Maternal smoking does not affect the amount of cell-free fetal DNA in maternal plasma during the 1st trimester of pregnancy.

CffDNA, from 344 non-smoking, 38 smoking and 33 ex-smoking pregnant women at 11 (+0)-13 (+6) gestational weeks, was extracted and quantified by the multicopy DYS14, as the fetal DNA marker and using the quantitative real-time PCR 7300 detection system. The smoking habit was based on maternal self-report, confirmed by cotinine levels and male fetuses were verified by phenotype at birth. The genders of newborns were compared with DYS14-cffDNA analysis, achieving a 100% diagnostic accuracy of the test. A total of 177 non-smokers, 18 smokers and 22 ex-smoker pregnancies with male fetuses were identified by the cffDNA concentration. Results showed that smoking status was not associated with different amounts of DYS14-cffDNA (p = 0.159), suggesting the possibility of offering cffDNA testing to all pregnant women, even if they are active smokers or ex-smokers, and the test can be unadjusted for smoking status.

Caspase 3 activation and PARP cleavage in lymphocytes from newborn babies of diabetic mothers with unbalanced glycaemic control.

Several epidemiological studies showed that gestational diabetes mellitus is the most frequent metabolic disorder of pregnancy, the pathogenesis of which has yet to be completely clarified. The aim of this study was to investigate the presence and processing of caspase 3 (Casp3) and poly(ADP-ribose) polymerase 1 (PARP1) in cord blood lymphocytes as markers of apoptosis in relation to glycaemic control during intrauterine life. Our results showed a specific positive correlation between the levels of active Casp3 (17-19 kDa) and the inactive form of PARP1 (89 kDa) in lymphocytes isolated from newborn babies of diabetic women with unbalanced glycaemic control, with a direct correlation between the activation of casp3 and the inactivation of PARP1, that makes lymphocytes unresponsive towards lipopolysaccharide stimulation, highlighting an altered functional response. Besides more studies are required to fully correlate the activation of the apoptotic process during the intrauterine life with the foetal health later in life, our study indicates that a cord blood lymphocyte, an easily accessible source, is informative about the activation of apoptotic stimuli in circulating cells of newborn babies in relation to the glycaemic control reached by the mother during pregnancy.

[Identification of preterm labor: the role of the fibronectin and ultrasound cervicometry and their association]. / La identificazione del travaglio pretermine: ruolo della fibronectina e della cervicometria ecografica e loro associazione.

OBJECTIVE: The aim of this study was to evaluate the effectiveness of the fetal fibronetcin (fFN) test and ultrasonographic cervical length measurement used alone or in combination with each other in order to further improve the identification of patients in preterm labor. METHODS: Prospective multicenter observational study on patients between 24 and 32 weeks of gestation with symptoms of preterm labor (total patients = 132). The endpoint was the delivery at 34 weeks or more. The screening methods used were: the fFN test (group 1), the cervical length measurement by transvaginal ultrasound (group 2) or a combination of both tests (group 3) according to an established protocol. The statistical analysis was performed using the χ2 test using the SPSS software. RESULTS: Group 1: positive fFN test in 25.7% of cases, incidence of preterm birth (<34 weeks) of 18%. Group 2: cervical length <25 mm in 56.2% of cases, incidence of preterm birth (<34 weeks) of 18.5%. The negative predictive value is equivalent to 99.0% for the fFN test and 95.2% for cervicometry; combined use reaches 100%, compared to 54% positive predictive value. CONCLUSION: The identification of women at high risk of preterm delivery carried out with the fFN test or with transvaginal ultrasound cervicometry is clinically valid. The study also showed that the contextual use of biochemical and biophysical tests reaches a high negative predictive value (100%), making it a very useful method to identify patients truly at risk and to further reduce the incidence of non adequate treatment.

Soluble adhesion molecules: marker of pre-eclampsia and intrauterine growth restriction.

