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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 wa...

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: ...

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. Nasl. z nasl. zaslona. Opis vira z dne 18. 9. 2020. Članek št. 2836. Abstract. Bibliografija: str. 15-18. ARRS ARRS ARRS ARRS

Purpose: To determine whether high-dose intravenous vitamin C reduces oxidative stress in patients with severe preeclampsia in the first days postpartum. Methods: Biomarkers of oxidative stress were assessed as secondary outcomes of a single-center, randomized, placebo-controlled trial. Thirty-four patients with singleton pregnancies complicated by severe features of preeclampsia were randomized into two groups: intravenous vitamin C (1.5 g/6 h) (n = 17) or placebo (n = 17). Urinary concentrations of dityrosine, 8-hydroxy-2-deoxyguanosine (8-OHdg), 8-isoprostane, and N epsilon-(hexanoyl) lysine (HEL) were measured at days one and three after delivery and normalized for urinary creatinine in 22 of patients included (12 in vitamin C and 10 in placebo group). The Mann&ndash, Whitney U-test was used to compare values of oxidative stress biomarkers at days one and three after delivery in vitamin C vs. placebo groups (p &le, 0.05 significant). Results: Dityrosine and 8-OHdg values did not differ significantly between the two study groups at day one after delivery (p = 0.23 and p = 0.77, respectively), but were significantly lower in the vitamin C group compared to the placebo group at day three after delivery (p = 0.04 and p = 0.03, respectively). Values of 8-isoprostane and HEL did not differ significantly between the two study groups at day one (p = 0.41 and p = 0.42, respectively), as well as at day three, after delivery (p = 0.25 and p = 0.24, respectively). Conclusion: High-dose intravenous vitamin C treatments in patients with severe preeclampsia reduced urinary levels of dityrosine and 8-OHdg (markers of protein and DNA oxidative damage, respectively) on day three after delivery. Vitamin C treatment had no significant effect on lipid peroxidation biomarkers, i.e., 8-isoprostane and HEL.

Objective To explore correlations between the sFlt-1/PlGF ratio and uterine arteries (UtA) Doppler indexes in placental dysfunction-related disorders (PDD). Methods We prospectively included women with a singleton pregnancy with preeclampsia (PE) only (n = 22), preeclampsia with fetal growth restriction (FGR) (n = 32), FGR only (n = 12), or normal pregnancy (n = 29). Results In PDDs, significantly positive correlations between the sFlt-1/PlGF ratio and the mean UtA pulsatility (mPI-UtA), as well as the resistance index (mRI-UtA) were found (p = 0.015, p = 0.019, respectively), but not in normal pregnancies. PDD with signs of impaired placentation, evidenced by the increased sFlt-1/PlGF ratio and mPI-UtA, was found in 50.0%, and, by the increased sFlt-1/PlGF ratio and mRI-UtA, in 65.2%. PDD without signs of impaired placentation, evidenced by the increased sFlt-1/PlGF ratio but normal mPI-UtA, was found in 24.2%, and, by the increased sFlt-1/PlGF ratio but normal mRI-UtA, in 7.6%. A substantial proportion of women with signs of impaired placentation were diagnosed with FGR with or without PE. Conclusion In PDD, the sFlt-1/PlGF ratio and UtA Doppler indexes increase proportionally. Correlations between the sFlt-1/PlGF ratio and UtA Doppler indexes might help to distinguish between PDDs with and without impaired placentation. However, further studies are needed to explore the correlations in different phenotypes of PDD.

The objective of this study was to identify new environmental and genetic risk factors for orofacial clefts that arise during early foetal development. In this retrospective, case-control, mother-child pair study, 172 orofacial clefts cases and 199 healthy controls, and their respective mothers, were genotyped for common variants in relevant genes obtained by text and database mining using STRING 10.0. Exposure to environmental risk factors was evaluated using questionnaires. Variant glycine N-methyltransferase (odds ratio (OR) 2.1, 95% confidence interval (95% CI) 1.0-4.4) and dihydrofolate reductase (OR 2.4, 95% CI 1.3-4.5) genotypes were identified as risk factors for cleft lip with or without cleft palate formation. Furthermore, synergy was detected between variant glycine N-methyltransferase and dihydrofolate reductase genotypes in promoting cleft lip with or without cleft palate formation (OR 7, 95% CI 2-23). This study is novel in finding that common glycine N-methyltransferase variant genotypes increase the risk of cleft lip with or without cleft palate.

