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.

Correction: Vicic et al. Assessment of Vitamin D Status in Slovenian Premenopausal and Postmenopausal Women, Using Total, Free, and Bioavailable 25-Hydroxyvitamin D (25(OH)D). Nutrients 2022, 14, 5349.

In the original publication [...].

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.

Vitamin D Fortification of Eggs Alone and in Combination with Milk in Women Aged 44-65 Years: Fortification Model and Economic Evaluation.

Introduction: For almost nine decades, the fortification of foods with vitamin D has been proven effective in preventing rickets. This study aims to build and economically evaluate a fortification model based on egg biofortification and milk (including yoghurt) fortification. Methods: A cross-sectional study was carried out between 1. March and 31. May 2021. Three hundred and nineteen healthy women from the Central Slovenian region aged between 44 and 65 were recruited for the study, with 176 participants included in the final analysis. For the fortification model calculations, the vitamin D contents of unenriched milk (including yoghurt) and eggs were replaced by enriched foods containing vitamin D. The economic evaluation was done using available drug and food supplement prices. Fortification costs were calculated using vitamin D prices provided by suppliers. Results: Mean vitamin D intake from food was 2.19±1.34 µg/d. With fortification Model 1 (enriched eggs), it would be: 6.49±4.45 µg/d, and with Model 2 (enriched eggs and milk): 10.53±6.49 µg/d. Without fortification, none of the participants would reach a daily vitamin D intake >10 µg. With fortification Model 1 (egg fortification), 15.3% would reach >10 µg and with Model 2 (egg and milk fortification) 46.2% would reach >10 µg. The economic comparison of the annual cost of 10 µg vitamin D/d/person was EUR 6.17 for prescription drugs, EUR 6.37 for food supplements, EUR 0.09 for direct milk fortification and EUR 0.12 for egg biofortification with vitamin D. Conclusions: Egg and milk (including yoghurt) fortification could cost-effectively increase vitamin D intake in the Slovenian population of women between 44 and 65 by almost five-fold, and could significantly lower the prevalence of vitamin D deficiency. Additional research and changes to legislation are needed before this can be introduced.

Effect of In Vitro Maturation of Human Oocytes Obtained After Controlled Ovarian Hormonal Stimulation on the Expression of Development- and Zona Pellucida-Related Genes and Their Interactions.

In an in vitro fertilization program, approximately 10-15% of oocytes obtained after controlled ovarian stimulation are immature, with germinal vesicles (GVs). These oocytes are usually discarded in clinical practice; however, an in vitro maturation (IVM) procedure can be applied to mature them. There are scarce data in the literature on the effect of IVM on the expression of important development- and zona pellucida (ZP)-related genes in human oocytes; therefore, we wanted to determine this. One hundred nine human oocytes were collected from patients enrolled in an intracytoplasmic sperm injection program. The expression of the BMP4, GDF9, ZP1, ZP2, ZP3, and ZP4 genes was analyzed using RT-qPCR in oocytes matured in vitro with different reproductive hormones in the IVM medium (AMH, FSH + hCG, FSH + hCG + AMH), in in vivo matured oocytes and in immature oocytes with GVs. No statistically significant differences in the expression of selected genes in oocytes were observed among groups with different reproductive hormones in IVM medium. However, several interesting significant correlations were found between BMP4 and GDF9, and ZP1 and ZP4; between GDF9 and ZP1, and ZP2 and ZP4; and between ZP1 and ZP3 and ZP4 in the in vitro matured oocytes, while no such correlations were present in other groups of oocytes. The type of reproductive hormone in the maturation medium does not affect the expression of the analyzed genes in oocytes during the maturation process. However, the in vitro maturation procedure itself generated correlations among analyzed genes that were otherwise not present in in vivo matured and immature oocytes.

Assessment of Vitamin D Status in Slovenian Premenopausal and Postmenopausal Women, Using Total, Free, and Bioavailable 25-Hydroxyvitamin D (25(OH)D).

