A Comprehensive Genetic Analysis of Slovenian Families with Multiple Cases of Orofacial Clefts Reveals Novel Variants in the Genes IRF6, GRHL3, and TBX22.

Although the aetiology of non-syndromic orofacial clefts (nsOFCs) is usually multifactorial, syndromic OFCs (syOFCs) are often caused by single mutations in known genes. Some syndromes, e.g., Van der Woude syndrome (VWS1; VWS2) and X-linked cleft palate with or without ankyloglossia (CPX), show only minor clinical signs in addition to OFC and are sometimes difficult to differentiate from nsOFCs. We recruited 34 Slovenian multi-case families with apparent nsOFCs (isolated OFCs or OFCs with minor additional facial signs). First, we examined IRF6, GRHL3, and TBX22 by Sanger or whole exome sequencing to identify VWS and CPX families. Next, we examined 72 additional nsOFC genes in the remaining families. Variant validation and co-segregation analysis were performed for each identified variant using Sanger sequencing, real-time quantitative PCR and microarray-based comparative genomic hybridization. We identified six disease-causing variants (three novel) in IRF6, GRHL3, and TBX22 in 21% of families with apparent nsOFCs, suggesting that our sequencing approach is useful for distinguishing syOFCs from nsOFCs. The novel variants, a frameshift variant in exon 7 of IRF6, a splice-altering variant in GRHL3, and a deletion of the coding exons of TBX22, indicate VWS1, VWS2, and CPX, respectively. We also identified five rare variants in nsOFC genes in families without VWS or CPX, but they could not be conclusively linked to nsOFC.

Recombinant anti-Müllerian hormone in the maturation medium improves the in vitro maturation of human immature (GV) oocytes after controlled ovarian hormonal stimulation.

BACKGROUND: In vitro maturation (IVM) of oocytes is a laboratory method that allows the maturation of immature (GV) oocytes retrieved from patients enrolled in the in vitro fertilization (IVF) programme. However, this method is still sparsely researched and used in clinical practice, leading to suboptimal clinical results. Anti-Müllerian hormone (AMH) is an important hormone with known effects on human ovaries, especially on follicles (follicular cells) during folliculogenesis. In contrast, the effect of AMH on the human oocyte itself is unknown. Therefore, we wanted to determine whether human oocytes express AMH receptor 2 (AMHR2) for this hormone. Recombinant AMH was added to the IVM medium to determine whether it affected oocyte maturation. METHODS: In total, 247 human oocytes (171 immature and 76 mature) were collected from patients enrolled in the intracytoplasmic sperm injection (ICSI) programme who were aged 20 to 43 years and underwent a short antagonist protocol of ovarian stimulation. The expression of AMHR2 protein and AMHR2 gene was analysed in immature and mature oocytes. Additionally, maturation of GV oocytes was performed in vitro in different maturation media with or without added AMH to evaluate the effect of AMH on the oocyte maturation rate. RESULTS: Immunocytochemistry and confocal microscopy revealed that AMHR2 protein is expressed in both immature and mature human oocytes. AMHR2 was expressed in a spotted pattern throughout the whole oocyte. The IVM procedure revealed that AMH in maturation medium improved GV oocyte maturation in vitro, as all oocytes were successfully matured in maturation medium containing recombinant AMH only. Furthermore, antagonism between AMH and follicle-stimulating hormone (FSH) during the maturation process was observed, with fewer oocytes maturing when both AMH and FSH were added to the maturation medium. Finally, AMHR2 gene expression was found in immature and in vitro matured oocytes but absent in mature oocytes. CONCLUSIONS: The positive AMHR2 protein and AMHR2 gene expression in human oocytes shows that AMH could directly act on human oocytes. This was further functionally confirmed by the IVM procedure. These findings suggest the potential clinical application of recombinant AMH to improve IVM of human oocytes in the future.

Comparison of Oxytocin vs. Carbetocin Uterotonic Activity after Caesarean Delivery Assessed by Electrohysterography: A Randomised Trial.