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

Estrogens and antioxidants indirectly alleviate telomere attrition. However, available clinical data on the association between hormone exposure and telomere length are inconclusive. In the present study, we examined the effects of exogenous estrogen use and of some genetic factors implicated in estrogen metabolism and oxidative stress response on mean leukocyte telomere length. We studied 259 postmenopausal women. Genotyping was conducted for CYP1B1 (rs1056836), COMT (rs4680), GSTP1 (rs1695), MnSOD (rs4880), KRAS (rs61764370), and MTHFR (rs1801133 and rs1801131) polymorphisms. Mean leukocyte telomere length was measured using a quantitative real-time PCR assay. In multivariate analysis we found no association between oral contraceptives or hormone replacement therapy (HRT) and mean leukocyte telomere length. The presence of variant alleles in CYP1B1, KRAS and MTHFR genes was statistically significantly associated with shorter mean leukocyte telomere length. Further, the data provided evidence for the effect modification of the association between HRT and mean leukocyte telomere length by the CYP1B1, KRAS and MTHFR genotypes. Our findings suggest that functionally relevant genetic variants within estrogen and folate metabolic pathways may influence telomere length. We propose these genetic factors should be taken into consideration when interpreting associations between hormone exposure and telomere length.

Estrogens are known to selectively influence cell proliferation. Physiological variations of blood hormone concentration might play a role in regulating the level of X chromosome aneuploidy. In this study we observed the percentages of X aneuploid cells in standard lymphocyte cultures from blood samples obtained in relation to the menstrual cycle, noting whether collection occurred during either the follicular or the luteal phase.A study consisting of 28 women with X mosaicism and recurrent pregnancy loss, and 28 age-matched healthy controls. Cytogenetic studies were carried out on peripheral blood samples according to standard procedures.A significant difference in the percentage of X aneuploidy was found in blood samples obtained during different phases of the menstrual cycle. In the case group, the mean value of aneuploid cells in the follicular and luteal phase samples was 10.0 and 6.3 % respectively and in the control group, it was 2.8 and 1.0 % (P 0.0001). The difference in the case group varied between 0 and 8 % (3.6 ± 2.1 %) and in the control group between 0 and 4 % (1.7 ± 1.1 %). The specificity for detecting true X mosaicism was 0.875. We estimate that the initial diagnosis of X mosaicism could be correct in 68 % of patients with recurrent pregnancy loss.This observational study establishes that the time of blood sampling in relation to the menstrual cycle can influence lymphocyte X chromosome mosaicism. The results, further proven by additional controlled studies, would have practical implications for genetic counselling and fertility treatment.

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.

Hyperandrogenemia has been the most consistent feature of polycystic ovary syndrome (PCOS). Androgens exert their effects through androgen receptors (ARs). The expansion of the codon CAG trinucleotide repeat polymorphism in exon 1 of the AR gene represents a type of genetic alteration associated with changes in the AR gene function. The purpose of this study was to establish a possible association of the AR gene CAG repeat length polymorphism with PCOS, and its influence on clinical and biochemical androgen traits. Two hundred and fourteen Croatian women with PCOS and 209 healthy control women of reproductive age were enrolled. Phenotypic hyperandrogenism, BMI and waist to hip ratio were recorded. Hormonal profiles, fasting insulin and glucose levels were measured on cycle days 3–5. Genotyping of the CAG repeat polymorphism in the AR gene was performed. We found no significant difference in the mean CAG repeat number between the PCOS patients and controls (22.1 ± 3.4 vs. 21.9 ± 3.2, P = 0.286). There was a positive correlation between the CAG repeat length and total testosterone (TT) in the PCOS group (R = 0.225, P = 0.015). A multiple linear regression model using mean CAG repeat length, BMI, age and HOMA-IR as predictors explained 8.5% (adjusted R2) of the variability in serum TT levels. In this model the CAG repeat polymorphism was found to be a significant predictor of serum TT levels in PCOS patients (P = 0.015). The logistic regression analysis revealed that the CAG repeat length is not a significant predictor of hirsutism and acne status (P = 0.921 and P = 0.437, respectively). The model was adjusted for serum TT, free testosterone, androstendione and DHEAS levels as independent variables, which were also not found to be significant predictors of hirsutism (P = 0.687, P = 0.194, P = 0.675 and P = 0.938, respectively) or acne status (P = 0.594, P = 0.095, P = 0.290 and P = 0.151, respectively). In conclusion, the AR CAG repeat polymorphism is not a major determinant of PCOS in the Croatian population, but it is a predictor of serum TT level variability in women with PCOS.

