to determine which circadian clock gene variants might be associated with the circadian phenotypes in patients with MI. In the present study, we analyzed the allele frequencies of 10 single nucleotide polymorphisms in four circadian clock genes in two independent samples: MI patients and controls. Chronotype was assessed using the Morningness - Eveningness Questionnaire (MEQ) and daytime sleepiness using the Epworth Sleepiness Scale (ESS). Results Chronotype was associated with the ARNTL genetic variant rs12363415 in MI patients. The polymorphisms rs11932595 of the CLOCK gene and rs934945 of the PER2 gene were associated with daytime sleepiness in the patient group. Conclusion Our data suggest that genetic variations in some circadian clock genes might be related to circadian phenotype (i.e., chronotype and daytime sleepiness) in patients with myocardial infarction.

Interleukin (IL)-10 is an anti-inflammatory cytokine, and a decrease in its secretion is associated with obesity, metabolic syndrome and type 2 diabetes. However, it has not been established whether the intensity of the immune response during diabetes-associated chronic inflammation affects the devel-opment and/or progression of type 2 diabetes and its microvascular complications. The aim of this study was to investigate the role of single nucleotide polymorphism (SNP)-1082G/A for IL-10 gene in development of diabetes type 2 and its complications. DNA was extracted from blood cells of 240 overweight/obese subjects for IL-10 genotyping. Based on the presence of diabetes type 2, patients were divided in two groups: experimental group of 144 patients with diabetes type 2 and control group of 96 age- and gender-matched subjects without diabetes. Compared to control group, diabetic group had higher levels of leukocytes (p=0.012), fibrinogen (p=0.049) and plasminogen activator inhibitor-1 (PAI-1) (p=0.009), and lower levels of albumin (p=0.001). There were no differences in the frequency of SNP-1082G/A for IL-10 gene between the two groups (p=0.654). When considering diabetes related traits in all subjects in relation to specific genotype, a group with homozygous (AA) genotype had higher values of the mean fasting glucose (p, Smanjena sekrecija protuupalnog citokina interleukina (IL)-10 opisana je u debljini, metaboličkom sindromu i tipu 2 šećerne bolesti (T2ŠB). Međutim, nije utvrđeno može li intenzitet imunog odgovora tijekom kronične upale utjecati na razvoj i/ili progresiju dijabetesa kao i razvoj mikrovaskularnih komplikacija. Cilj ove studije bio je istražiti povezanost polimorfizma gena (SNP) -1082G/A za IL-10 s razvojem T2ŠB i kroničnih komplikacija. DNK za genotipizaciju se ekstrahirala iz krvnih stanica 240 adipoznih ispitanika koji su podijeljeni u dvije skupine: u eksperimentalnoj skupini bilo je 144 bolesnika s T2ŠB, a u kontrolnoj skupini 96 ispitanika usklađenih prema spolu i dobi. U skupini s T2ŠB nađene su više vrijednosti leukocita (p=0,012), fibrinogena (p=0,049) i inhibitora aktivacije plazminogena-1 (PAI-1) (p=0,009) te niže koncentracije albumina (p=0,001) nego u kontrolnoj skupini. Nije bilo statističke razlike u frekvencijama -1082G/A polimorfizma gena za IL-10 (p=0,654). Kada su se gledali ishodi za specifični genotip u svih ispitanika, ispitanici s homozigotnim genotipom (AA) imali su značajno više koncentracije glukoze natašte (p

