Reference values for variables of fetal cardiocirculatory dynamics at 11-14 weeks of gestation.

OBJECTIVE: Despite the increasing popularity of first-trimester fetal echocardiography, the evaluation of fetal heart function during this period remains challenging. The parameters of normal cardiac function at 11-14 weeks' gestation are not well defined and appropriate reference values have not yet been established. The purpose of this study was to evaluate the fetal cardiocirculatory dynamics during routine first-trimester screening and establish cross-sectional reference ranges for 11-14 weeks' gestation. METHODS: Fetal echocardiography was performed on 202 women with singleton pregnancies at 11 + 0 to 13 + 6 weeks' gestation. Global cardiac function was evaluated using the heart : chest area ratio and Tei index of the left (LV) and right (RV) ventricles. The proportion of isovolumic contraction (ICT%) and ejection (ET%) times of the cardiac cycle, and the outflow velocities described the systolic function. Diastolic function was evaluated by the proportion of relaxation (IRT%) and filling (FT%) times, the ratio of the blood velocity through the atrioventricular valves during early filling (E) and atrial contraction (A) phases of the cardiac cycle, and ductus venosus pulsatility index for veins (DV-PIV). All participants had additional fetal echocardiography in the second trimester and neonatal clinical examination after birth to confirm normality. RESULTS: The mean heart : chest area ratio (0.203 +/- 0.04) and the Tei indices of both ventricles did not vary significantly during weeks 11-14, but the mean Tei index of the LV (0.375 +/- 0.092) was significantly higher than that of the RV (0.332 +/- 0.079) (P = 0.001). The fetal heart rate (FHR) decreased with increasing crown-rump length (CRL) (P < 0.00001). The LV-ICT% did not vary significantly (P = 0.27), LV-IRT% (P = 0.03) and LV-ET% decreased (P = 0.01), whereas the LV-FT% increased (P = 0.02) with CRL. The RV-ET% (P = 0.84) and RV-FT% (P = 0.60) remained relatively stable. The LV-ET% was lower than the RV-ET% (P = 0.0001). The LV (P = 0.004) and RV (P < 0.00001) outflow velocities and E : A ratios of both ventricles (P < 0.0001) increased with advancing gestation. The E-velocity of the LV (P = 0.003) and RV (P = 0.002) increased significantly but the increase in A-velocity was not significant. The outflow velocity (P = 0.008) and E-velocity (P = 0.005) of the RV were higher than that of the LV but the A-velocities were similar (P = 0.066). The mean DV-PIV was 0.97 +/- 0.23 and did not change significantly (P = 0.95) during weeks 11-14. The FHR and DV-PIV did not correlate with the Tei index of either ventricle. CONCLUSION: We have established reference ranges for the noninvasive evaluation of fetal cardiocirculatory dynamics at 11-14 weeks' gestation.

Microvessel density assessment in benign and malignant endometrial changes.

Tumor angiogenesis is believed to be a prognostic indicator associated with tumor growth and metastasis. Microvessel density (MVD) assessment with common endothelial markers such as CD34 has been found to influence prognosis among endometrial carcinoma patients. The CD105/endoglin antibody has been reported to preferentially bind to proliferated endothelial cells in tissues participating in angiogenesis. The aim of this study was to evaluate the quantification of angiogenesis by assessing MVD in endometrial lesions when comparing the performance of anti-CD34 and anti-CD105 in women with benign and malignant endometrial changes. The study included 58 women (37 postmenopausal) with normal, hyperplastic and malignant endometrium in which preoperative transvaginal sonography was performed. Histological results of the removed endometrium were correlated with MVD assessed in "hot areas" where high densities of microvessels were detected within tumoral tissue. Endometrial cancer was confirmed in 37 women (3 premenopausal). Benign hyperplasia (14 cases), secretory or proliferative endometrium (5 cases) or endometrial atrophy (2 cases) was found in the remaining women. Malignant changes were mostly noted as FIGO stage I and II (28 cases) and had a low (1 or 2) histological grade (29 cases). Median MVD's assessed with CD105 and CD34 were 10.4 and 32.3, respectively. Median MVD assessed with CD34 was almost twice higher in women with endometrial cancer than in women with benign endometrium (CD34 MVD = 41.8 vs. 27.6, p=0.004). In cases of CD105 MVD significant differences between women with benign and malignant endometrial changes were also found (CD105 MVD = 11.8, vs. 6.4; p=0.00007). The menopausal status, but not the clinical stage or histological grading was significantly correlated with both CD34 MVD (p=0.02) and CD105 MVD (p=0.0003). A significant correlation was also found between CD34 and CD105 measured MVD (p=0.000001). In conclusion, transition from endometrial hyperplasia to endometrial cancer appears to be accompanied by microvessel density changes. MVD assessed with both CD34 and CD105 antibodies could be used as a potential prognostic factor in women with endometrial cancer. Our study showed that endoglin, by staining the proliferating microvessels could be more specific and sensitive marker for tumor neoangiogenesis than the more commonly used marker, CD34.