The study aimed to investigate the influence of some generally recognized risk factors for hormone receptor (HR)- and human epidermal growth factor receptor 2 (HER2)-defined breast cancer among Slovenian postmenopausal women.Eligible women diagnosed with breast cancer were compared with 709 controls of the same age and ethnicity. Immunohistochemistry and FISH analyses were used to classify cases into molecular subtypes: 454 HR(+), 106 HR(-), 81 HER2(+) and 603 HER2(-). Adjusted odds ratios and 95% confidence intervals were estimated using multivariate logistic regression analysis.Overweight and obese women were at increased risk of HR(+), HER2(-) and of HR(+), HR(-), HER2(-) tumors, respectively. Women who started menstruating at the age of 11 years or earlier were at decreased risk of ER(-)PR(-) tumors. Users of hormone replacement therapy (HRT) were negatively associated with HR(+) and HER2(-) tumors. The inverse effect was most pronounced with the use of estrogen-only HRT, and longer duration of HRT use did not result in a significant change in risk. In contrast, combined HRT decreased the risk of HER2(+) tumors. Having a first-degree relative with breast and/or ovarian cancer increased the risk of HR(+) and HER2(-) tumors.We conclude that certain breast cancer risk factors may vary by molecular subtypes. According to our results, HRT use may have a greater influence on HR (+) and HER2(-) breast cancers and the risk of HER2-defined breast cancer may differ with respect to the regimen of HRT.

Objectives To present cases exhibiting possible effects of angiotensin II receptor blockers (ARBs) received in pregnancy on the fetus. Methods Retrospective analysis included women who delivered between 2002 and 2006 at Department of Obstetrics and Gynaecology Ljubljana. Results Antihypertensive medications were prescribed to 346 of the 26,735 women. ARBs were given in only five pregnancies: two women received losartan, and three irbesartan. The therapy was stopped between 5 and 23 weeks of gestation. Two women delivered healthy babies at term; another term baby had one additional finger and toe. Other two pregnancies were complicated with oligohydramnios and ended in preterm delivery. One preterm infant had transient abnormal renal function tests. Conclusion Women should be informed that ARB-antihypertensive therapy must be replaced/stopped before planning their pregnancy or at least as soon as the pregnancy is confirmed. Fetal morphology scan and monitoring of amniotic fluid volume should be obligatory, if ARBs are prescribed accidentally.

Literature data concerning thyroid enlargement during pregnancy are not conclusive. Our aim was to systematically follow the thyroid volume changes during pregnancy and after delivery in an iodine-sufficient area.Prospective study of healthy pregnant women living in an iodine-sufficient area. We followed 118 pregnant women with the mean age 30.9+/-4.1 years in the first trimester (mean 11.2+/-2.5 weeks of pregnancy), in the third trimester (mean 31.6+/-1.7 weeks of pregnancy), and 4 months after delivery (mean 15.9+/-3.9 weeks). Additionally, 71 women were also evaluated 14 months after delivery (mean 13.3+/-1.1 months). All women were negative for thyroid autoantibodies. We measured urinary iodine concentration (UIC), thyroid volume, serum TSH, and body mass index (BMI). After delivery, in a subgroup of women we also estimated the colour flow Doppler sonography (CFDS) patterns 0, I, II and III, where thyroid vascularity increased from pattern 0 to III, and the peak systolic velocity (PSV) using a 7.5 mHz linear transducer.Median UIC in the third trimester (176 microg/g creatinine) was significantly higher than 4 and 14 months after delivery (P=0.030, P0.001, respectively). Thyroid volume in the third trimester (11.3+/-3.1 mL) was significantly greater (P0.001) than in the first trimester (8.7+/-2.5 mL), 4 months after delivery (8.6+/-2.5) and 14 months after delivery (7.8+/-2.4 mL). TSH concentration was significantly higher in the third trimester than in the first trimester and 4 months after delivery (P=0.007, P=0.006, respectively). As expected, BMI was the highest in the third trimester. CFDS pattern I was more frequent 4 months after delivery than 14 months after delivery (P0.001). Similarly, PSV was significantly higher 4 months after delivery than 14 months after delivery (P0.001). Linear regression analysis revealed TSH and BMI as significant independent predictors for thyroid volume.In an iodine-sufficient area, thyroid volume increases during pregnancy and decreases after delivery, and the changes in volume are associated with changes in TSH and BMI. They may be viewed as indicators for metabolic and haemodynamic changes during pregnancy.

Pregnancy is a physiological state accompanied by excessive levels of oxidative stress (OS), due to the increased demand and utilisation of oxygen. There is increasing evidence that maternally augmented OS exerts an adverse effect on pregnancy outcome. The aim of the present prospective study was to determine the association between the urinary concentration of relatively novel OS markers measured in the second trimester of pregnancy and the infant characteristics at birth. The maternal levels of urinary hexanoyl-lysine (HEL), propanoyl-lysine (PRL), dityrosine (DiY) and 3-nitrotyrosine (NY) were evaluated in generally healthy pregnant subjects to determine their association with birth weight, gestation at delivery and Apgar score. The observed levels of the markers were in agreement with those measured in healthy non-pregnant subjects in a previous study. A positive correlation was detected between HEL and PRL, as well as between HEL and DiY. Although the absence of a correlation between NY and the other markers has been previously noted in a non-pregnant population, a positive correlation in the pair PRL-NY (r=0.367; P