Hereditary hemochromatosis (HH) is a disorder of iron accumulation in tissues, which is related to coronary heart diseases. Free radicals and reactive oxygen species, created because of iron deposition, promote oxidation of LDL cholesterol and could lead to the development of atherosclerosis. Studies have shown that HFE gene mutation carriers might be at higher risk of developing cardiovascular diseases compared with non-carriers. This study aimed to determine the frequency of HFE gene mutations in patients with myocardial infarction compared to a healthy group in eastern Slavonia. A retrospective case-control study was carried out on a population of 400 participants. In the first group there were 200 patients (114 males and 86 females) with myocardial infarction. The second group consisted of 200 controls (103 males and 97 females) without a history of cardiovascular diseases. All patients were genotyped for the three most common mutations of the HH in the HFE gene: C282Y, H63D, and S65C, by real-time PCR. The difference in the frequency of carriers of these mutations between the patients and the controls was not significant (C282Y: 4.5 vs. 8.1%; H63D: 19 vs. 24.5%; S65C: 3.5 versus 4%), and neither was the frequency and distribution of possible HFE gene genotypes and compound heterozygotes. There were no statistically significant associations of cardiovascular risk factors and HFE gene mutations in patients with myocardial infarction. In this study, no association was found between the HFE gene mutation for HH and myocardial infarction in the population of eastern Slavonia.

Cell-free DNA has been associated with outcome in several acute conditions including two reports concerning the outcomes after cardiac arrest that found association of circulating DNA quantities at admission with mortality. The origins of cell-free DNA are primarily necrosis and apoptosis, which in cardiac arrest occur during ischaemia ("no-flow" and "low-flow" period), during reperfusion injury and as a consequence of post-arrest inflammatory response. Respecting the facts that significant cellular damage may occur during the post-arrest period, and that damage might be reduced by mild therapeutic hypothermia, we investigated the prognostic value of cell free DNA at ICU admission and 24h after admission. A prospective study was conducted in three university associated intensive care units and included patients resuscitated from non-traumatic out-of-hospital cardiac arrest. Patient data were collected in accordance with the Utstein protocol. Therapeutic hypothermia was performed according to ICU policies. Blood for cell-free DNA quantification was sampled at admission and at 24±1h after admission. Outcome measures were hospital morality and cerebral performance expressed with CPC scale at discharge. Inclusion criteria were met in 67 patients ; 24-h mortality was 37.3% and hospital mortality was 71.6%. The following variables were associated with 24-h mortality in univariate analysis: asystole as the presenting rhythm, "no-flow" time, "low-flow" time and cell-free DNA at admission (median 0.081 in survivors vs. 0.160ng/μl in non- survivors ; P=0.038). Multivariate analysis that included the above variables showed that no-flow time and low-flow time were independently associated with 24-h mortality. Hospital mortality was associated with the following factors: "low flow" time, coronary intervention, cell-free DNA at ICU admission and at 24h after admission (0.042 vs. 0.188ng/μl ; P=0.048). ROC curve for cell-free DNA 24h post-admission showed sensitivity of 81.0% and specificity of 78.3% for the cut-off value of 0.115ng/μl. Multivariate analysis showed that "low-flow" time and cell-free DNA at 24h after ICU admission were independently associated with hospital mortality. Cell free DNA showed different dynamics in patients who were and who were not treated with mild therapeutic hypothermia: it decreased in treated patients and slightly increased in non-treated patients. Cell-free DNA quantity at ICU admission and 24h after admission is associated with hospital mortality. Further studies will need to additionally investigate possible practical use of this new laboratory marker in patients resuscitated from cardiac arrest.

Diabetes is associated with certain environmental exposures, heritable factors, and metabolic conditions of intrauterine development due to diabetes in the mother. We evaluated genomic damage, cell-free DNA, N-glycosylation of umbilical cord plasma proteins (PG), and nuclear division index (NDI) as possible prognostic biomarkers of health risk in the newborns of mothers with treated pregestational diabetes(NBDM ; 22 mothers), compared these parameters with those from newborns of healthy mothers (NBHM ; 89 mothers), and associated the results with the mothers’ lifestyle in both groups, based on a detailed questionnaire. Genomic damage was estimated by the in vitro micronucleus (MN) assay. NDI was detectedon MN slides. Glycans were analyzed by ultra-performance liquid chromatography that separates theplasma N-glycome into 46 glycan peaks. Cell-free DNA was analyzed by real-time PCR. For the association between biomarkers and individual characteristics, generalized linear/nonlinear analysis was performed. No significant difference was found between NBHM and NBDM for cell-free DNA levels. There was no association between cell-free DNA levels and lifestyle. MN frequency was significantly higher in NBDM than in NBHM (median, 0.6 vs. 0.3%, p < 0.001). MN frequency and NDI were significantly associated withresidence (urban vs. rural). PG differed significantly between NBHM and NBDM (p < 0.001). A significant association was found between PG and increase of MN frequency (p < 0.001). As both MN frequency and altered N-glycosylation are associated with cancer risk, our study indicates need for further investigations.