Microvessel density and CpG island methylation of the THBS2 gene in malignant ovarian tumors.

We aimed to investigate the role of thrombospondin-2 (THBS2) related angiogenic activity in malignant ovarian tumors and to determine if aberrant methylation associated inactivation is involved in down-regulating THBS2 expression in ovarian cancer. The methylation status of the THBS2 promoter region and microvessel density (MVD) was studied in 70 malignant ovarian tumors and in 15 control ovarian samples. A methylation specific PCR (MSP) method was used to distinguish methylated from unmethylated DNA in the promoter regions of the THBS2 gene. MVD was assessed with anti-CD34 antibodies and the results were compared between tumors with average (AVD) and high (HVD) microvessel density. Alterations in the expression of trombospondin-2 were more often seen in early (FIGO stage I and II ) than in late stage tumors (66% vs. 30%, p=0.01). Age, menopausal status, the histological type and tumor grade did not correlate with thrombospondin-2 expression, however, silencing of THBS2 gene was more often seen in higher rather than in lower grade (50% vs. 28%) cancers and in nonserous rather than in serous (43% vs. 32%) tumors. In 81% of THBS2 mRNA-negative tumors, ahypermethylated promoter region of THBS2 was found (p=0.00003). An unmethylated product of the MSP reaction was more often detected in high grade tumors (93% vs. 76%, p=0.04). The incidence of THBS2 hypermethylation was not related to the tumor histological type, but unmethylated THBS2 was more often found in serous rather than in nonserous tumor (96% vs. 74%, p=0.01). The median MVD in malignant the tumor samples was 21,7 (range: 7.6-55.2). In the group with HVD, 54% were THBS2 mRNAnegative, conversely, in the group with AVD tumors only 26% of the cases had undetectable THSB2 mRNA. A significant correlation between microvessel density and the expression of trombospondin-2 (p=0.009) was found. In the samples with HVD, 51% had hypermethylated THBS2, however methylation pattern had no significant influence on microvessel density. In conclusion, hypermethylation might be responsible for altered expression of thrombospondin-2 in ovarian cancer. The THSB2 methylation pattern had no significant influence on microvessel density.

Doppler study of the peripheral flows in early gestation.

The aim of this study was to determine the first trimester human peripheral arterial and venous blood flow between 5 - 10 weeks of gestation. Two hundred twenty four women with singleton, uncomplicated pregnancies were prospectively studied with transvaginal ultrasound. Ductus venosus, umbilical artery waveforms and pulsatility indexes (PI) were assessed as well as the waveform of the umbilical vein and the mean velocity (V(mean)) of the umbilical artery flow. The heart rate was also obtained and analyzed. The fetal heart rate showed a positive correlation with increasing gestational age R=0.76 (p<0.000001). Recordings from the umbilical artery, umbilical vein and ductus venosus were obtained starting from 7 weeks of gestation. The signal from the ductus venosus presented always as antegrade flow during atrial contractions. The pulsatility index (PI) of DV as well as PI of the umbilical artery remained unchanged during the study (statistically non-significant). The umbilical artery, using Doppler tracing was investigated and an absent diastolic flow was documented in every case. Umbilical artery V(mean) increased from 3.8 + 0.32 cm/s to 9.0 + 0.21 cm/s from 7 to 10 weeks of gestation (p< 0.005). Recordings from the umbilical vein showed the pulsation during atrial contractions. Ductus venosus blood velocity and waveform patterns did not change significantly during the study period. Pulsation in the umbilical vein is a typical Doppler finding at the embryonic time. Placental volume blood flow increased significantly with no change in the placental vascular impedance.