OBJECTIVE: This study monitored circulating plasma levels of soluble vascular cellular adhesion molecule-1 (sVCAM-1), intracellular adhesion molecule-1 (sICAM-1) and soluble E-selectin (sE-selectin) in women with healthy pregnancies, with pregnancy-induced hypertension (PIH), with pre-eclampsia and with pregnancies with isolated intrauterine growth restriction (IUGR) in order to determine whether elevated concentrations have a predictive value for the clinical signs of those pregnancy-induced disorders. METHODS: Plasma concentrations of sVCAM-1, sICAM-1 and sE-selectin were determined in healthy pregnant women at each trimester of pregnancy and in pregnant women with PIH, pre-eclampsia and IUGR using commercial kits. RESULTS: In the group of healthy pregnant women, plasma levels of sVCAM-1, sICAM-1 and sE-selectin did not change throughout pregnancy. No significant differences in the levels of these molecules were observed between healthy pregnant women at the third trimester of pregnancy and women with PIH. In addition, concentrations of soluble adhesion molecules were significantly higher in women with pre-eclampsia than in the group of women with healthy pregnancies. Only sVCAM-1 and sE-selectin levels were significantly higher in women with IUGR compared to healthy pregnant women. CONCLUSIONS: Abnormally circulating levels of sVCAM-1, sICAM-1 and sE-selectin may have a predictive value for pre-eclampsia and IUGR, as they may be linked with endothelial activation and/or damage.

Prenatal diagnosis of genetic abnormalities using fetal CD34+ stem cells in maternal circulation and evidence they do not affect diagnosis in later pregnancies.

In the present study, we report a new method for enrichment and analysis of fetal CD34+ stem cells after culture in order to determine whether it is feasible for noninvasive prenatal diagnosis. We also determined whether fetal CD34+ stem cells persist in maternal blood after delivery and assessed whether they have an impact on noninvasive prenatal diagnosis of genetic abnormalities. Peripheral blood samples were obtained from 35 pregnant women, 13 non-pregnant women who had given birth to male offsprings, 12 women who had never been pregnant, and eight pregnant women with male fetuses. CD34+ stem cells were enriched and either cultured for prenatal diagnosis or analyzed with fluorescence in situ hybridization (FISH)/polymerase chain reaction (PCR) to determine peristance in maternal blood. Fetal/maternal cells can be isolated and grown "in vitro" to provide enough cells for a more accurate fetal sex or aneuploid prediction than is provided by unenriched and uncultured CD34+ stem cells. The presence of fetal cells in maternal blood samples from mothers who had given birth to male offspring was found in 3 of 13 blood samples. PCR was positive for Y chromosome in one woman who had never been pregnant. Analysis of cultured CD34+ stem cells from mothers with Y PCR positivity did not detect any male cells in any samples. Even if PCR positivity is due to persistence of fetal stem cells from previous pregnancies, it does not seem to affect this new system of enrichment, culture, and FISH analysis of CD34+ fetal stem cells.

A new methodology of fetal stem cell isolation, purification, and expansion: preliminary results for noninvasive prenatal diagnosis.

We developed a combined methodological approach to enrich and to proliferate in vitro fetal CD34+ stem progenitor cells. Using a magnetic cell-sorting technique, CD34+ cells from pregnant women at the early-second trimester were isolated and enriched and compared to those isolated from blood of nonpregnant women. The number and frequency of CD34+ cells were significantly higher (p < 0.001) in the pregnant women. Unenriched peripheral blood mononuclear cells (PBMC) and enriched CD34+ cells were cultured in a methylcellulose system to evaluate the cloning potential of progenitor cells. After culture, the numbers of burst-forming units erythroid/colony-forming units erythroid (BFU-E/CFU-E) and colony-forming units granulocyte-macrophage (CFU-GM) colonies were increased by 33 and 16 times, respectively. Finally, to distinguish between fetal and maternal cells, four cases of cultured cells were hybridized with specific probes for X and Y chromosomes and two cases with a specific probe for chromosome 21. In normal pregnancies, we identified a high number of male fetal cells and an elevated fetal/maternal ratio. When we analyzed blood samples from pregnancies with trisomic fetuses, we scored a high ratio of trisomic cells respect to maternal cells that was significantly different from the ratio of pregnancies with normal fetuses. Our results demonstrate fetal progenitor cells may be cultured and detected successfully with an appropriate combined methodological approach, which may significantly increase the feasibility of noninvasive prenatal diagnosis.