Electrohysterography has been used for monitoring uterine contractility in pregnancy and labour. Effective uterine contractility is crucial for preventing postpartum haemorrhage. The objective of our study was to compare postpartum electrohysterograms in women receiving oxytocin vs. carbetocin for postpartum haemorrhage prevention after caesarean delivery. The trial is registered at ClinicalTrials.gov with the identifier NCT04201665. We included 64 healthy women with uncomplicated singleton pregnancies at term scheduled for caesarean section after one previous caesarean section. After surgery, a 15 min electrohysterogram was obtained after which women were randomised to receive either five IU of oxytocin intravenously or 100 µg of carbetocin intramuscularly. A 30 min electrohysterogram was performed two hours after drug application. Changes in power density spectrum peak frequency of electrohysterogram pseudo-bursts were analysed. A significant reduction in power density spectrum peak frequency in the first two hours was observed after carbetocin but not after oxytocin (median = 0.07 (interquartile range (IQR): 0.87 Hz) compared to median = -0.63 (IQR: 0.20) Hz; p = 0.004). Electrohysterography can be used for objective comparison of uterotonic effects. We found significantly higher power density spectrum peak frequency two hours after oxytocin compared to carbetocin.

Apgar Score and Risk of Cerebral Palsy in Preterm Infants: A Population-Based Cohort Study.

A low Apgar score is associated with increased risk of cerebral palsy (CP) in term infants, while such association remains controversial in preterm neonates. The objective of this study was to assess association between 5-minute Apgar scores and CP in different subcategories of preterm birth based on gestational age. The Slovenian National Perinatal Information System was used to identify singleton children without congenital malformations live-born at 22 to 37 weeks of gestation between 2002 and 2010. Data were linked to the Slovenian Registry of Cerebral Palsy in children born between 2002 and 2010. CP was diagnosed at a minimum of 5 years of age. Of 11,924 children included, 241 (2.0%) died before discharge and 153 (1.3%) were diagnosed with CP. Five-minute Apgar scores <7 were significantly associated with higher risk of death or CP (compared with scores ≥9) at all preterm gestations. CP alone was associated with Apgar scores <7 only at moderately or late preterm gestation (32-36 weeks) (adjusted relative risk [aRR]: 8.27; 95% confidence interval [CI]: 1.87-36.64 for scores 0-4 and aRR: 4.96; 95% CI 1.89-13.06 for scores 5-6). In conclusion, a low 5-minute Apgar score was associated with combined outcome of neonatal death or CP in all preterm births, while in surviving preterm infants at >32 weeks a low 5-minute Apgar score was associated with CP.

Simultaneous quantification of intracellular concentrations of clinically important metabolites of folate-homocysteine cycle by LC-MS/MS.

Inadequate folate status is detrimental to human development. Deficiency has been implicated in congenital birth defects and cancer, whereas excess has been linked to various negative neurocognitive development outcomes. We developed a method for translational studies involving lymphoblastoid cell models for studying role of folates in vital cell processes. We describe a simple, sensitive, and fast liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the simultaneous quantification of intracellular concentrations of clinically important metabolites of folate-homocysteine cycle; namely, folic acid (FA), 5-methyltetrahydrofolate (5-Me-THF), and homocysteine (Hcy). The method was validated for specificity, linearity, limits of quantification, repeatability, reproducibility, matrix effects, and stability. Method had a wide linear range between 0.341 and 71.053 ng Hcy/mg protein for Hcy, 0.004-0.526 ng FA/mg protein for FA and 0.003-0.526 ng 5-Me-THF/mg protein for 5-Me-THF. The method overcomes challenges associated with the quantification of endogenous molecules, poor stability, and extremely small amounts of the analytes. The method was successfully applied to evaluate the effects of FA and 5-Me-THF treatment of cells in vitro mimicking supplement therapy with various metabolically active species, and showed that 5-Me-THF is more effective than FA in increasing intracellular levels of the biologically active form of folate.

Assessing Velocity and Directionality of Uterine Electrical Activity for Preterm Birth Prediction Using EHG Surface Records.

The aim of the present study was to assess the capability of conduction velocity amplitudes and directions of propagation of electrohysterogram (EHG) waves to better distinguish between preterm and term EHG surface records. Using short-time cross-correlation between pairs of bipolar EHG signals (upper and lower, left and right), the conduction velocities and their directions were estimated using preterm and term EHG records of the publicly available Term-Preterm EHG DataSet with Tocogram (TPEHGT DS) and for different frequency bands below and above 1.0 Hz, where contractions and the influence of the maternal heart rate on the uterus, respectively, are expected. No significant or preferred continuous direction of propagation was found in any of the non-contraction (dummy) or contraction intervals; however, on average, a significantly lower percentage of velocity vectors was found in the vertical direction, and significantly higher in the horizontal direction, for preterm dummy intervals above 1.0 Hz. The newly defined features-the percentages of velocities in the vertical and horizontal directions, in combination with the sample entropy of the EHG signal recorded in the vertical direction, obtained from dummy intervals above 1.0 Hz-showed the highest classification accuracy of 86.8% (AUC=90.3%) in distinguishing between preterm and term EHG records of the TPEHGT DS.