Doppler study of the embryonic heart in normal pregnant women.

OBJECTIVE: To describe normal fetal cardiac and hemodynamic development in normal early first trimester pregnancies. MATERIALS AND METHODS: Eighty-eight women with singleton, uncomplicated pregnancies were prospectively studied with transvaginal ultrasound, pulsed and color Doppler. Heart diameter, heart rate, and inflow and outflow waveforms with valve signals were documented. The proportion of the cardiac cycle of isovolumetric relaxation time (IRT%) and isovolumetric contraction time (ICT%) as well as Tei index were calculated. RESULTS: Ninety-one percent of studies were successful. Heart diameter and the fetal heart rate showed a positive correlation with increasing gestational age: R = 0.80 (p < 0.000001), R = 0.76 (p < 0.000001), respectively. Mean heart diameter at 6 weeks was 1.28 +/- 0.26 mm and mean fetal heart rate was 117 +/- 6 bpm compared to 3.88 +/- 0.54 mm and 171 +/- 6 bpm at 10 weeks. The inflow waveform was monophasic (atrial contraction) in all cases from 6 to 9 weeks. Eight pregnancies (9%) miscarried between 6 and 12 weeks of gestation and the heart exams were characterized by increased IRT% compared with the survivors. In survivors, IRT% decreased between 7 and 8 weeks, from 32.9 +/- 10.7% to 20.8 +/- 5.7% (p < 0.0001). ICT% decreased from 18.6 +/- 4.4% of the cardiac cycle at 8 weeks to 12.6 +/- 4.4% at 9 weeks (p < 0.0008) (after heart development period). CONCLUSIONS: Doppler examination of the fetal cardiac function is possible after 5 weeks of gestation. After 8 weeks of gestation, the fetal heart is morphologically mature but has not yet achieved effective myocardial compliance. The embryonic human heart is dependent on the atrial contraction for ventricular filling throughout the period of cardiac development. Non-survivors manifest myocardial dysfunction.

Basal and postprandial gut peptides affecting food intake in lean and obese pregnant women.

Maternal obesity has been reported as a risk factor for various maternal and fetal complications. The aim of the present study was to examine the patterns of basal and postprandial plasma concentrations of certain gut hormones affecting food intake such as acylated ghrelin, peptide YY(3-36) (PYY(3-36)), cholecystokinin (CCK), insulin and glucose in pregnant women with varying body mass gain during physiological pregnancy. The study included 34 women with singleton pregnancies in the 2(nd) trimester of gestation. The examined pregnant women were divided into 4 groups; I. control pregnancy (CP) with weight gain below 0.5 kg/week; II. overweight low weight gain <1 kg/week (OLWG), III. overweight high weight gain >1 kg/week (OHWG); morbidly obese pregnant with weight gain >1.5 kg/week (MOP). The basal acylated-ghrelin levels in MOP subjects were significantly higher than those in CP and no usual suppression of acylated ghrelin after the meal observed in CP as well as in OLWG and OHWG was found in MOP women. Basal PYY(3-36) plasma levels were similar in CP, OLWG and OHWG but in MOP was significantly reduced and no significant increase in hormone level, typically observed in CP, was detected after a meal in overweight or obese women studied. The fasting CCK and C-reactive protein (CRP) levels in MOP subjects were significantly higher than those in CP and other overweight women. In conclusion, we found that pregnant women with overweight and obesity exhibit significant changes in fasting and postprandial gut hormones affecting food intake such as acylated ghrelin, PYY(3-36) and CCK as well as in CRP and these changes might contribute, at least in part, the development of obesity in pregnancy.