Nitric oxide-releasing NSAIDs inhibit interleukin-1beta converting enzyme-like cysteine proteases and protect endothelial cells from apoptosis induced by TNFalpha.

BACKGROUND: Nitric oxide (NO)-releasing NSAIDs are a new class of NSAID derivatives with markedly reduced gastrointestinal toxicity. Although it has been demonstrated that NO-NSAIDs spare gastric mucosal blood flow, molecular determinants involved in this effect are unknown. AIM: To investigate the effect of aspirin, naproxen and flurbiprofen, and their NO-derivatives, on gastric apoptosis and endothelial cell damage induced by tumour necrosis factor-alpha (TNFalpha). In other systems, TNFalpha-induced apoptosis is mediated by caspases, a growing family of cysteine proteases similar to the IL-1beta converting enzyme (ICE), and so we have investigated whether NO-NSAIDs modulate ICE-like endopeptidases. METHODS: Rats were treated orally with aspirin, naproxen and flurbiprofen, or their NO-releasing derivatives in equimolar doses, and were killed 3 h later to assess mucosal damage and caspase activity. Endothelial cells (HUVECs) were obtained from human umbilical cord by enzymatic digestion. Caspase 1 and 3 activities were measured by a fluorimetric assay using selective peptides as substrates and inhibitors. Apoptosis was quantified by ELISA specific for histone-associated DNA fragments and by the terminal transferase nick-end translation method (TUNEL). RESULTS: In vivo NSAID administration caused a time-dependent increase in gastric mucosal damage and caspase activity. NCX-4016, NO-naproxen and NO-flurbiprofen did not cause any mucosal damage and prevented cysteine protease activation. NSAIDs and NO-NSAIDs stimulated TNFalpha release. Exposure to TNFalpha resulted in a time- and concentration-dependent HUVEC apoptosis, an effect that was prevented by pretreating the cells with NCX-4016, NO-naproxen, NO-flurbiprofen, SNP or Z-VAD.FMK, a pan-caspase inhibitor. The activation of ICE-like cysteine proteases was required to mediate TNFalpha-induced apoptosis of HUVECs. Exogenous NO donors inhibited TNFalpha-induced cysteine protease activation. Inhibition of caspase activity was due to S-nitrosylation of ICE/CPP32-like proteases. NO-NSAIDs prevented IL-1beta release from endotoxin-stimulated macrophages. CONCLUSIONS: NO-releasing NSAIDs are a new class of non-peptide caspase inhibitors. Inhibition of ICE-like cysteine proteases prevents endothelial cell damage induced by pro-inflammatory agents and might contribute to the gastro-protective effects of NO-NSAIDs.

Doppler velocimetry of different sections of the fetal middle cerebral artery in relation to perinatal outcome.