Total gestational weight gain and the risk of preeclampsia by pre-pregnancy body mass index categories: a population-based cohort study from 2013 to 2017.

Background Identifying the risk factors for preeclampsia (PE) is essential for the implementation of preventive actions. In the present study, we aimed at exploring the association between total gestational weight gain (GWG) and PE. Methods We performed a population-based cohort survey of 98,820 women with singleton pregnancies who delivered in Slovenia from 2013 to 2017. Aggregated data were obtained from the National Perinatal Information System (NPIS). The main outcome measure was the incidence of PE. The main exposure variable was total GWG standardized for the gestational duration by calculating the z-scores. The associations between total GWG and PE stratified by pre-pregnancy body mass index (BMI) categories adjusted for a variety of covariates were determined using multivariable logistic regression. We calculated the crude odds ratio (OR) and adjusted odds ratio (aOR) with a 95% confidence interval using a two-way test. Results Excessive GWG was associated with increased odds of PE in all pre-pregnancy BMI categories. The increase in the odds of PE by 445% was the highest in underweight women and by 122% was the lowest in obese women. Low GWG was associated with decreased odds of PE in all pre-pregnancy BMI categories except in normal-weight women with a GWG below -2 standard deviation (SD) and underweight women. The decrease in the odds of PE by 67% was the highest in obese women and by 41% was the lowest in normal-weight women. Conclusion Excessive GWG is a significant risk factor for PE, especially in underweight women, while low GWG is an important protective factor against PE, especially in obese women.

Infant mortality and causes of death by birth weight for gestational age in non-malformed singleton infants: a 2002-2012 population-based study.

OBJECTIVE: To explore the associations between birth weight for gestational age (GA) and infant mortality as well as causes of infant death. STUDY DESIGN: A population-based observational study conducted between 2002 and 2012 included 203,620 non-malformed singleton live births from Slovenia. Poisson regression analyses were performed to estimate the crude relative risk (RR) and adjusted RR (aRR) for infant mortality by birth weight percentiles stratified by the GA subgroups term, moderate-to-late preterm, very preterm and extremely preterm. RESULTS: Compared with appropriate for GA (AGA) term infants (referent-AGA), infant mortality was significantly higher in small for GA (SGA) term infants [aRR=2.79 (1.41-5.50)], with significant cause-specific infant mortality risk for neuromuscular disorders [RR=10.48 (2.62-41.91)]. The differences in infant mortality and cause-specific infant mortality in preterm subgroups between referent-AGA and SGA were insignificant. CONCLUSIONS: In the Slovenian population, birth weight for GA is significantly associated with infant mortality only in infants born at term.

Effects of vaginal progesterone for maintenance tocolysis on uterine electrical activity.

AIM: The effectiveness of vaginal progesterone for maintenance tocolysis after arrested preterm labor remains controversial. Myometrial contractility can be assessed objectively and non-invasively after progesterone treatment by monitoring uterine electromyography (EMG). We examined the effects of vaginal progesterone on uterine EMG after successful acute tocolysis. METHODS: This was a randomized, double-blind, single-center study performed between 2012 and 2015. Thirty women who experienced preterm labor between 24 0/7 and 33 6/7 weeks were randomly allocated to groups administered either 400 mg vaginal progesterone or a placebo 48 h after acute tocolysis. EMG measurements were taken prior to and 1 h and 2 h following treatment. Mann-Whitney U tests were used to compare EMG power density spectrum peak frequency and peak amplitude, propagation velocity of EMG signals, and duration and number of EMG bursts in 30 min recordings between the groups (P < 0.05). RESULTS: EMG propagation velocity was higher in patients receiving the placebo compared to those treated with progesterone at 1 h (27.83 ± 10.66 vs 15.60 ± 2.94 cm/s) and 2 h (26.97 ± 13.39 vs 15.12 ± 2.58 cm/s) following treatment (P = 0.001). PDS peak frequencies were higher in the placebo compared to the progesterone group at 2 h following treatment (0.54 ± 0.11 vs 0.44 ± 0.06 Hz; P = 0.003). CONCLUSIONS: Treatment of 400 mg of vaginal micronized progesterone as maintenance tocolysis significantly reduces the propagation velocity of electrical signals within the myometrium and is associated with a shift toward lower uterine electrical signal frequencies.