The objective of our study was to evaluate vitamin D status and its predictors in Slovenian premenopausal and postmenopausal women. A cross-sectional study was carried out between 1 March 2021 and 31 May 2021. A total of 319 healthy women from the Central Slovenian region aged between 44 and 65 were recruited; 176 were included in the final analysis. The vitamin D status was determined by measuring the total 25-Hydroxycholecalciferol (25(OH)D) concentration, vitamin D binding protein (DBP), and albumin and calculating the bioavailable 25(OH)D and free 25(OH)D. For the calculation of bioavailable and free 25(OH)D, we developed a new online calculator. The Endocrine Society's thresholds for vitamin D deficiency and insufficiency were used; 29.0% of premenopausal and 24.4% of postmenopausal subjects were found to be vitamin D deficient (total 25(OH)D < 50 nmol/L); 76.8% of the premenopausal and 61.7% of postmenopausal subjects were found to have insufficient levels (total 25(OH)D < 75 nmol/L). Premenopausal women had 11.8% lower total 25(OH)D, 32.2% lower bioavailable 25(OH)D, and 25.2% higher DBP than postmenopausal women. The most important predictors of vitamin D status were vitamin D supplementation and time spent in the sun. Contrary to similar studies, the vitamin D status in Slovenian postmenopausal women was significantly better than in premenopausal women. In postmenopausal women, the measurement of free or bioavailable 25(OH)D instead of the total 25(OH)D could be advantageous.

Characterization and separation of preterm and term spontaneous, induced, and cesarean EHG records.

To improve the understanding of the underlying physiological processes that lead to preterm birth, and different term delivery modes, we quantitatively characterized and assessed the separability of the sets of early (23rd week) and later (31st week) recorded, preterm and term spontaneous, induced, cesarean, and induced-cesarean electrohysterogram (EHG) records using several of the most widely used non-linear features extracted from the EHG signals. Linearly modeled temporal trends of the means of the median frequencies (MFs), and of the means of the peak amplitudes (PAs) of the normalized power spectra of the EHG signals, along pregnancy (from early to later recorded records), derived from a variety of frequency bands, revealed that for the preterm group of records, in comparison to all other term delivery groups, the frequency spectrum of the frequency band B0L (0.08-0.3 Hz) shifts toward higher frequencies, and that the spectrum of the newly identified frequency band B0L' (0.125-0.575 Hz), which approximately matches the Fast Wave Low band, becomes stronger. The most promising features to separate between the later preterm group and all other later term delivery groups appear to be MF (p=1.1⋅10-5) in the band B0L of the horizontal signal S3, and PA (p=2.4⋅10-8) in the band B0L' (S3). Moreover, the PA in the band B0L' (S3) showed the highest power to individually separate between the later preterm group and any other later term delivery group. Furthermore, the results suggest that in preterm pregnancies the resting maternal heart rate decreases between the 23rd and 31st week of gestation.

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.

Association of Zn and Cu Levels in Cord Blood and Maternal Milk with Pregnancy Outcomes among the Slovenian Population.

Trace elements, including zinc (Zn) and copper (Cu), are known to play important roles in human health. The present study aimed to assess the levels of Zn and Cu in cord blood and maternal milk and to analyze their association with maternal and infant characteristics and pregnancy outcomes in a Slovenian study population of mothers and their neonates recruited within the PHIME prospective cohort study. The study included 324 mothers, but the data on Zn and Cu levels in both cord blood and maternal milk was available for 243 mothers. Questionnaires were used to assess the socio-demographic and health status of the mothers, their lifestyle habits (including detailed nutritional habits), and their residential and occupational histories. Inductively Coupled Plasma Mass Spectrometry (ICP-MS) was used to measure Zn and Cu levels in cord blood and maternal milk. Low Zn levels in cord blood were associated with lower gestational age and birth weight and were correlated with an increased probability of the birth of small for gestational age (SGA) infants. Maternal smoking influenced the Cu levels in both cord blood and maternal milk. Cord blood Cu levels were higher and Cu levels in maternal milk were lower in smoking compared to non-smoking mothers. Most importantly, a decreased Zn/Cu ratio in cord blood was associated with lower gestational age and lower birth weight. This indicates the overall positive effects of Zn and negative effects of Cu on pregnancy outcomes.