To assess the standard curves of pulsatility index (PI) in different segments of middle cerebral artery (MCA): initial segment (MI) and subcortical segment (M2); to determine the variation of the flow velocity waveforms (FVW) of the M1 and M2 segments of MCA in presence of fetal distress and to establish the possible correlation between the two segments of MCA. 50 normal pregnancies from 25 weeks of gestation to term and 20 pregnancy with alteration of fetal growth rate were investigated with serial records of the FVW of the M1 and M2 segments of the MCA and of the umbilical artery (UA) with a colour Doppler system. Severe fetal distress was associated to cerebral-placental ratio below 1 (C/P < 1). The perinatal outcome was established on the basis: 1) abnormal intrapartum CTG, 2) emergency cesarean section, 3) Apgar score at 1st and 5th minute after birth and 4) birth eight centiles. In normal pregnancy P1 of M2 was always higher than that of M1: therefore M2/M2 resulted below 1, with a maximum peak near 32 weeks of gestation. In presence of moderate fetal distress only P1 of M2 was reduced (M1/M2 > 1). It exists a significant difference of PI in M1 and M2 segments of fetal MCA during gestation: thus MCA so it is important to identify the tract of fetal MCA when recording its FVW. Moreover we suppose that an initial "cerebral sparing" effect exists in order to protect the cortex by the initial hypoxic injury: this is shown by a M1/M2 > 1. The progression of fetal distress results in a greater haemodynamic modification, the so called "brain sparing" which is usually present when C/P < 1.

Effect of low-dose oral triphasic contraceptives on blood viscosity, coagulation and lipid metabolism.

The purpose of the study was to determine the relationship between hemorheological profile, i.e. blood viscosity, and other risk factors for cardiovascular and thrombotic diseases in women taking oral contraceptives and if blood viscosity may be considered a marker of cardiovascular risk in OC users. Plasma levels of coagulation parameters, serum lipids, blood viscosity and RBC deformability were determined in a group of 10 women taking OC vs. 10 controls. The blood parameters were evaluated before OC use and thereafter at 3 and 6 months. A significant change in the partial thromboplastin time, fibrinogen, HDL and apolipoprotein A-I was observed, while the other parameters remained unchanged. Plasma viscosity was significantly increased during OC treatment; whole blood viscosity and RBC deformability remained unchanged. However, although some parameters were significantly modified during OC treatment, all alterations remained within the normal range of laboratory values. The data confirm that low-dose triphasic OC therapy does not affect significantly the coagulation system, serum lipid metabolism and blood viscosity. Plasma viscosity measurement may be considered as a marker for monitoring women using OC because it is apparently the most sensitive parameter.

PIP: Previous studies have documented an association between blood viscosity and risk factors for cardiovascular and thromboembolic disease. The present study assessed the impact of use of a low-dose, triphasic oral contraceptive (OC) containing ethinyl estradiol in combination with gestodene on the coagulation system, serum lipid metabolism, and blood viscosity. Enrolled were 10 OC users with no history of OC use before the study and 10 non-users. At 3 and 6 months after initiation of OC use, significant changes were recorded in partial thromboplastin time, fibrinogen, high density lipoprotein, and apolipoprotein A-1. Plasma viscosity was significantly increased during OC use, while whole blood viscosity and erythrocyte deformability remained unchanged. All alterations associated with OC treatment remained within the normal range of laboratory values, however. Thus, these findings suggest an absence of any significant OC effects on the hemostatic balance or lipid metabolism that might represent a risk factor for cardiovascular disease. Moreover, the measurement of blood viscosity may be a promising marker for monitoring thrombotic risk in women taking OCs given its apparent high sensitivity.

Management of postmenopausal osteoporosis.

More than 25% of postmenopausal women are at risk of osteoporosis. In order to avoid its consequences, it is necessary to find an appropriate prevention and/or treatment. We studied: (1) 15 postmenopausal women treated with percutaneous estradiol (50 micrograms/24 h) plus MPA (10 mg/10 days/month); (2) 15 postmenopausal women treated with synthetic calcitonin nasal spray at the daily dose of 100 IU; (3) 10 postmenopausal women treated with nandrolone decanoate (50 mg every 3 weeks); (4) 10 postmenopausal women treated with ipriflavone (600 mg/day); and (5) 10 postmenopausal women treated with sodium fluoride (20 mg) plus calcium (600 mg). Clinical examination, bone mass measurement (total BMD), hematochemical and urinary parameters of bone metabolism (calcium, urinary hydroxyproline, PTH) and growth factors (as IGF-I and TNF-beta) were evaluated. After 6 months of therapy, a complete prevention of bone resorption was achieved. In agreement with current literature, we observed that the various therapeutic approaches have all some positive effect on BMD, with different results on pain, blood biochemical parameters and growth factors' concentrations.