Correlation between markers of DNA and lipid oxidative damage in maternal and fetoplacental compartment in the mid-trimester of pregnancy.

OBJECTIVE: To determine the levels of 8-isoprostane (8-IP) and 8-hydroxy-2'-deoxyguanosine (8-OHdG) in urine and in amniotic fluid (AF) of pregnant women and to assess the correlation between oxidative status in the maternal and fetal compartment in the second trimester of pregnancy. METHODS: One hundred and forty-six women with singleton pregnancies, undergoing amniocentesis at the Department of Obstetrics and Gynaecology at the University Medical Centre Ljubljana, were prospectively enrolled. AF and maternal urine were collected in the second trimester of pregnancy. Paired urinary and AF 8-IP and 8-OHdG were measured and evaluated cross-sectionally. RESULTS: 8-IP and 8-OHdG concentrations were higher in maternal urine compared to AF and the ratios were 47:1 and 50:1, respectively. AF 8-OHdG was very low and in 74% was below the limit of detection (LOD). We found a positive correlation between 8-IP in maternal and fetal compartment (ρ=0.217, P=0.008), which stayed unchanged also after adjustment for possible confounding factors. CONCLUSIONS: Oxidative damage to lipids and DNA is also a part of physiologic processes during healthy pregnancy. 8-IP and 8-OHdG are constantly present in urine and AF. A weak positive correlation between maternal and fetal unit suggests a weak reflection of fetal oxidative status in maternal urine in the mid-trimester.

Mutations in LARS2, encoding mitochondrial leucyl-tRNA synthetase, lead to premature ovarian failure and hearing loss in Perrault syndrome.

The genetic causes of premature ovarian failure (POF) are highly heterogeneous, and causative mutations have been identified in more than ten genes so far. In two families affected by POF accompanied by hearing loss (together, these symptoms compose Perrault syndrome), exome sequencing revealed mutations in LARS2, encoding mitochondrial leucyl-tRNA synthetase: homozygous c.1565C>A (p.Thr522Asn) in a consanguineous Palestinian family and compound heterozygous c.1077delT and c.1886C>T (p.Thr629Met) in a nonconsanguineous Slovenian family. LARS2 c.1077delT leads to a frameshift at codon 360 of the 901 residue protein. LARS2 p.Thr522Asn occurs in the LARS2 catalytic domain at a site conserved from bacteria through mammals. LARS2 p.Thr629Met occurs in the LARS2 leucine-specific domain, which is adjacent to a catalytic loop critical in all species but for which primary sequence is not well conserved. A recently developed method of detecting remote homologies revealed threonine at this site in consensus sequences derived from multiple-species alignments seeded by human and E. coli residues at this region. Yeast complementation indicated that LARS2 c.1077delT is nonfunctional and that LARS2 p.Thr522Asn is partially functional. LARS2 p.Thr629Met was functional in this assay but might be insufficient as a heterozygote with the fully nonfunctional LARS2 c.1077delT allele. A known C. elegans strain with the protein-truncating alteration LARS-2 p.Trp247Ter was confirmed to be sterile. After HARS2, LARS2 is the second gene encoding mitochondrial tRNA synthetase to be found to harbor mutations leading to Perrault syndrome, further supporting a critical role for mitochondria in the maintenance of ovarian function and hearing.

Uterine electromyography during active phase compared with latent phase of labor at term.