Purpose :Women with ultrasonic evidence of polycystic ovaries are at higher risk of ovarian hyperstimulation syndrome (OHSS). We focused on investigating a possible association of the (TTTTA) n microsatellite polymorphism in the promoter of the CYP11A gene with OHSS during controlled ovarian hyperstimulation (COH). Methods :We evaluated 58 patients at high risk of OHSS (study group) and 58 control patients undergoing controlled ovarian hyperstimulation. Results :The difference in the allele distribution between both groups of patients was not statistically significant. The genotype distribution of 4+ (with at least one copy of the four-repeat-unit allele) and 4− (without the four-repeat-unit allele) genotypes was identical in the two groups. Conclusion :An association between the (TTTTA) n microsatellite polymorphism in the promoter of the CYP11A gene and the pathogenesis of OHSS could not be confirmed.

Objective: To determine whether hCG and IGFBP-1 appear in the same or different cells and in what sequence. Design: Retrospective analysis of laboratory data. Setting: University medical center. Patient(s): Twenty-five women undergoing IVF-ET with natural cycles and 25 women having stimulated IVF-ET. Intervention(s): Cells were obtained from dominant follicles in women with natural cycles and from the follicles from hMG- and hCG-stimulated cycles. Main Outcome Measure(s): Detection and localization of hCG and IGFBP-1 in granulosa-luteal cells using double immunocytochemical staining. Measurement of hCG and IGFBP-1 in follicular fluid and serum. Result(s): Three types of hCG staining were found: on the cell surface, on the cell surface and in the cytoplasm, and in the cytoplasm alone. IGFBP-1 stained diffusely in the cytoplasm and was found only in those cells that were luteinized and contained hCG. IGFBP-1 and hCG were colocalized in the same cells. There was a positive correlation between follicular fluid hCG and IGFBP-1 levels, but only in natural IVF-ET cycles. Conclusion(s): HCG-driven luteinization is required for IGFBP-1 synthesis to take place in granulosa cells.

Purpose: Our purpose was to find the differences in granulosa—luteal cells obtained from gonadotropin- versus gonadotropin-releasing hormone (GnRH) agonist/gonadotropintreated follicles in in vitro fertilization-embryo transfer (IVF-ET) cycles. Methods: Granulosa-luteal cells were obtained from 45 follicles of women undergoing IVF-ET with gonadotropin releasing hormone (GnRH) agonist and human menopausal gonadotropin (hMG) and from 45 follicles of women with hMG IVF-ET cycles. Subpopulations of granulosa-lut eal cells were observed by computerized image analysis in which human chorionic gonadotropin (hCG) was localized using immunoperoxidase staining. Results: The luteinized granulosa-luteal cells from hMGtreated follicles were larger than those from GnRH agonist/ hMG-treated follicles. The hMG-treated follicles contained more hCG-stained cells, particularly those with cytoplasmic hCG localization. Conclusions: We found differences in morphometric characteristics and hCG localization in granulosa-lut eal cells obtained from hMG- versus GnRH agonist/hMG-treated follicles. We presume that the results indicate the influence and importance of luteal-phase support on the clinical pregnancy rate in GnRH agonist/hMG-treated IVF-ET cycles.

Polycystic ovary syndrome (PCOS) is possibly the most common endocrine disorder in premenopausal women. In this study, we aimed to investigate the association of the -108 C>T polymorphism in the PON1 gene, which encodes the antioxidant enzyme paraoxonase-1, with PCOS. A total of 118 women with PCOS and 108 control subjects were included in this case-control study. The PON1 polymorphism was genotyped, biochemical and clinical parameters were determined and the correlations between the parameters were statistically evaluated. The differences in the PON1 allele and genotype distributions between PCOS patients and controls did not reach a statistical significance. The serum fasting glucose (GLU) levels did not differ significantly between the PCOS patients and the controls. However, the serum fasting insulin (INS) concentration, INS/GLU ratio and homeostasis model assessment (HOMA) index, although within the normal range, were significantly higher in the PCOS group. When considering PCOS patients and controls as separate groups or as a single group of patients, none of the analyzed biochemical or clinical parameters were found to be significantly correlated with the PON1 polymorphism. Therefore, the -108 C>T PON1 polymorphism was not found to be significantly associated with the presence of PCOS or with its particular clinical and biochemical characteristics in non-insulin resistant, non-obese patients.