[Prenatal diagnostics of Toxoplasma gondii invasion by quantitative method of PCR-TaqMann]. / Prenatalna diagnostyka zarazenia pierwotniakiem Toxoplasma gondii metoda ilosciowej polimerazowej reakcji lancuchowej PCR-TaqMann.

Routine serological diagnosis of toxoplasmosis provides high sensitivity, but not high specificity. The high sensivity combined with high specifity offered by PCR-TaqMann as well as the degree of infection led us to investigate the presence and levels of T. gondii DNA in amniotic fluid, maternal and neonatal blood in cases of pregnancy where infection with this agent was suspected. Samples of amniotic fluid and blood were taken from pregnant women. Postnatal blood samples were also taken from their infants. Presence and levels of toxoplasma DNA was investigated using PCR-TaqMann. PCR products were detected by electrophoresis on polyacrylamide gel. The PCR-TaqMann test is highly sensitive, specific and useful method allowing detection of the parasite genome and assessement of its level.

[Detection of Toxoplasma gondii DNA by PCR in mother's blood, amniotic fluid and child's blood in selected cases of pathological pregnancy]. / Wykrywanie DNA Toxoplasma gondii metoda polimerazowej reakcji lancuchowej (PCR) we krwi matki, plynie owodniowym i krwi noworodka w wybranych przypadkach patologii ciazy.

Prenatal screening of Toxoplasma gondii infection is controversial. The diagnosis is based on serological tests detecting IgM and IgG antibodies against T. gondii, but interpretation of these tests results is often confusing. It is commonly made retrospectively when serological screening indicates a possibility of recent infection. Most women have antibodies against T. gondii and serial testing is required only in monitoring of pregnancies where initial screening is negative. The introduction of Polymerase Chain Reaction (PCR) assay for detection of Toxoplasma gondii DNA in selected cases of pathologic pregnancies has permitted a more accurate and faster diagnosis of congenital toxoplasmosis infections.

[Predictive usefulness of lipoproteins a -Lp (a) in cases of preeclampsia]. / Przydatnosc predykcyjna oznaczenia stezen Lipoproteiny a--Lp (a) u ciezarnych z stanami przedrzucawkowymi.

The causes of Pregnancy Induced Hypertension are still under study. A genetic cause has been suggested as well as raised activity of the blood lipid system, which is however determined genetically. One element of the lipid system is lipoprotein a, which may have a key role in prediction of preeclampsia. The aim of our study was to determine lipoprotein (a) concentration in women with preeclampsia and in healthy pregnancies, and to assess the usefulness of lipoprotein (a) concentration as prognostic factor of eclampsia. Sera from 19 women with preeclampsia and from 19 with healthy pregnancies were tested. Our results show that determination of lipoprotein (a) levels in preeclampsia may be significant as predictive factor of eclampsia.

[Usefulness of quantitative assessment of Toxoplasma gondii genome using PCR-TaqMan in amniotic fluid, maternal and neonatal blood in selected complications in pregnancy]. / Przydatnosc ilosciowej oceny genomu Toxoplasma gondii przy uzyciu metody PCR TaqMan w plynie owodniowym, krwi matki oraz krwi noworodków w wybranych przypadkach patologii ciazy.

UNLABELLED: Routine serological diagnosis of Toxoplasmosis provides high sensitivity, but not high specificity. The high sensitivity combined with high specificity offered by PCR-TaqMan as well as the degree of infection led us to investigate the presence and levels of Toxoplasma gondii genome in amniotic fluid, maternal and neonatal blood in cases of pregnancy where infection with this agent was suspected. MATERIALS AND METHODS: Samples of amniotic fluid and blood were taken from 28 women between the 16th and 40th week of gestational age. Postnatal blood samples were also taken from their infants. Included in the study group were women with IUGR, PROM, preterm delivery imminent, Toxoplasmosis in previous pregnancies, raised IgG or IgM anti-Toxoplasma antibody titers and with amniotic fluid disturbances (oligohydramnios and polyhydramnios). Presence and levels of Toxoplasma genome was investigated using PCR-TaqMan. PCR products were detected by electrophoresis on polyacrylamide gel. RESULTS: Toxoplasma gondii genome was detected in blood from 13 women, 3 newborns and in amniotic fluid from one other women. Toxoplasma genome was detected in blood from one newborn, but was not detected in sample from its mother. CONCLUSIONS: The PCR-TaqMan test is highly sensitive and specific method allowing detection of the parasite genome and assessment of its level. Limitations of this method are its relatively high cost and poor access to ABI PRISM 7700 (TaqMan) sequence detector. The PCR TaqMan is useful in cases, where serological tests for the presence of infections are ambiguous.