A Common Polymorphism in the MTHFD1 Gene Is a Modulator of Risk of Congenital Heart Disease.

Several environmental and genetic factors may influence the risk of congenital heart defects (CHDs), which can have a substantial impact on pediatric morbidity and mortality. We investigated the association of polymorphisms in the genes of the folate and methionine pathways with CHDs using different strategies: a case-control, mother-child pair design, and a family-based association study. The polymorphism rs2236225 in the MTHFD1 was confirmed as an important modulator of CHD risk in both, whereas polymorphisms in MTRR, FPGS, and SLC19A1 were identified as risk factors in only one of the models. A strong synergistic effect on the development of CHDs was detected for MTHFD1 polymorphism and a lack of maternal folate supplementation during early pregnancy. A common polymorphism in the MTHFD1 is a genetic risk factor for the development of CHD, especially in the absence of folate supplementation in early pregnancy.

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.

Genetic markers for non-syndromic orofacial clefts in populations of European ancestry: a meta-analysis.

To date, the involvement of various genetic markers in the aetiopathogenesis of non-syndromic orofacial cleft (nsOFC) has been extensively studied. In the present study, we focused on studies performed on populations of European ancestry to systematically review the available literature to define relevant genetic risk factors for nsOFC. Eligible studies were obtained by searching Ovid Medline and Ovid Embase. We gathered the genetic markers from population-based case-control studies on nsOFC, and conducted meta-analysis on the repeatedly reported markers. Whenever possible, we performed stratified analysis based on different nsOFC phenotypes, using allelic, dominant, recessive and overdominant genetic models. Effect sizes were expressed as pooled odds ratios (ORs) with 95% confidence intervals (CIs), and p ≤ 0.05 were considered statistically significant. A total of 84 studies were eligible for this systematic review, with > 700 markers included. Of these, 43 studies were included in the meta-analysis. We analysed 47 genetic variants in 30 genes/loci, which resulted in 226 forest plots. There were statistically significant associations between at least one of the nsOFC phenotypes and 19 genetic variants in 13 genes/loci. These data suggest that IRF6, GRHL3, 8q24, VAX1, TGFA, FOXE1, ABCA4, NOG, GREM1, AXIN2, DVL2, WNT3A and WNT5A have high potential as biomarkers of nsOFC in populations of European descent. Although other meta-analyses that included European samples have been performed on a limited number of genetic variants, this study represents the first meta-analysis of all genetic markers that have been studied in connection with nsOFC in populations of European ancestry.

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.

Elevated soluble-St2 concentrations in preeclampsia correlate with echocardiographic parameters of diastolic dysfunction and return to normal values one year after delivery.

Objectives: To compare soluble-ST2 (sST2) concentrations in patients with severe features of preeclampsia and healthy pregnant controls before as well as 1 year after delivery. Another objective was to assess potential correlation between sST2 concentrations and myocardial function.Methods: Patients with singleton pregnancy complicated by severe features of preeclampsia and healthy controls were included in a prospective observational study. Plasma sST2 concentrations were measured within 24 h before delivery and 1 year after delivery. Standard two-dimensional and Doppler echocardiography was performed at the time of first sST2 measurement before delivery. Mann-Whitney U test was used to compare sST2 values in preeclamptic patients versus controls. Kendall's tau was used to assess correlation between sST2 values and echocardiographic measures of left ventricular systolic and diastolic function (p < .05 significant).Results: We included 24 patients with severe preeclampsia and 29 controls. One year after delivery, sST2 concentrations were available for 24 (45%) participants (13 in preeclampsia group and 11 controls). Concentrations of sST2 were markedly elevated in patients with severe preeclampsia compared to healthy controls before delivery (p = .04), but not 1 year after delivery (p = .80). There was no significant correlation between sST2 and parameters of systolic function. In preeclamptic patients, we found a significant inverse correlation between sST2 and markers of diastolic function: peak early mitral inflow velocity E (Kendall's tau = -0.40; p = .02), peak early diastolic myocardial velocities at septal and lateral mitral annulus (e') (Kendall's tau = -0.354, p = .04) and ratio between e' and peak systolic myocardial velocities at the septal and lateral mitral annulus (e'/s') (Kendall's tau = -0.362, p = .04).Conclusions: Preeclampsia with severe features is associated with increased maternal plasma concentrations of sST2, which return to normal values in the first year after delivery. Higher sST2 levels in preeclamptic patients correlate with impaired parameters of left ventricular diastolic function.