Insulin resistance is one of the key factors in the pathogenesis of polycystic ovary syndrome (PCOS). The peroxisome proliferator-activated receptor gamma (PPARG) plays a role in the regulation of insulin sensitivity. The aim of the present study was to establish a possible association of the PPARG Pro12Ala polymorphism with PCOS and its effect on family and personal history, as well as on the metabolic and endocrine parameters in PCOS patients. A total of 151 PCOS patients and 179 healthy women of reproductive age were enrolled. History, body mass index (BMI), waist-to-hip ratio and the presence of phenotypic hyperandrogenism were recorded. Hormonal, metabolic and biochemical profiles were assessed. A molecular analysis for the genetic polymorphism was performed. One third (29.8%) of the PCOS patients were found to be carriers of at least one variant of the Ala allele (X/Ala), while 70.2% carried two wild-type Pro alleles (Pro/Pro), with an equal distribution observed in the control group. The PCOS patients carrying the X/Ala alleles exhibited lower serum fasting insulin levels, homeostatic model assessment of insulin resistance (HOMA-IR) and BMI compared to Pro/Pro carriers. This finding was significant only in the lean PCOS group. The polymorphic genotype exerted no effect on history, hormonal and clinical hyperandrogenism, lipid status or C-reactive protein, leptin, adiponectin, resistin and ghrelin serum levels in women with PCOS. In conclusion, although the PPARG Pro12Ala polymorphism is not a major determinant of PCOS in the Croatian population, it may exert a positive effect on insulin sensitivity and BMI. As these associations were recorded exclusively in the lean group of patients with PCOS, this polymorphism potentially contributes to a protective role against hyperinsulinemia and obesity.

Background A single nucleotide polymorphism located in the 3'-untranslated region of the KRAS oncogene (KRAS variant; rs61764370) disrupts a let-7 miRNA binding and was recently reported to act as a genetic marker for increased risk of developing human cancers. We aimed to investigate an association of the KRAS variant with sporadic and familial breast cancer and breast tumor characteristics. Methods Genotyping was accomplished in 530 sporadic postmenopausal breast cancer cases, 165 familial breast cancer cases (including N = 29, who test positive for BRCA1/2 mutations) and 270 postmenopausal control women using the flurogenic 5' nuclease assay. Information on hormone replacement therapy (HRT) use and tumor characteristics in sporadic breast cancer cases was ascertained from a postal questionnaire and pathology reports, respectively. Associations between the KRAS genotype and breast cancer or breast tumor characteristics were assessed using chi-square test and logistic regression models. Results No evidence of association was observed between the KRAS variant and risk of sporadic and familial breast cancer - either among BRCA carriers or non-BRCA carriers. The KRAS variant was statistically significantly more often associated with human epidermal growth factor receptor 2 (HER2) - positive tumors and tumors of higher histopathologic grade. However, both associations were detected only in HRT users. Conclusion Our data do not support the hypothesis that the KRAS variant rs61764370 is implicated in the aetiology of sporadic or of familial breast cancer. In postmenopausal women using HRT, the KRAS variant might lead to HER2 overexpressed and poorly-differentiated breast tumors, both indicators of a worse prognosis.

The aim of the study was to evaluate ovarian response to gonadotrophin stimulation, with and without premedication with gonadotrophin-releasing hormone (GnRH) agonist, in patients with polycystic ovary syndrome. In all, 40 women included in the in-vitro fertilization/embryo transfer programme were divided into two groups. In the first group, buserelin, 500 micrograms/day s.c., was given until pituitary desensitization was achieved. Ovarian stimulation was performed by the combination of GnRH agonist and human menopausal gonadotrophin (HMG). The second group was treated using a conventional HMG and human chorionic gonadotrophin (HCG) protocol. Desensitization was achieved in 15.2 +/- 6.3 days (mean +/- SD) and the luteinizing hormone:follicle stimulating hormone ratio decreased from 2.84 +/- 1.54 to 0.60 +/- 0.35. Comparing the duration of stimulation, the number and size of all observed and aspirated follicles, oocytes recovered and fertilized and the number of embryos replaced, no statistically significant differences were found between the groups. The average oestradiol concentration on the day of HCG administration was lower in the group treated with premedication (P < 0.05). These data suggest that short pre-treatment with GnRH agonist can temporarily correct endocrine abnormalities of polycystic ovary syndrome but do not change the ovarian response to gonadotrophin stimulation and multiple follicular development.

Association between long-term hormone replacement therapy (HRT) use and increased risk of breast cancer is still under debate. Functionally relevant genetic variants within the estrogen metabolic pathway may alter exposure to exogenous sex hormones and affect the risk of postmenopausal breast cancer. We investigated the associations of common polymorphisms in 4 genes encoding key proteins of the estrogen metabolic pathway, duration of HRT use and their interactions with breast cancer risk. We studied 530 breast cancer cases and 270 controls of the same age and ethnicity participating in a case-control study of postmenopausal women. Duration of HRT use was ascertained through a postal questionnaire. Genotyping was conducted for CYP1B1 (rs1056836), COMT (rs4680), GSTP1 (rs1695) and MnSOD (rs4880) polymorphisms by PCR-based RFLP and TaqMan® allelic discrimination method. Adjusted odds ratios and 95% confidence intervals were calculated using logistic regression analysis. HRT use was significantly associated with decreased breast cancer risk (p0.001). None of the polymorphisms studied was associated with breast cancer risk. A significant interaction was observed between MnSOD 47TC and HRT use (pinteraction=0.036); the risk of breast cancer associated with long-term vs. short-term HRT use was decreased in women homozygous for the wild-type allele and increased in women with at least one variant allele of the MnSOD 47TC polymorphism. Our results suggest that MnSOD 47TC polymorphism in interaction with long-term HRT use may modify the risk of breast cancer.

