Ksenija Gersak

INVESTIGATING THE ASSOCIATION BETWEEN INHIBIN ALPHA GENE PROMOTER POLYMORPHISMS AND PREMATURE OVARIAN FAILURE

OBJECTIVE To determine whether variants in the promoter region of the inhibin alpha gene (INHA) are associated with premature ovarian failure (POF). DESIGN Mutational analysis of the INHA gene promoter in women with POF. SETTING Academic institution. PATIENT(S) Patients with POF (n = 194) and controls (n = 162) from New Zealand and Slovenia. INTERVENTION(S) Peripheral blood samples were screened for known polymorphisms in the INHA promoter (c.-16C-->T, c.-124A-->G, and an imperfect TG repeat at approximately -300 base pairs). Genotyping was performed by restriction fragment length polymorphism, forced restriction fragment length polymorphism, and nondenaturing high-performance liquid chromatography analysis. MAIN OUTCOME MEASURE(S) Genotypic status of INHA promoter polymorphisms. RESULT(S) Significant differences in INHA promoter allele frequencies were observed between POF patient populations and controls. Significant reductions in allele frequency were observed for the -16T allele (New Zealand POF) and -124G allele (total POF) and for INHA promoter haplotypes C (New Zealand POF) and D (Slovenian POF). CONCLUSION(S) We conclude that INHA promoter variants are associated with the development of POF.

Androgen receptor gene (CAG)n polymorphism in patients with polycystic ovary syndrome

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.

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

Abstract 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.

Association between serum levels and pentanucleotide polymorphism in the sex hormone binding globulin gene and cardiovascular risk factors in females with polycystic ovary syndrome.

The objective of the present study was to evaluate the influence of TAAAA repeat allele length on the levels of serum sex hormone binding globulin (SHBG) and cardiovascular risk factors in patients with polycystic ovary syndrome (PCOS). The study included 91 females with PCOS and 99 healthy controls. Phenotypic hyperandrogenism, body mass index and waist‑to‑hip ratio (WHR) were recorded. Hormonal profiles, fasting insulin and glucose levels, lipid profiles and C‑reactive protein (CRP) levels were measured. Genotyping of TAAAA repeat polymorphisms in the SHBG gene was performed. No significant difference was found in the frequency and distribution of TAAAA repeat alleles between PCOS patients and controls (P=0.739). In PCOS patients, SHBG levels were inversely correlated with serum C‑reactive protein (CRP) levels (R=-0.489, P<0.001). PCOS patients with long TAAAA repeat alleles had significantly lower serum SHBG and free testosterone levels, yet higher CRP levels than patients with short allele repeats. A multiple linear regression model using the number of TAAAA repeats, waist‑to‑hip ratio, a homeostatic model assessment of insulin resistance and age as independent predictors explained 44.8% of the variability in serum SHBG levels. In this model, TAAAA repeat polymorphism was found to be the only reliable predictor of serum SHBG levels (P<0.001). In conclusion, the TAAAA repeat polymorphism was shown to not be a major determinant of the PCOS status, although it influenced serum SHBG levels in females with PCOS. A strong independent association existed between serum SHBG and CRP levels. CRP is an established risk factor of cardiovascular disease and a marker of low‑grade inflammation, typical of atherogenesis. This may be one of the pathways by which low SHBG levels affect cardiovascular risk.

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.

Influence of follicular phase duration on human granulosa-luteal cell subpopulations in natural and stimulated IVF-ET cycles

ObjectivesTo observe the granulosa-luteal cell subpopulations presented within follicular aspirates concerning duration of the follicular phase and the type of IVF protocol.DesignCells 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.Results(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.ConclusionsDuration of the follicular phase influences the immunocytochemical hCG localization and the morphometric characteristics of granulosa-luteal cell sub-populations presented within natural developing follicles and stimulated ones.

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

Abstract 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.

DNA ploidy of human granulosa cells from natural and stimulated in vitro fertilization cycles

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.

No Association Between the Microsatellite Polymorphism (TTTTA) n in the Promoter of the CYP11A Gene and Ovarian Hyperstimulation Syndrome

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.

Subpopulations of Human Granulosa-Luteal Cells Obtained from Gonadotropin- or Gonadotropin-Releasing Hormone Agonist/ Gonadotropin-Treated Follicles in In Vitro Fertilization- Embryo Transfer Cycles

Purpose:Our purpose was to find the differences in granulosa–luteal cells obtained from gonadotropin- versus gonadotropin-releasing hormone (GnRH) agonist/gonadotropin-treated 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–luteal 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 hMG-treated 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–luteal 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.