The aim of the study was to assess the frequency of SNP896A/G in the Toll-like receptor (TLR) 4 gene and SNP1350T/C in the TLR2 gene in patients with acute myocardial infarction (AMI) and to analyse the association of these SNPs with risk factors for atherosclerosis and clinical aspects of AMI in a sample of the Croatian population. We included 240 participants in the study: 120 AMI patients and 120 sex- and age-matched healthy blood donor controls. The SNP1350T/C variant in the TLR2 gene showed a lower frequency in the AMI patient group than in the control group (P = 0.033). The frequency of SNP896A/G variants in the TLR4 gene between the patients and the controls did not differ (P = 0.286). Significantly, fewer people had SNP1350T/C in the TLR2 gene (P = 0.003) among the participants with arterial hypertension than those without it. The frequency of SNP896A/G in TLR4 was the same in hypertensive patients compared with normotensive subjects (P = 0.088). SNP1350T/C in TLR2 was less frequent in the AMI patients and in those with hypertension. Thus, SNP1350T/C in TLR2 might play a protective role against AMI and arterial hypertension. The frequency of SNP896A/G in the TLR4 gene was not associated with AMI and arterial hypertension. Other risk factors for atherosclerosis and clinical aspects of myocardial infarction were not associated with the genotype distribution of the examined genes.

The existence of cell free DNA in the human circulatory system has been known since the 1950s, however, intensive research in this area has been conducted for the last ten years. This review paper brings a short overview of the existing literature concerning the cell free DNA research in various cli-nical fields and pathological states and considers the application possibilities of this new analyte in clinical laboratory diagnostics. At the moment, cell free DNA is most widely used for the purpose of non-invasive prenatal diagnosis of fetal sex or fetal RhD status. The recent discovery of epigenetic changes in placental/fetal DNA and the detection of fetal/placental-specific RNAs have made it possible to use this technology in all preg-nancies irrespective of the gender of the fetus. With the application of new techniques such as next generation sequencing, digital PCR and mass spectrometry, it is now possible to detect very small amounts of specific DNA in the presence of excess of other nonspecific nucleic acids. Second most probable application is in oncology, where detection and monitoring of tumors is now possible by the detection of tumor-derived nucleic acids. Third promising field for near future implementation of this analyte is transplantation medicine, where free DNA level could serve as a marker of transplant rejec-tion. Before any further utilization of this new biomarker, pre-analytical and analytical aspects of free DNA analysis remain to be standardized. In the field of noninvasive prenatal diagnosis, important ethical, legal and social questions remain to be discussed.

The aims of the present study were to assess (1) the parental origin of trisomy 21 and the stage in which nondisjunction occurs and (2) the relationship between altered genetic recombination and maternal age as risk factors for trisomy 21. The study included 102 cases with Down syndrome from the Croatian population. Genotyping analyses were performed by polymerase chain reaction using 11 short tandem repeat markers along chromosome 21q. The vast majority of trisomy 21 was of maternal origin (93%), followed by paternal (5%) and mitotic origin (2%). The frequencies of maternal meiotic I (MI) and meiotic II errors were 86% and 14%, respectively. The highest proportion of cases with zero recombination was observed among those with maternal MI derived trisomy 21. A higher proportion of telomeric exchanges were presented in cases with maternal MI errors and cases with young mothers, although these findings were not statistically significant. The present study is the first report examining parental origin and altered genetic recombination as a risk factor for trisomy 21 in a Croatian population. The results support that trisomy 21 has a universal genetic etiology across different human populations.