Published data on the association between methylenetetrahydrofolate reductase (MTHFR) gene polymorphisms and breast cancer risk are inconclusive. We investigated the independent and the combined effects of two commonly occurring polymorphisms, MTHFR 677C>T (rs1801133) and MTHFR 1298A>C (rs1801131), as well as their interaction with the use of hormone replacement therapy (HRT), to determine their potential contribution to breast cancer risk. We studied 530 breast cancer cases and 270 controls of the same age and ethnicity participating in a case-control study of postmenopausal women. The duration of HRT use was ascertained through a postal questionnaire. Genotyping was conducted by TaqMan® allelic discrimination. Adjusted odds ratios and 95% confidence intervals were calculated using logistic regression. No significant association was observed between either the individual or the combined MTHFR genotypes and the risk of postmenopausal breast cancer. Additionally, no effects resulting from the interaction between MTHFR genotypes and HRT use were detected. Therefore, our data do not support the hypothesis that genetic variation in the MTHFR gene is implicated in the aetiology of postmenopausal breast cancer.

To establish whether the higher thyroid stimulating hormone (TSH) levels and lower levels of the 2 free thyroid hormones noted toward the end of pregnancy are in relation with iodine supply.We compared these hormones' levels in the third trimester of pregnancy and 4 months after delivery in 116 consecutive women without thyroid disease and otherwise healthy. The study was conducted in Slovenia, an iodine-sufficient area. The Mann-Whitney test, the Kruskal-Wallis rank test, and Spearman analysis were used for statistical analysis.In the third trimester TSH was significantly higher and both free thyroid hormones were significantly lower than after delivery (P=0.003 and P0.001), but the free thyroxine to free triiodothyronine ratios in the third trimester and 4 months after delivery did not significantly differ. Urinary iodine concentration (UIC) was significantly higher during pregnancy than after delivery (P=0.044). We found no significant correlations between UIC and TSH or between UIC and both free thyroid hormones during pregnancy or after delivery.The decrease of both free thyroid hormones in the third trimester of pregnancy is most likely due to reasons that are not related to iodine supply.

Insulin resistance is one of the main characteristics of polycystic ovary syndrome (PCOS) and is probably genetically predisposed. Possible associations of variable nucleotide tandem repeat (VNTR) polymorphism of the insulin gene ( INS) with insulin resistance and PCOS in Slovene patients were investigated. A total of 117 PCOS patients and 108 age-matched female controls were genotyped for the INS VNTR polymorphism using real-time polymerase chain reaction and measurement of appropriate biochemical and clinical parameters. Serum fasting insulin ( I0) levels and the homeostasis model assessment index were significantly elevated in PCOS patients compared with controls. Class III INS VNTR alleles were significantly more frequent in the PCOS group. The interaction between body mass index and INS VNTR genotype was a significant predictor of serum I0 level. The interaction of obesity and the III/III INS VNTR genotype might be a risk factor for the development of PCOS.

BACKGROUND: Hyperandrogenaemia is a common feature of polycystic ovary syndrome (PCOS). The sex hormone-binding globulin (SHBG) gene was proposed as being a PCOS candidate gene. A possible influence of the microsatellite polymorphism (TAAAA)n in the SHBG gene on serum SHBG levels in PCOS patients was investigated. METHODS: One hundred and twenty-three PCOS patients and 110 age-matched controls were included in the study. Peripheral blood samples were obtained. Genotyping of the (TAAAA)n polymorphism in the SHBG gene was performed. Serum LH, FSH, SHBG and total testosterone concentrations were determined. RESULTS: SHBG alleles with 6‐11 TAAAA repeats were found. None of the SHBG alleles or genotypes were present at a significantly more frequent rate in PCOS patients compared with controls. Serum SHBG levels were significantly lower ( P< 0.001) in PCOS patients compared with controls and were found to be strongly influenced by the (TAAAA)n SHBG polymorphism, in both the PCOS (55.3%) and control (33.1%) groups of patients. CONCLUSIONS: The (TAAAA)n SHBG gene polymorphism might be an important predictor for serum SHBG levels and, consequently, for hyperandrogenaemic clinical presentation of PCOS.

