[Extracellular chromosome 21--derived microRNAs in maternal circulation: evaluation of their diagnostic potential for screening of Down syndrome]. / Chromozom 21 - specifické mikroRNA v materské cirkulaci: zhodnocení jejich významu pro screening Downova syndromu u plodu.

OBJECTIVE: Initially, we focused on the detection of extracellular microRNAs in maternal circulation, whose genes are located on human chromosome 21 (miR-99a, let-7c, miR-125b-2, miR-155 and miR-802). Subsequently, we studied if plasmatic concentrations and/or expression profile of extracellular chromosome 21-derived microRNAs would distinguish between pregnancies bearing euploid foetuses and those affected with Down syndrome. DESIGN: Pilot study. SETTING: Division of Molecular Biology and Cell Pathology, Department of Gynaecology and Obstetrics, Third Faculty of Medicine, Charles University, Prague. METHODS: 12 women with normal course of gestation (mean 16.4 weeks, median 16.0 weeks), 12 pregnancies bearing Down syndrome foetus (mean 18.2 weeks, median 18.5 weeks) and 6 non-pregnant individuals were involved in the retrospective study. RNA enriched for small RNAs (including microRNAs) was isolated from 1ml of plasma sample. Consequently relevant microRNA was transcribed into cDNA using specific stem-loop primer and detected by specific real-time PCR assay. RESULTS: Commercial systems enabled reliable detection of 4 out of 5 extracellular chromosome 21-derived microRNAs (miR-99a, let-7c, miR-125b-2 and miR-155). Expression profile of extracellular miR-99a, miR-125b-2 and miR-155 was significantly higher in the cohort of pregnant women than in non-pregnant individuals. Also plasmatic levels of miR-99a and miR-125b-2 were significantly increased in pregnant women. Unfortunately, the concentrations and gene expression of extracellular chromosome 21-derived microRNAs (miR-99a, let-7c, miR-125b-2 and miR-155) did not differ between the cohorts of pregnancies bearing euploid foetuses and those affected with Down syndrome. CONCLUSION: Analysis of extracellular chromosome 21-derived microRNAs does not distinguish between pregnancies with euploid and aneuploid foetuses and has no benefit for screening programmes.

Feasibility of fetal-derived hypermethylated RASSF1A sequence quantification in maternal plasma--next step toward reliable non-invasive prenatal diagnostics.

We determined the feasibility of universal fetal marker detection in maternal circulation. Using real-time PCR, we compared the levels of fetal (SRY and hypermethylated RASSF1A) and total (GLO gene and total RASSF1A) extracellular DNA and fractions of extracellular fetal DNA (SRY/GLO vs. hypermethylated RASSF1A/total RASSF1A) in maternal circulation. Sensitivity and specificity reached 100% as the fetal-specific hypermethylated RASSF1A sequence was detected in all 151 examined plasma samples derived from 70 normal pregnancies with a singleton male (n=51) or female (n=19) fetus sampled throughout gestation and absent in non-pregnant individuals (n=29). A strong positive correlation was observed between fetal-derived hypermethylated RASSF1A and SRY (ρ=0.66, P<0.001), total RASSF1A and GLO (ρ=0.65,P<0.001), SRY/GLO vs. hypermethylated RASSF1A/total RASSF1A ratio (ρ=0.62, P<0.001) in maternal plasma. The results indicate that a universal fetal marker could be useful not only for the confirmation of the presence of fetal cell-free DNA in maternal plasma but could enable quantification of cell-free fetal DNA in pregnancy associated disorders, independently of the sex of the fetus.

[Detection of placenta-specific microRNAs in maternal circulation]. / Detekce placentárne specifických mikroRNA v materské cirkulaci.

OBJECTIVE: We focused on the detection of microRNAs in maternal circulation during the course of physiological pregnancy. We tested initially microRNAs (mir-135b and miR-517a) which presence in maternal circulation had been previously demonstrated and those microRNAs (miR-518b and miR-517a) with up-regulated expression profile in placentas derived from pregnancies during the onset of preeclampsia. Further we selected those microRNAs, which were reported to be placenta specific according to the miRNAMap database (these microRNAs were significantly expressed in the placenta and simultaneously showed no or minimal expression in other tissues). SETTING: Division of Molecular Biology and Cell Pathology, Department of Gynaecology and Obstetrics, Third Faculty of Medicine, Charles University, Prague. METHODS: RNA enriched for small RNAs (including microRNAs) was isolated from 1 ml of maternal plasma during the 12th, 16th and 36th week of gestation and 200 microl of peripheral blood derived from healthy non-pregnant women. Consequently relevant microRNA was transcribed into cDNA using specific stem-loop primer and detected by specific real-time PCR assay. RESULTS: From the cohort of tested microRNAs we excluded those ones, which were not detectable in maternal circulation during pregnancy (miR-136 and miR-519a) and/or were demonstrated in peripheral blood of healthy non-pregnant women (miR-34c, miR-224, miR-512-5p, miR-515-5p, miR-516-5p, miR-518f*, miR-519d, miR-519e). CONCLUSION: On the base of the current study results we finally selected 6 most suitable microRNAs (miR-517*, miR-518b, miR-520a*, miR-520h, miR-525 and miR-526a) for subsequent studies concerning the follow-up of placenta specific microRNAs in maternal circulation during pregnancy and the differentiation between normal and pathologic pregnancies (preeclampsia, IUGR) within the same gestational age.