Cell-free DNA has been investigated as a diagnostic marker in many diseases, including acute conditions. Our hypothesis was that in acute pancreatitis free serum DNA correlates with the extent of pancreatic necrosis and that it may be an early marker of severity.Free DNA was measured in sera from 30 patients with acute pancreatitis at admission, on the first, fourth and seventh day following admission.On the first day following admission patients who would develop severe pancreatitis had significantly higher serum DNA levels than those with mild disease (median 0.271 ng/microL vs. 0.059 ng/microL respectively; P0.001). This parameter showed very good characteristics as a potential severity predictor (area under ROC curve 0.97). Free serum DNA was in correlation with the extent of pancreatic necrosis.Free serum DNA correlates with the extent of pancreatic necrosis and is a potential early marker of severe acute pancreatitis.

Prenatal paternity analysis can be performed only after invasive sampling of chorionic villi or amnionic fluid. Aiming to enable noninvasive paternity testing, we attempted to amplify fetal alleles from maternal plasma. Cell-free DNA was isolated from plasma of 20 pregnant women and amplified with ampFLSTR Identifiler and ampFLSTR Yfiler kits. Unfortunately, autosomal fetal alleles were heavily suppressed by maternal DNA, and the only locus that was reliably amplified with AmpFLSTR Identifiler kit was amelogenin, which revealed only fetal gender. Much better success was obtained with AmpFLSTR Yfiler kit, which, in the case of male fetuses, successfully amplified between six and 16 fetal loci. All amplified fetal alleles matched the alleles of their putative fathers, confirming the tested paternity. To the best of our knowledge, this is a first report of noninvasive prenatal paternity testing.

Aim to determine whether maternal leptin (LEP) and leptin receptor (LEPR) gene polymorphisms are associated with idiopathic recurrent spontaneous abortion (IRSA). Methods: this case-control association study conducted from 2010 to 2012 at the Department of Gynecology and Obstetrics, University Hospital Center Osijek and Clinical Institute of Medical Genetics Ljubljana included 178 women with a history of three or more IRSAs before the 22nd week of gestation and 145 women with at least two live births and no history of pathologic pregnancies during reproductive period. Polymorphisms of maternal LEP (rs7799039, rs2122627, rs11761556, rs10244329) and LEPR (rs1137101, rs7516341, rs1186403, rs12062820) were assessed by allele specific real-time polymerase chain reaction. Genotype distribution, allele frequencies, and frequency of haplotypes at LEP and LEPR genetic loci were determined. Results: we observed more frequent genotype for rs7516341 (nominal P = 0.034, odds ratio [OR] 0.61, 95% confidence interval [CI] 0.38-0.97) and rs1137101 (nominal P = 0.048, OR 1.66, 95% CI 1.00-2.80) in the LEPR gene in patients than in controls, but these results did not remain significant after correction for multiple testing according to Bonferroni (adjusted P value threshold was set at 0.05). We did not observe differential distribution of genotype frequencies in the LEP gene between cases and controls. In patients with IRSA, GTCC haplotype in the LEPR gene locus was significantly less frequent than in controls (P = 0.00865, OR 0.45), contrary to ACTC haplotype (P = 0.0087, OR 1.98). Conclusions: we showed that genetic variability in the LEPR gene was associated with IRSA, warranting confirmation on a greater number of patients and pathogenesis investigation.