Premature ovarian failure (POF) is a common condition affecting 1% of women worldwide. There is strong evidence for genetic involvement in POF as many cases are familial, and mutations in several genes have been associated with POF. We investigated variation in FOXE1 polyalanine tract length, following the observation that polyalanine tract deletions are seen in the closely related FOXL2 in patients with POF. In addition, polyalanine tract expansions in FOXL2 are often seen in patients with blepharophimosis-ptosis-epicanthus inversus syndrome (BPES), a rare eyelid disorder often associated with POF. The FOXE1 polyalanine tract shows marked variation in its length between POF patients and normal controls, existing as an allele of 12, 14, 16, 17 or 19 alanine residues. We found evidence to suggest that variation in FOXE1 polyalanine tract length predisposes to POF.

The aim of the present case–control study was to evaluate the incidence of the (CAG) n AR polymorphism in Slovene polycystic ovary syndrome (PCOS) patients. The polymorphism was not found to be a major risk factor for the presence of PCOS and for hyperandrogenemia in PCOS.

FOXO3A and FOXO1A are excellent candidate genes for the development of premature ovarian failure and have not been analyzed previously in POF patients. Potentially causal mutations in FOXO3A (2/90; 2.2%) and FOXO1A (1/90; 1.1%) were identified in POF patients; however, the pathological role of these mutations will be determined only by screening increased numbers of patients and controls, or by functional studies.

We determined the frequency of galactose-1-phosphate uridyl transferase gene mutations: Q188R, K285N, and the Duarte allelle in 86 patients with idiopathic premature ovarian failure (POF) and 95 controls. No association of the mutations with POF was found.

In a retrospective case-control study, the frequencies of Q188R, K285N, N314D, and IVS5-24G>A mutations were determined with the use of polymerase chain reaction and restriction fragment length polymorphism in the group of infertile women and the controls. No statistically significant differences were observed in the allele frequencies between the infertile women and control groups.

Premature ovarian failure (POF) occurs in 1% of all women, and in 0.1% of women under the age of 30 years. The mechanisms that give rise to POF are largely unknown. Inhibin has a role in regulating the pituitary secretion of FSH, and is therefore a potential candidate gene for ovarian failure. Using single-stranded conformation polymorphism (SSCP) and DNA sequencing, DNA samples were screened from 43 women with POF for mutations in the three inhibin genes. Two variants were found: a 1032C-->T transition in the INHssA gene in one patient, and a 769G-->A transition in the INHalpha gene in three patients. The INHssA variant appears to be a polymorphism, as there was no change in the amino acid sequence of the gene product. The INHalpha variant resulted in a non-conservative amino acid change, with a substitution from alanine to threonine. This alanine is highly conserved across species, and has the potential to affect receptor binding. The INHalpha variant is significantly associated with POF (3/43 patients; 7%) compared with control samples (1/150 normal controls; 0.7%) (Fisher's exact test, P < 0.035). Further analysis of the inhibin gene in POF patients and matched controls will determine its role in the aetiology of POF.

Objective: To analyze and compare the DNA ploidy of granulosa cells from natural and gonadotropin-stimulated follicles obtained during IVF. Design: Retrospective analysis of laboratory data. Setting: University medical center. Patient(s): Seventy-three aspirates of dominant follicles from natural IVF cycles and 113 aspirates from gonadotropin-stimulated cycles were analyzed. Intervention(s): Cytospins were prepared and stained by the Feulgen-thionine method. Main Outcome Measure(s): Image DNA analysis was performed on an automated high-resolution image cytometer. DNA content and the number of nuclei with DNA content >5c were measured. Result(s): All samples from natural and gonadotropin-stimulated follicles were found to be diploid. Single cells with DNA content >5c were found in follicular fluid samples of four women with natural IVF cycles and in samples of nine women with gonadotropin-stimulated cycles. Conclusion(s): DNA ploidy of granulosa cells from natural follicles has not been studied before. In natural samples, granulosa cells were only diploid, without euploid polyploidization. We were unable to confirm DNA aneuploidy of granulosa cells in gonadotropin-stimulated follicles of women undergoing IVF.