INTRODUCTION: In a prospective study in a tertiary university hospital we wanted to determine whether uterine electromyography (EMG) can differentiate between the active and latent phase of labor. MATERIAL AND METHODS: Thirty women presenting at ≥37(0/7) weeks of gestation with regular uterine contractions, intact membranes, and a Bishop score <6. EMG was recorded from the abdominal surface for 30 min. Latent phase was defined as no cervical change within at least 4 h. Student's t-test was used for statistical analysis (p ≤ 0.05 significant). Diagnostic accuracy of EMG was determined by receiver operator characteristics (ROC) analysis. The integral of the amplitudes of the power density spectrum (PDS) corresponding to the PDS energy within the "bursts" of uterine EMG activity was compared between the active and latent labor groups. RESULTS: Seventeen (57%) women were found to be in the active phase of labor and 13 (43%) were in the latent phase. The EMG PDS integral was significantly higher (p = 0.02) in the active (mean 3.40 ± 0.82 µV) compared with the latent (mean 1.17 ± 0.33 µV) phase of labor. The PDS integral had an area under the ROC curve (AUC) of 0.80 to distinguish between active and latent phases of labor, compared with number of contractions on tocodynamometry (AUC = 0.79), and Bishop score (AUC = 0.78). The combination (sum) of PDS integral, tocodynamometry, and Bishop score predicted active phase of labor with an AUC of 0.90. CONCLUSIONS: Adding uterine EMG measurements to the methods currently used in the clinics could improve the accuracy of diagnosing active labor.

Changes in incidence of iatrogenic and spontaneous preterm births over time: a population-based study.

OBJECTIVE: To examine the proportion of iatrogenic births among all preterm births over a 26-year period. PATIENTS AND METHODS: A registry-based survey of preterm deliveries between 1987 and 2012 analyzed by the onset of labor: spontaneous with intact membranes, preterm premature rupture of membranes (PPROM) or iatrogenic. Stratification into categories by gestation (22 weeks to 27 weeks and 6 days, 28 weeks to 31 weeks and 6 days, 32 weeks to 33 weeks and 6 days, 34 weeks to 36 weeks and 6 days) was performed. Preterm birth rates were analyzed using the Mantel-Haenszel linear-by-linear association χ2-test (P<0.05 significant). Logistic regression was used to account for potential confounders. RESULTS: Overall preterm birth rate was 5.9% (31328 deliveries) including 2358 (0.4%) before 28 completed weeks, 3388 (0.6%) between 28 weeks and 31 weeks 6 days, 3970 (0.8%) between 32 weeks and 33 weeks and 6 days, and 21611 (4.1%) between 34 weeks and 36 weeks and 6 days There was an increase in overall preterm birth rate (P<0.001). The rate of iatrogenic preterm births and PPROM increased over time (P<0.001 and P<0.014, respectively). Rates of spontaneous preterm birth decreased (P<0.001). After accounting for potential confounders, year of birth remained an independent risk factor for iatrogenic preterm delivery in all four gestational age categories (P<0.001). CONCLUSION: The incidence of iatrogenic preterm birth is increasing with a concomitant decrease in the incidence of spontaneous preterm birth. Attempts to analyze, interpret and decrease preterm birth rates should consider spontaneous and iatrogenic preterm births separately.

Determination of HEL (Hexanoyl-lysine adduct): a novel biomarker for omega-6 PUFA oxidation.

Published evidences indicate that reactive oxygen species (ROS) can induce lipid peroxidation, which plays important role in the pathophysiology of numerous diseases including atherosclerosis, diabetes, cancer and aging process. Monitoring of oxidative modification or oxidative damages of biomolecules may therefore be essential for the understanding of aging, and age-related diseases. N-epsilon-Hexanoyl-lysine (HEL) is a novel lipid peroxidation biomarker which is derived from the oxidation of omega-6 unsaturated fatty acid. In this chapter, development of HEL ELISA and its applications are reported. Assay range of HEL ELISA was 2-700 nmol/L, and showed good linearity and reproducibility. Accuracy of this assay was validated by recovery test and absorption test. HEL concentration in human urine was 22.9 ± 15.4 nmol/L and it was suggested that HEL exists as low molecular substances, in a free or in the peptide-attached form. In contrast with the urine sample, serum HEL was suggested to exist in the protein-attached form, and hydrolysis by protease might be essential for the accurate measurement of HEL in protein containing samples such as serum and cultured cells. By sample pretreatment with proteases, HEL was successfully detected in oxidized LDL, oxidized serum, and rat serum. In conclusion, HEL ELISA can be applied to measure urine, serum, and other biological samples independent of the animal species, and may be useful for the assessment of omega-6 PUFA oxidation in the living bodies.

SEMA3A, a gene involved in axonal pathfinding, is mutated in patients with Kallmann syndrome.