Clinical features in patients with segmental aneuploidy often vary depending on the size of the chromosomal region involved. Monosomy 2p is usually observed as a part of more complex syndromes among proband of balanced reciprocal translocation carriers. Partial monosomy 2p (2p25.1→pter) has also been shown to cause a characteristic phenotype associated with severe mental retardation. Trisomy of the long arm of chromosome 4 is well studied pathology. Patients with dup4q syndrome have variable clinical features, which are both related to the size of duplicated segment of the chromosomal arm 4q and specific associated monosomy. Clinical findings were compatible with those previously reported in dup4q and del2p patients. Although partial trisomy 4q and partial monosomy 2p vary in their phenotypes, they also have many features in common such as developmental delay, mental retardation, hypertelorism, low set ears, epicanthic folds and hand anomalies. Herein are presented the clinical and cytogenetic findings in a 4-years-old female with karyotype 46, XX, der(2)t(2 ; 4)(p25.1 ; q31.3)pat. Clinical phenotypes in these translocation cases are variable, because the involved breakpoints vary case-by-case. We compare in this report similarity of the clinical features of our patient and other patients carrying a duplication of the distal part of 4q and patients carrying a deletion of distal part of 2p as described in the literature. To our knowledge, this is the first case of partial trisomy 4q accompanied with partial monosomy for 2p.

Phenotypes in patients with segmental aneuploidy often vary in their clinical manifestation depending on the size of the chromosomal region involved. Recently defined microdeletion syndrome 8p23.1 is characterized by congenital heart disease, developmental delay and a characteristic behavior profile with hyperactivity and impulsiveness. On the other hand, more than 75 cases of trisomy 4p have been reported thus far, most of them due to unbalanced translocations. Trisomy 4p has been shown to cause a characteristic phenotype associated with characteristic facial appearance, postnatal growth retardation, severe psychomotor retardation with or without seizures, microcephaly and various major and minor anomalies. The large variability of the phenotype in trisomy 4p syndrome may be explained by the variation in length of the duplicated segments. Here we present clinical and cytogenetic findings in a 5-month old boy with a duplication of a chromosomal segment 4p16.1→4pter and a deletion of a chromosomal segment 8p23.1→8pter. His karyotype is determined by applying classical GTG banding and FISH probes (WHSCR region, cen 4, cen 8, tel 8p): 46, XY, der(8)t(4 ; 8) (4pter→4p16.1::8p23.1→8qter)dn. Patient’s parents are not related and have normal karyotypes. We have compared similarity of the clinical features of our patient to other patients carrying a duplication of the distal part of 4p and patients carrying a deletion of distal part of 8p as described in the literature. To the best of our knowledge, until today there has been only one report of a combination of dup4p and del8p syndrome. Compared to mentioned case, our patient has less severe manifestations, probably due to a more distal 4p trisomy.

To investigate associations between the postoperative immune response and the levels of extracellular circulating DNA (cDNA), C-reactive protein (CRP), neutrophil/lymphocyte (N/L) ratio, and regulatory T (Treg) cells in the peripheral blood and their role as potential predictors of postoperative septic complications. This was a prospective observational study involving 115 adult patients who underwent elective surgery. Patients were divided into three groups: with benign disease, with malignant disease, and with malignant disease and administration of dexamethasone. Serum CRP levels, N/L ratio, monocyte human leukocyte antigen-DR (HLA-DR) expression, proportion of Treg cells, and cDNA levels were measured at different time points before and after surgery. All patients had increased CRP levels after surgery. Septic patients had higher serum CRP levels at baseline. Compared with the other groups, the dexamethasone group had significantly higher CRP levels before and after surgery, a significantly higher N/L ratio before surgery, a significantly lower rise in the N/L ratio after surgery, and a significantly lower HLA-DR expression at baseline, which remained stable after surgery. In the malignant-disease group, we observed a significant postoperative decrease in the HLA-DR expression. Our results suggest that the immunosuppressive effect of surgery and the presence of a malignant disease may contribute to a higher risk of postoperative sepsis. Preoperative CRP levels may be a reliable predictor of sepsis in oncological patients.