Production of PEG-modified bovine hemoglobin: economics and feasibility.

Bovine hemoglobin has many advantages as a blood substitute: a) it's ready availability; b) it's low cost; c) it's oxygen carrying capacity; and d) the ease with which it can be modified with polyethylene glycol (PEG) to improve its pharmacokinetic profile. This study investigates the potential of PEG-modified bovine hemoglobin as a cost-effective blood substitute.

Structural characterization of cholylcoenzyme A:glycine-taurine N-acyltransferase and a covalent substrate intermediate.

Bile acid-CoA:glycine-taurine N-acyltransferase was found to catalyze a reaction in the absence of glycine or taurine in which the substrate cholyl-CoA is cleaved with the release of CoA and the formation of a covalently bound enzyme-cholate intermediate. This unstable intermediate was trapped by a rapid mixing and denaturation procedure. The denatured protein was digested with trypsin and the cholate-labeled tryptic peptide was isolated. This cholate-peptide is considered to originate from the active site region of the enzyme based on the following criteria: cholyl-CoA does not react with any of the 20 common amino acids, the hydrolysis of cholyl-CoA is known to occur on the enzyme, the lack of reaction of the enzyme with just cholate, and the fact that labeling is extensive even at low (substrate level) concentrations of cholyl-CoA. The isolated cholate-peptide was submitted to amino acid analysis. It contained 32 amino acid residues and was devoid of cysteine, methionine, and tyrosine. Amino acid analysis of the N-acyltransferase was conducted. The enzyme was also shown to possess a blocked N terminus.

The effect of bile acid structure on the activity of bile acid-CoA:glycine/taurine-N-acetyltransferase.

Bile acid-CoA:glycine/taurine N-acetyltransferase from bovine liver was analyzed for its ability to conjugate a variety of different bile acid-CoA analogues. A complete steady state bisubstrate kinetic analysis was conducted for each analogue. The enzyme demonstrated strict specificity for the normal 4-substituted pentanoic acid side chain; shortening the side chain by 1 methylene group (norcholyl-CoA) completely eliminates enzymatic activity, and extending the side chain by 1 methylene group (homocholyl-CoA) causes a 30-fold decrease in activity at Vmax. These effects of side chain modification were not related to decreased binding affinity as much as to decreases in the rates of the bond-breaking and bond-making steps. Bile acid-CoA analogues with a variety of ring substitutions involving keto and hydroxyl groups were also examined. Varying the position of substitution and the nature of the substituent had major effects on both the Km and Vmax terms. The analogues with the highest activities at Vmax were 7-dehydrocholyl-CoA and the "allo" bile acid 5 alpha,6-ketolithocholyl-CoA. However, in both cases, the high activity is obtained at the expense of binding energy. The most efficient substrates were 7-ketolithocholyl-CoA and 3-dehydrocholyl-CoA. The more common analogue, chenodeoxycholyl-CoA, was a surprisingly inefficient substrate. The relative rates of formation of glycine versus taurine conjugates were also found to vary with changes in structure. This indicates that certain bile acids are more likely to be conjugated with taurine, and others with glycine.

Identification of a unique mammalian species of cholyl-CoA: amino acid N-acyltransferase.

The N-acyltransferase enzyme which catalyzes the conjugation of the CoA adducts of bile acids with amino acid has been partially purified from dog liver. Unlike the N-acyltransferase from five other mammalian species, the enzyme from dog liver was unable to synthesize glycine conjugates from cholyl-CoA in vitro even in the presence of 0.1 M glycine. The enzyme did catalyse the synthesis of taurine conjugates. Thus, the enzyme from dog liver resembles non-mammalian forms of the enzyme which also synthesize only taurine conjugates. However, the molecular weight of the enzyme from dog liver was found to be approximately 45 700, which is similar to values reported for other mammalian forms and considerably smaller than the molecular weight of 65 000 reported previously for a non-mammalian form of the enzyme.