Kallmann syndrome (KS) associates congenital hypogonadism due to gonadotropin-releasing hormone (GnRH) deficiency and anosmia. The genetics of KS involves various modes of transmission, including oligogenic inheritance. Here, we report that Nrp1(sema/sema) mutant mice that lack a functional semaphorin-binding domain in neuropilin-1, an obligatory coreceptor of semaphorin-3A, have a KS-like phenotype. Pathohistological analysis of these mice indeed showed abnormal development of the peripheral olfactory system and defective embryonic migration of the neuroendocrine GnRH cells to the basal forebrain, which results in increased mortality of newborn mice and reduced fertility in adults. We thus screened 386 KS patients for the presence of mutations in SEMA3A (by Sanger sequencing of all 17 coding exons and flanking splice sites) and identified nonsynonymous mutations in 24 patients, specifically, a frameshifting small deletion (D538fsX31) and seven different missense mutations (R66W, N153S, I400V, V435I, T688A, R730Q, R733H). All the mutations were found in heterozygous state. Seven mutations resulted in impaired secretion of semaphorin-3A by transfected COS-7 cells (D538fsX31, R66W, V435I) or reduced signaling activity of the secreted protein in the GN11 cell line derived from embryonic GnRH cells (N153S, I400V, T688A, R733H), which strongly suggests that these mutations have a pathogenic effect. Notably, mutations in other KS genes had already been identified, in heterozygous state, in five of these patients. Our findings indicate that semaphorin-3A signaling insufficiency contributes to the pathogenesis of KS and further substantiate the oligogenic pattern of inheritance in this developmental disorder.

Exposure to endocrine disrupting chemicals (bisphenols, parabens, and triclosan) and their associations with preterm birth in humans.

Preterm birth in humans (PTB), defined as birth prior to 37 weeks of gestation, is one of the most important causes of neonatal morbidity and mortality and is associated with adverse health outcomes later in life. Attributed to many different etiological factors, estimated 15.1 million or 11.1% of births each year are preterm, which is more than 1 per 10 livebirths globally. Environmental pollution is a well-established risk factor that could influence the pathogenesis of PTB. Increasing evidence has shown an association between maternal exposure to endocrine disrupting chemicals (EDCs) and PTB. This scoping review aims to summarize current research on the association between EDC exposure and PTB in humans. Database PubMed was used to identify articles discussing the effect of selected EDCs, namely bisphenol A, bisphenol S, bisphenol F, parabens, and triclosan, found in plastics, cosmetics and other personal care products, on PTB occurrence. Regardless of some inconsistences in the findings across studies, the reviewed studies suggest a potential association between involuntary exposure to reviewed EDCs and the risk of PTB. However, further studies are needed to delineate exact correlations and mechanisms through which EDC exposure causes PTB so that efficient preventative measures could be implemented. Until then, health care providers should inform women about possible EDC exposure thus empowering them to make healthy choices and at the same time decrease the EDC negative effects.

Circadian characteristics of term and preterm labors.

The labor is a physiological event considered to have its own circadian (diurnal) rhythm, but some of the data remain conflicting, especially for preterm births. In this retrospective study, we analyzed the circadian trends of labor onset times in the Slovenian birth cohort from 1990 to 2018 with over 550,000 cases of singleton births. The number of term and preterm labor onsets was calculated for each hour in a day and circadian trends were evaluated for each of the study groups by modeling with a generalized Poisson distribution linked with the cosinor regression model using logarithmic link function. The induced labors were taken as the control group since the timing of labor depends mostly on the working schedule of personnel and not on the intrinsic rhythmic characteristics. For induced labors, the main peak in the number of labor cases was observed in the late morning hours (around 10 AM) for all gestational ages. The prominence of this peak becomes smaller in spontaneous premature labors with gradually disrupting rhythmicity in very preterm and extremely preterm cases. Labors starting with spontaneous contractions peak between 6 and 7 AM and lose the rhythmicity at 35 weeks of gestation while labors starting with a spontaneous rupture of membranes peak at 1 AM and lose the rhythmicity at 31 weeks of gestation, suggesting differences in underlying mechanisms. According to our knowledge, this is the first study that shows differences of circadian trends between different types of spontaneous labors, i.e., labors initiated with contraction and labors initiated with a spontaneous rupture of membranes. Moreover, the obtained results represent evidence of gradual disruption of rhythmicity from mild to extreme prematurity.

Can prenatal detection of Down syndrome be improved by enhancing obstetricians' skills of performing adequate foetal cardiac examination at the primary level?