Although, deletion of short arm of chromosome 18 is one of the most frequent autosomal terminal deletions, mosaic form of 18p deletion is infrequently observed. Furthermore, prenatally detected cases of 18p deletion and isochromosome 18q mosaicism are extremely rare. Herein, we present a case of del(18p)/i(18q) mosaicism, prenatally detected after chorionic villus sampling. A 37-year-old woman was referred for prenatal diagnosis because of fetal septated cystic hygroma measuring 4.3 mm. Cytogenetic analysis showed a mosaic 46, XX, del(18)(p11.2)/46, XX, i(18)(q10) karyotype in both, short- and long-term culture. Parents elected to terminate the pregnancy. Fetal mosaic karyotype was confirmed by chromosomal analysis of cultured skin fibroblasts. Molecular characterization of chromosome 18 structural aberrations was performed by fluorescence in situ hybridization (FISH). Considering variable ultrasound findings among cases with del(18p)/i(18q) mosaicism, we emphasized that first and second trimester ultrasound screening examinations for fetal malformations, followed by cytogenetic and molecular evaluations, are very important in the management of prenatally detected cases.

Mosquitoes (Culicidae) are widespread insects and can be important in forensic context as a source of human DNA. In order to establish the quantity of human DNA in mosquitoes’ gut after different post-feeding interval and for how long after taking a bloodmeal the human donor could be identified, 174 blood-engorged mosquitoes (subfamily Anophelinae and Culicinae) were captured, kept alive and sacrificed at 8 hour intervals. Human DNA was amplified using forensic PCR kits (Identifiler, MiniFiler, and Quantifiler). A full DNA profiles were obtained from all Culicinae mosquitoes (74/74) up to 48 h and profiling was successful up to 88 h after a bloodmeal. Duration of post-feeding interval had a significant negative effect on the possibility of obtaining a full profile (p

Acute pancreatitis (AP) is, in most cases, a mild disease, but approximately 20% of patients have severe disease with systemic and local complications and high mortality. Early identification of such patients is critical because timely treatment reduces morbidity and mortality but remains one of the major problems in practice. Cell-free (circulating) DNA in serum and plasma has been investigated as a diagnostic tool in chronic diseases and is already applied in prenatal diagnostics. To confirm the value of this new potential marker and test its practical usage, we quantified circulating DNA in both serum and plasma samples of a larger and unselected population of AP patients. Our results suggest that DNA quantification could be used as an early marker of severity in AP. The growing availability of the methods for DNA quantification could make this new marker for predicting severity of pancreatitis widely used in practice.

Leberova nasljedna optička neuropatija (Leber hereditary opticoneuropathy-LHON) manifestira se bilateralnim akutnim ili subakutnim gubitkom centralnoga vida. LHON je povezan s nekoliko točkastih mutacija mitohondrijalne DNA i nasljeđuje se preko majke. Prikazujemo bolesnika u dobi od 44 godine kod kojeg se LHON manifestirao u 14. godini života. Bolesnikova dva brata, tri sestre i njihova djeca nisu razvili znakove bolesti. U dobi od 42 i 44 godine, klinički oftalmološki pregled potvrdio je postojanje LHON-a i posljedične atrofije vidnoga živca iako mitohondrijalne DNA mutacije LHON 3460 G>A, 11778 G>A i 14484 T>C nisu detektirane., Leber’s hereditary optic neuroretinopathy (LHON) is manifested by bilateral acute or subacute loss of the central vision. LHON is linked to several point mutations of the mitochondrial DNA and is maternally inherited. The patient is a male, 44 years old, who manifested LHON at the age of 14. The patient’s two brothers, three sisters, and their children have no signs of LHON. Ophthalmic examination confirmed LHON at the age of 42 and 44, while LHON 3460 G>A, 11778 G>A and 14484 T>C mitochondrial DNA mutations have not been detected.