Purification and characterization of cholyl-CoA: taurine N-acetyltransferase from the liver of domestic fowl (Gallus gallus).

The enzymological basis for the ability of mammalian liver to conjugate bile acids with both glycine and taurine, and for non-mammalian liver to make only taurine conjugates, was investigated. The taurine-conjugating enzyme has been purified 1200-fold from the liver of domestic fowl and its properties compared with those of the glycine/taurine-conjugating enzyme from bovine liver [Czuba & Vessey (1980) J. Biol. Chem. 255, 5296-5299]. The enzyme from both species followed a Ping Pong mechanism. The enzymes were also similar with respect to their affinity for taurine, although the enzyme from domestic fowl would not bind glycine. The affinity of both for cholyl-CoA was quite similar, too, and both enzymes were inhibited reversibly by p-mercuribenzoate. The enzymes, however, were quite different in size. The enzyme from domestic fowl had a mol.wt. of 63000-65000 by both gel filtration and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. This is approx. 15 000 mol.wt. units larger than the enzyme from bovine liver, and suggests a loss of genome over the course of evolution as the basis for the altered specificity at the amino-acid binding site.

Kinetic characterization of cholyl-CoA glycine-taurine N-acyltransferase from bovine liver.

The bile acid-conjugating enzyme cholyl-coenzyme A:glycine/taurine N-acyltransferase previously purified from bovine liver was subjected to a bisubstrate kinetic analysis. Double reciprocal plots of reaction rates as a function of variable concentrations of aminoa acid and cholyl-CoA yielded the nonintersecting type of plots typical of ping-pong reaction mechanisms. A Tetra Uni ping-pong mechanism was supported by further kinetic analysis including demonstration that the enzyme will catalyze the release of CoA from cholyl-CoA without glycine being present. This half-reaction is shown not to be the result of an acyl-CoA thiolase contaminating the N-acyltransferase preparation. The specificity of the enzyme for amino acid was found to be quite narrow; no detectable rate was found for D-Ala, L-Ala, L-Ser, L-Glu-NH2, L-ornithine, or beta-alanine. Activity toward various acyl-CoA derivatives was also tested. Phenylacetyl-CoA, benzoyl-CoA, and acetyl-CoA were not substrates. CoA derivatives of the other common bile acids were very good substrates. Conjugated bile acids were efficient competitive inhibitors of cholyl-CoA binding. Cholic acid also inhibited; however, its inhibition pattern was complex. The enzyme could be reversibly inhibited by p-mercuribenzoate and both cholyl-CoA and glycine protected. N-Ethylmaleimide and iodoacetate were not inhibitory.

Manganous ion binding to tubulin.

The assembly of pure tubulin into microtubules is induced by Mn(II), and the polymerized product displays the characteristic sensitivity of normal microtubules to low temperature. The interaction of Mn(II) with tubulin purified by phosphocellulose chromatography to remove microtubule-associated proteins was investigated using electron paramagnetic resonance and atomic absorption spectroscopy. Mn(II) interacts with tubulin at about one high affinity site with an apparent dissociation constant (KD) of 1.6 +/- 0.3 microM and at 8 +/- 2 low affinity (KD = 0.38 +/- 0.18 mM) sites. The binding of Mn(II) to tubulin at the high affinity site was accompanied by release of tubulin-bound Mg(II) from the protein. Thus, Mn(II) substitutes for Mg(II) at the high affinity site and in promoting the assembly of tubulin. Mg(II), Co(II), and Zn(II) can displace Mn(II) from manganese-tubulin, but Ca(II) cannot. The electron paramagnetic resonance spectrum at 9.1 GHz of Mn(II) bound to the high affinity divalent cation site of tubulin is characterized by broadened hyperfine structure and the absence of solid state spectral features, suggesting that the bulk solvent is accessible to the bound metal ion.