Simultaneous flue gas bioremediation and reduction of microalgal biomass production costs.

A flue gas originating from a municipal waste incinerator was used as a source of CO(2) for the cultivation of the microalga Chlorella vulgaris, in order to decrease the biomass production costs and to bioremediate CO(2) simultaneously. The utilization of the flue gas containing 10-13% (v/v) CO(2) and 8-10% (v/v) O(2) for the photobioreactor agitation and CO(2) supply was proven to be convenient. The growth rate of algal cultures on the flue gas was even higher when compared with the control culture supplied by a mixture of pure CO(2) and air (11% (v/v) CO(2)). Correspondingly, the CO(2) fixation rate was also higher when using the flue gas (4.4 g CO(2) l(-1) 24 h(-1)) than using the control gas (3.0 g CO(2) l(-1) 24 h(-1)). The toxicological analysis of the biomass produced using untreated flue gas showed only a slight excess of mercury while all the other compounds (other heavy metals, polycyclic aromatic hydrocarbons, polychlorinated dibenzodioxins and dibenzofurans, and polychlorinated biphenyls) were below the limits required by the European Union foodstuff legislation. Fortunately, extending the flue gas treatment prior to the cultivation unit by a simple granulated activated carbon column led to an efficient absorption of gaseous mercury and to the algal biomass composition compliant with all the foodstuff legislation requirements.

Influence of processing parameters on disintegration of Chlorella cells in various types of homogenizers.

The following bead mills used for disruption of the microalga Chlorella cells were tested: (1) Dyno-Mill ECM-Pilot, grinding chamber volume 1.5 L; KDL-Pilot A, chamber volume 1.4 L; KD 20 S, chamber volume 18.3 L; KD 25 S, chamber volume 26 L of Willy A. Bachofen, Basel, Switzerland, (2) LabStar LS 1, chamber volume 0.6 L of Netzsch, Selb, Germany, (3) MS 18, chamber volume 1.1 L of FrymaKoruma, Neuenburg, Germany. Amount of disrupted cells decreased with increasing Chlorella suspension feed rate and increased up to about 85% of the beads volume in the grinding chamber of the homogenizers. It also increased with agitator speed and number of passes of the algae suspension through the chamber. The optimum beads diameter was 0.3-0.5 mm in the homogenizers Dyno-Mill and LabStar LS 1 and 0.5-0.7 mm in the homogenizer MS 18. While the degree of the cell disruption decreased with increasing cell density in Dyno-Mill and LabStar, the cell disruption in the MS 18 increased. Depending on processing parameters, more than 90% of algae cells were disrupted by passing through the bead mills and bacteria count in algae suspension was reduced to about two orders.

Salivary cortisol in low dose (1 microg) ACTH test in healthy women: comparison with serum cortisol.

To date, a single report has appeared on the use of salivary cortisol for adrenal function testing with a low dose ACTH, although 1 microg has become preferred as a more physiological stimulus than the commonly used 250 microg ACTH test. Our present study was aimed to obtain physiological data on changes of free salivary cortisol after 1 microg ACTH stimulation. This approach was compared with the common method based on the changes of total serum cortisol. Intravenous, low-dose ACTH test was performed in 15 healthy women (aged 22-40 years) with normal body weight, not using hormonal contraceptives, in the follicular phase of the menstrual cycle. Blood and saliva for determination of cortisol were collected before ACTH administration and 30 and 60 min after ACTH administration. Basal concentration of salivary cortisol (mean +/- S.E.M., 15.9+/-1.96 nmol/l) increased after 1 microg ACTH to 29.1+/-2.01 nmol/l after 30 min, and to 27.4+/-2.15 nmol/l after 60 min. The differences between basal and stimulated values were highly significant (p<0.0001). The values of salivary cortisol displayed very little interindividual variability (p<0.04) in contrast to total serum cortisol values (p<0.0001) A comparison of areas under the curve (AUC) related to initial values indicated significantly higher AUC values for salivary cortisol than for total serum cortisol (1.89+/-0.88 vs. 1.22+/-0.19, p<0.01). Correlation analysis of serum and salivary cortisol levels showed a borderline relationship between basal levels (r=0.5183, p=0.0525); correlations after stimulation were not significant. Low-dose ACTH administration appeared as a sufficient stimulus for increasing salivary cortisol to a range considered as a normal adrenal functional reserve.