Metode koje se primjenjuju u suvremenoj prenatalnoj dijagnostici mogu biti invazivne i neinvazivne. Uobičajene invazivne metode, poput amniocenteze i biopsije korionskih resica, primjenjuju se posljednjih tridesetak godina. Posljednjih dvadesetak godina intenzivno se razvijaju neinvazivne tehnike koje trebaju biti potpuno bezopasne, i za majku, a i za plod, te lako dostupne široj trudničkoj populaciji. U raduje dan pregled najčešće primjenjivanih rutinskih tehnika prenatalne dijagnostike te najnovija dostignuća u tom području, s naglaskom na mogućnosti primjene analize slobodne fetalne DNA iz krvne plazme majke., Current methods used for prenatal diagnosis can be invasive or noninvasive. Common invasive techniques, like amniocentesis or chorionic villus biopsy, have been used for the last thirty years. During the last twenty years, extensive work in development of noninvasive techniques has been done. General goal of noninvasive methods is to be completely safe and available to all pregnant women. In this review, most commonly used techniques of prenatal diagnosis as well as recent developments in the field are being presented, especially possible application of analysis of cell free fetal DNA from maternal plasma.

Almost all plasma proteins are glycosylated and their glycan parts can exist in various structural forms, resulting in different glycoforms of the same molecule. Glycan heterogeneity was shown to be connected to many diseases and glycosylation holds an unbelievable diagnostic potential due to main biological features and structural characteristics of glycans. Recently we observed high biological variability of human plasma N-glycome at population level. Thus, it was of great importance to standardize analytical methods and examine the temporal constancy of N-glycome in human individuals before routine implementation of techniques that determine glycosylation changes in diagnostic laboratories. Plasma samples were taken from 12 healthy individuals. The blood was drawn on seven occasions during five days. N-linked glycans, released from plasma proteins, were separated using hydrophilic interaction high performance liquid chromatography into 16 groups (GP1-GP16) and quantified. The results showed very small variation in all glycan groups, indicating very good temporal stability of Nglycome in individual. Coefficients of variation from 1.6% for GP8 to 11.4% for GP1, with average of 5.6%, were obtained. These variations were comparable to those due to limitations in the experimental methods when analytical procedure was tested for its precision. We conclude that plasma N-glycome in each individual has very good stability which implies that it is under genetic regulation. Changes occurring in glycan profiles are consequence of environmental influences and physiologic responses and therefore carry good potential diagnostic value.

Provedeno je istraživanje na brojlerima Ross 308 kako bi se istražio utjecaj različitih razina sirovih bjelančevina i metaboličke energije na proizvodne rezultate pilića u tovu. Pokus je obuhvatio 600 jednodnevnih tovnih pilića, podijeljenih u tri osnovne skupine s dva ponavljanja. Skupina K hranjena je na razini energije i bjelančevina 100%, P1 na razini 93,5%, a P2 na razini 87%. Odnos energije i bjelančevina u starter smjesama (1-3 tjedna) bio je 1:570, a u finišeru (4-6 tjedana) 1:650. Veći prirasti i tjelesne mase ostvareni su kod skupina pilića hranjenih s višim udjelom sirovih bjelančevina i metaboličke energije u krmnim smjesama. Cijena koštanja 1 kg prirasta neznatno je rasla sa smanjivanjem razine sirovih bjelančevina i metaboličke energije u krmnim smjesama. Biokemijski pokazatelji u serumu pilića pokazali su manju opterećenost organizma kod skupina hranjenih krmnim smjesama s manjom razinom sirovih bjelančevina i metaboličke energije., The research was conducted on broilers Ross 308, in order to investigate the influence of different levels of raw proteins and metabolic energy on production results of fattened chicken. The experiment was conducted on 600 one-day broilers, divided into three main groups, with two repetitions. Group K was fed on energy and protein level 100 %, P1 on level 93.5%, and P2 on level 87%. Proportion between energy and proteins in starter- mixtures (1-3 weeks) was 1:570, and in finisher (4-6 weeks) 1:650. Greater weight gain and body mass were realized in groups of chicken that were fed higher ratio of raw proteins and metabolic energy in fodder mixtures. The price value of 1kg of weight gain insignificantly rose with lowering the level of raw proteins and metabolic energy in fodder mixtures. Biochemical indicators in chicken serum showed lower organism encumbrance in groups fed with fodder mixtures with lower level of raw protein and metabolic energy.