Correlation between cerebral biomarkers and optic nerve sheath diameter in patients with severe preeclampsia.

Objective: To examine the correlation between plasma cerebral biomarkers (S100B and neuron-specific enolase (NSE)) and ultrasonographic optic-nerve-sheath-diameter (ONSD) in preeclampsia. Methods: Thirty preeclampsia patients and 27 controls were included. Mann-Whitney-U test was used for comparison of S100B, NSE, and ONSD in preeclampsia vs. controls. Kendall's tau was used to assess the correlation between biomarkers and ONSD (p < 0.05 significant). Results: ONSD, S100B and NSE were significantly higher in preeclampsia (p < 0.001, p = 0.004, and p < 0.001, respectively). There was significant correlation between NSE levels and ONSD: Kendall's tau = 0.26; p = 0.01. Conclusions: S100B and NSE are elevated in severe preeclampsia. NSE correlates with increased ONSD suggesting cerebral edema.

Folate Insufficiency Due to MTHFR Deficiency Is Bypassed by 5-Methyltetrahydrofolate.

Adequate levels of folates are essential for homeostasis of the organism, prevention of congenital malformations, and the salvage of predisposed disease states. They depend on genetic predisposition, and therefore, a pharmacogenetic approach to individualized supplementation or therapeutic intervention is necessary for an optimal outcome. The role of folates in vital cell processes was investigated by translational pharmacogenetics employing lymphoblastoid cell lines (LCLs). Depriving cells of folates led to reversible S-phase arrest. Since 5,10-methylenetetrahydrofolate reductase (MTHFR) is the key enzyme in the biosynthesis of an active folate form, we evaluated the relevance of polymorphisms in the MTHFR gene on intracellular levels of bioactive metabolite, the 5-methyltetrahydrofolate (5-Me-THF). LCLs (n = 35) were divided into low- and normal-MTHFR activity groups based on their genotype. They were cultured in the presence of folic acid (FA) or 5-Me-THF. Based on the cells' metabolic activity and intracellular 5-Me-THF levels, we conclude supplementation of FA is sufficient to maintain adequate folate level in the normal MTHFR activity group, while low MTHFR activity cells require 5-Me-THF to overcome the metabolic defects caused by polymorphisms in their MTHFR genes. This finding was supported by the determination of intracellular levels of 5-Me-THF in cell lysates by LC-MS/MS. FA supplementation resulted in a 2.5-fold increase in 5-Me-THF in cells with normal MTHFR activity, but there was no increase after FA supplementation in low MTHFR activity cells. However, when LCLs were exposed to 5-Me-THF, a 10-fold increase in intracellular levels of this metabolite was determined. These findings indicate that patients undergoing folate supplementation to counteract anti-folate therapies, or patients with increased folate demand, would benefit from pharmacogenetics-based therapy choices.

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.

Mapping premature ovarian insufficiency and potential environmental factors: A tool for triggering in-depth research of the problem in Slovenia.

Aiming at triggering in-depth research of the problem of Premature Ovarian Insufficiency (POI) in Slovenia, we assessed the regional differences in POI incidence emphasising the relationship with social and physical environmental factors at the population level using a mapping approach. The differences in POI incidence between regions were tested by goodness-of-fit chi-square test, while Pearson correlation coefficient was used to assess the ecological relationship between POI incidence and selected environmental indicators. Significant indicators were mapped. The results showed highly significant interregional differences in POI incidence (p<0.001). Statistically significant ecological relationships were observed between POI incidence and prevalence of active smoking (p=0.001), passive smoking (p=0.017) and consumption of vitamins (p=0.008). The results could be used in diminishing interregional differences in POI. It was concluded that mapping is an effective tool in public health research, especially in triggering new activities.