Quantification of fetal and total circulatory DNA in maternal plasma samples before and after size fractionation by agarose gel electrophoresis.

Fetal extracellular DNA is mainly derived from apoptotic bodies of trophoblast. Recent studies have shown size differences between fetal and maternal extracellular DNA. We have examined the quantification of fetal (SRY gene) and total (GLO gene) extracellular DNA in maternal plasma in different fractions (100-300, 300-500, 500-700, 700-900, and >900 bp) after size fractionation by agarose gel electrophoresis. DNA was extracted from maternal plasma samples from 11 pregnant women carrying male foetuses at the 16th week of gestation. Fetal circulatory DNA was mainly detected in the 100-300 bp fraction with the median concentration being 14.4 GE/ml. A lower median amount of 4.9 GE/ml was also found in the 300-500 bp fraction. Circulatory DNA extracted from the 100-300 bp fraction contained 4.2 times enriched fetal DNA when compared with unseparated DNA sample. Fetal DNA within the 300-500 bp fraction was 2.5 times enriched. Circulatory fetal DNA is predominantly present in a fraction with molecular size <500 bp, which can be used for the detection of paternally inherited alleles. However, the usage of size-separated DNA is not suitable for routine clinical applications because of risk of contamination.

[Prevalence of the female urinary incontinence. Results from a questionnaire study]. / Prevalence zenské mocové inkontinence. Výsledky dotazníkové studie.

OBJECTIVE: To evaluate the prevalence of urinary incontinence in patients of gynecological practise aged 31-60. DESIGN: Prospective questionnaire study. SETTING: Obstetric and Gynecologic Department, Charles University 2nd Medical Faculty and Teaching Hospital Motol, Prague. METHODS: Questionnaire study of 561 women aged 31-60 examined with gynecological problems (not for the symptoms of urinary incontinence) from November 2001 till October 2002 in standard gynecological practise. The questionnaire included history, evaluation of urinary continence, lasting of the symptoms, body mass index, obesity, age, parity. Stress, urgent and mixed incontinence and influence on the sexual life were also evaluated. Cochran Mantel-Haenszel test and chi2 test were used for the statistical analysis. RESULTS: The incontinence rate in the group of 533 evaluated patients (95% completed questionnaires from 561) of gynecological practise was 23.8%. 81.1% of incontinent patients in the study suffered from stress urinary incontinence. For an easy survey and analysis the patients were divided into three age groups (31-40, 41-50, 51-60). Prevalence of the urinary incontinence rised with age. Statistically significant lower prevalence of urinary incontinence was in the age group 31-40 (p<0.0005). Influence of parity on the prevalence of incontinence was statistically significant only in the age group 31-40 (p=0.002). Obesity had no statistical impact on prevalence of urinary incontinence (p=0.79). 5.5% of incontinent women suffered from negative effect of urinary incontinence on sexuality; the differencies among the age groups were not statistically significant. CONCLUSIONS: The results of the study show high prevalence of urinary incontinence in population of healthy women of gynecological practise. Low interest for the treatment is in contrast with high prevalence of this symptom. Higher quality of the enlightenment with attention to the prevention and therapy of urinary incontinence in population is the way how to improve quality of lives of the afflicted women.

[Ultrasonic diagnosis and mini-abortions]. / Ultrazvuková diagnostika a miniinterrupce.

PIP: The TOSHIBA SAL 22A ultrasound machine has been in use at the Gynecological and Obstetrics Department of the District and Regional Institute of National Health in Rakovnik, Czechoslovakia since 1981 for early ultrasound detections of pregnancies for the purpose of mini-abortion. With its resolution at 5-6 weeks after onset of amenorrhea, it is better than the human chorionic gonadotropin (HCG) test in preparing for mini-abortions because it shows objective proof of intrauterine gravidity with direct viewing of the amniotic cavity and the fertilized egg, while affording the possibility of photodocumenting the pregnancy. This eliminates the need for and the cost of histological tests, which are the only means of objective determination with the HCG test. Ultrasound diagnosis measures the diameter of the fetus and the real term of pregnancy, which allows selection of a suitable form of aspiration curettage while eliminating excessive amenorrhea in the mother's case history. Objective ultrasound determinations eliminate the infrequent but real danger of pathology to the trophoblast, arising from a lack of development in the fertilized egg, in mini-abortions performed on the basis of HCG testing. In addition to the economic advantages of the ultrasound diagnostic test over the HCG test, the former is more suitable because any negative or ambiguous findings lead to further examination of the patient, thus ruling out the possibility of overlooking ectopic pregnancy and obviating the one drawback of this type of diagnosis, i.e. a false negative finding. Though the result is termination of pregnancy 1 week later than otherwise, this effect is benign compared to the needless intrauterine intervention in the case of a false negative finding in the HCG test.^ieng