AIMS: To evaluate the prevalence of congenital heart defects (CHDs) in live-born infants with Down syndrome (DS) and to investigate whether these CHDs might be detected during routine second trimester ultrasound screening performed at the primary level. METHODS: A retrospective analysis of 66 cases of DS in live-born infants. The infants with DS underwent a detailed echocardiographic examination to evaluate cardiac morphological characteristics and function. RESULTS: Thirty-six live-born DS infants (54.5%) had associated CHDs. According to the apical four-chamber view at the first postnatal echocardiographic examination, we estimated that 20 (55.6%) of the 36 patients with associated CHDs should have been identified during the routine second-trimester prenatal scan [17 infants with complete atrioventricular septal defect (AVSD), two with partial AVSD, and one with non-restrictive perimembranous ventricular septal defect] if the results had been correctly interpreted. An additional seven patients with associated CHDs should have been identified if the evaluation of both outflow tracts had been included into the screening protocol. CONCLUSION: Our data suggest that the prenatal DS detection rate can be significantly increased by improving obstetricians' skills of performing adequate foetal cardiac examination as part of the routine 18- to 23-week ultrasound examination at the primary level.

Serum levels of sex hormones in males with Reinke's edema.

Smoking, laryngopharyngeal reflux and voice misuse/overuse are known possible etiological factors for the development of Reinke's edema (RE) on vocal folds. RE is found more frequently in women. The disparity between the incidence of RE in women and men suggests that endogenous sex hormones such as estrogens, progesterone and/or testosterone may have a significant influence on vocal folds. The aim of the study was to investigate the level of sex hormones such as estradiol (E), progesterone (P), and testosterone (T) in men with RE in comparison with men without laryngeal pathology. Fifty-six men with RE and 48 men without laryngeal pathology participated in the study. All participants received a questionnaire for assessing possible risk factors for the development of RE. The serum levels of T, E and P were determined and the ratios between hormones (T/E, T/P, P/E) were calculated. T and P serum levels were significantly higher in patients with RE (p = 0.002, p = 0.017). No differences were found in the hormone ratio values. Smoking was the only known risk factor for RE (p < 0.001). In conclusion, the difference in the level of sex hormones implies that hormones may affect both, the development and the maintenance of the edema in the lamina propria of vocal folds. The authors suppose that the possible mode of action of sex hormones is through enzymatic activity of nitric oxide synthase in the endothelial cell wall.

Genetic polymorphisms of INS, INSR and IRS-1 genes are not associated with polycystic ovary syndrome in Croatian women.

Obesity and insulin resistance is a common finding in patients with polycystic ovary syndrome (PCOS). Significant number of PCOS women experience insulin resistance that is irrespective of the degree of obesity suggesting possible genetic basis. Therefore, several polymorphisms of the genes encoding for the insulin (INS), insulin receptor (INSR) or insulin receptor substrates (IRS) involved in postreceptor signaling have been explored for their association with abnormal sensitivity to insulin in PCOS. The aim of the present study was to determine whether selected polymorphisms of INS, INSR and IRS-1 are associated with the development of PCOS as well as with increased insulin resistance in Croatian women with PCOS. The study enrolled 150 women with PCOS and 175 control women. The diagnosis of PCOS was based on Rotterdam consensus criteria. Each subject underwent an evaluation of body mass index (BMI), hirsutism, acne and menstrual cycle abnormalities as well as follicular stimulating hormone (FSH), luteinizing hormone (LH), total and free testosterone, androstendione, dehydroepiandrosterone sulphate (DHEAS), sex hormone binding globulin (SHBG), fasting glucose and fasting insulin. Insulin resistance (IR) was quantified using the homeostatic model assessment of IR (HOMA-IR). Molecular analyses for the genetic polymorphisms were preformed. There was a significant difference in clinical and biochemical characteristics of the studied groups except for BMI and fasting glucose levels. No significant differences were observed in the genotype and allele distribution of the VNTR INS, C/T INSR, Gly792Arg IRS-1 polymorphisms between cases and controls. Moreover, no association was found between VNTR INS, C/T INSR and Gly792Arg IRS-1 polymorphism and parameters of insulin resistance in PCOS patients. In conclusion, our data does not support an association between VNTR INS, C/T INSR and Gly792Arg IRS-1 polymorphism and susceptibility to PCOS or insulin resistance in Croatian women with PCOS.