Objective: To find the differences between human granulosa-luteal cells obtained during early timed follicular aspiration to prevent severe ovarian hyperstimulation syndrome (OHSS) and during normally timed follicular aspiration. Design: Retrospective analysis of clinical laboratory data. Setting: In vitro fertilization unit, University Department of Obstetrics and Gynecology, Ljubljana, Slovenia. Patient(s): Twenty women undergoing IVF-ET at high risk for OHSS. Intervention(s): Cells were obtained from the follicles of women who were stimulated with hMG and hCG during an early timed follicular aspiration of one ovary, 10–12 hours after hCG, and during a normally timed follicular aspiration of the contralateral ovary, 32–36 hours after hCG administration. Main Outcome Measure(s): Subpopulations of granulosa-luteal cells were observed by computerized image analysis in which hCG was localized using immunoperoxidase staining. Result(s): Early timed follicular aspirates contained no oocytes and only a scant number of granulosa cells. Granulosa-luteal cells were smaller than those from normally timed follicular aspirates. We identified three subpopulations in early timed follicular aspirates: nonluteinized, small luteinized, and medium luteinized cells. In normally timed follicular aspirates, four subpopulations were identified, including large luteinized cells. The normally timed follicular aspirates contained more hCG-stained cells. Three staining types of hCG localization were found: on the surface membrane, on the surface membrane and within the cytoplasm, and only within the cytoplasm of cells from normally timed follicular aspirates. Early timed follicular aspirates contained only cells with membrane hCG localization. Conclusion(s): We found differences in morphometric characteristics and hCG localization between human granulosa-luteal cells obtained during early timed follicular aspiration to prevent severe OHSS and during normally timed follicular aspiration.

Objective To find out the differences between human granulosa-luteal cells derived from natural and stimulated IVF-ET cycles. Design Cells were obtained from dominant follicles of 52 women with natural cycles in whom preovulatory hCG was given when the follicle was mature and from 50 dominant follicles of women undergoing IVF-ET with hMG and hCG. Setting In Vitro Fertilization Unit, University Department of Obstetrics and Gynecology Ljubljana, Slovenia. Main Outcome Measure Four subpopulations of cells were observed by computerized image analysis in which hCG was localized using immunoperoxidase staining. Results The nonluteinized granulosa cells from natural cycles were larger than those from the stimulated ones. In luteinized cell types, there was no difference in cell area between natural and stimulated cycles. Three staining types of hCG localization were found: on the surface membrane, on the surface membrane and within the cytoplasm, and within the cytoplasm alone. The hCG stained cells from natural cycles were larger than the ones from stimulated cycles. The natural developing follicles contained more hCG stained cells than the stimulated ones. The follicles with fertilizable oocytes had more cells with cytoplasmic hCG localization. Only in natural cycles was there was a correlation between follicular fluid hCG levels and the percentage of the hCG stained cells. Conclusion We found differences in morphometric characteristics and hCG localization between human granulosa and granulosa-luteal cells obtained from natural and stimulated IVF-ET cycles.

To observe the granulosa-luteal cell subpopulations presented within follicular aspirates concerning duration of the follicular phase and the type of IVF protocol.Cells were obtained from dominant follicles of 40 women with natural IVF-ET cycles, in which preovulatory hCG was given when the follicle was mature, and from 40 follicles of 32 women with hMG and hCG stimulated IVF-ET cycles. Granulosa-luteal cell subpopulations were observed by computerized image analysis in which hCG was localized using immunoperoxidase staining.(1) The nonluteinized granulosa cells from natural developing follicles were larger than those from stimulated ones regardless of the follicular phase duration. (2) The size of each luteinized cell subpopulations was influenced neither by the two IVF protocols nor by the follicular phase duration. (3) The hCG stained cells from natural developing follicles were larger than the ones from stimulated follicles and their relative number in aspirates was higher. Cell areas and distribution were not influenced by the duration of follicular phase. (4) In stimulated conditions, hCG stained cells became larger if follicular phase was longer.Duration of the follicular phase influences the immunocytochemical hCG localization and the morphometric characteristics of granulosa-luteal cell subpopulations presented within natural developing follicles and stimulated ones.

Objective Estrogen plays a key role in breast cancer development and functionally relevant genetic variants within the estrogen metabolic pathway are prime candidates for a possible association with breast cancer risk. We investigated the independent and the combined effects of commonly occurring polymorphisms in four genes encoding key proteins of estrogen metabolic pathway on their potential contribution to breast cancer risk. Methods We studied 530 breast cancer cases and 270 controls of the same age and ethnicity participating in a case-control study of postmenopausal women. Genotyping was conducted for CYP1B1 (rs1056836), COMT (rs4680), GSTP1 (rs1695), and MnSOD (rs4880) polymorphisms by polymerase chain reaction based restriction fragment length polymorphism and TaqMan allelic discrimination method. Adjusted ORs and 95% CIs were calculated using logistic regression. Results None of the 4 genetic variants examined contributed to breast cancer risk individually. When the combined effects of the risk genotypes were investigated, significant associations were observed among women with two high-risk genotypes in CYP1B1 and COMT (OR, 2.0; 95% CI, 1.1 to 3.5) and two high-risk genotypes in COMT and MnSOD (OR, 2.0; 95% CI, 1.0 to 3.8), compared to those with low-risk genotypes. Conclusion Our results suggest that individual susceptibility to breast cancer incidence may be increased by combined effects of the high-risk genotypes in CYP1B1, COMT, and MnSOD estrogen metabolic genes.