Doppler studies of uterine arteries in spontaneous and artificially induced menopausal women.

In the postmenopausal period, women's genital organs undergo several changes and female hormones, as well as modification of vascular reactivity to various stimuli, play a major role in this process. We studied the modification of uterine vascularity consequent to spontaneous or artificially induced menopause, with gonadotropin releasing hormone (GnRH) analogs. Flow velocity waveforms of the uterine arteries were detected using a color Doppler imaging system with a transvaginal probe; the pulsatility index was calculated. The study group consisted of 15 women observed before and after 6 months of treatment with GnRH analogs (triptorelin 3.75 mg depot every 4 weeks intramuscularly) for uterine fibromyomata; 20 women after 5 years of spontaneous menopause; and 20 healthy and normally menstruating women in the late luteal phase. We found a significant difference among the three groups with a progressive increase of the pulsatility index in spontaneous menopausal women. This phenomenon may be due to a different hormonal pattern which exists in spontaneous and artificially induced menopause (increase of gonadotropin level in the former and decrease in the latter), and to a decrease of vascular compliance caused by progressive sclerosis of the vasal walls.

Platelet thromboxane A2 receptor number and function in normal and hypertensive pregnancy.

PROBLEM: Our study evaluated the number and function of platelets for thromboxane A2/prostaglandin H2 (TxA2/PGH2) and the platelet response to TxA2 receptor agonists in normal and hypertensive pregnancy. In addition, correlations between platelet membrane lipid composition and TxA2 receptor number and function were evaluated. METHODS: Ten normotensive healthy pregnant women (GC), 10 hypertensive pregnant women (PIH), and 10 normotensive nonpregnant healthy women (C) were examined. Radioligand binding and aggregation studies were performed. Lipid composition of platelet membrane was assessed as cholesterol to phospholipids ratio (CH/PL) and normalized to protein content. RESULTS: TxA2/PGH2 receptor binding sites and affinity did not differ among the groups. An enhanced response to TxA2 receptor agonist U46619 in GC and in PIH compared to C was observed, while platelet response to thrombin was not different among the groups. CH/PL was altered in PIH respect to GC and C. No significant correlation was found between CH/PL and number and function of platelet TxA2 receptors in PIH. CONCLUSIONS: In PIH there is no alteration of number or function of TxA2 receptors on platelet membrane. However, the enhanced TxA2 production associated with the increased sensitivity of platelet to TxA2 in pregnancy may contribute to the microcirculatory thrombotic changes of PIH.

Evidence for an altered dopamine beta-hydroxylase activity in migraine and tension-type headache.

Sympathetic dysfunction is often present in migraine. It has been suggested that serum dopamine-beta-hydroxylase (D beta H) can be taken as an index of peripheral sympathetic activity. We studied the serum D beta H activity in migraine with and without aura and in tension-type headache patients compared with healthy control subjects. The serum D beta H activity was significantly lower in migraine and tension-type headache patients than in the control group. No significant difference was observed among the three groups of patients studied. These findings suggest that patients with migraine and tension-type headache have a sympathetic hypofunction that may play an important role in the pathogenesis.

Erythrocyte membrane composition in pregnancy-induced hypertension: evidence for an altered lipid profile.

OBJECTIVE: To investigate whether the increased membrane fluidity postulated as a possible contributing factor to the hypertensive states of pregnancy is related to the lipid composition of the erythrocyte membrane. DESIGN: An observational case control study. SUBJECTS: 30 women with pregnancy induced hypertension, 26 normotensive pregnant women matched for gestational age, and 10 normotensive non pregnant nulliparous women. INTERVENTIONS: Erythrocyte membranes were prepared from venous blood samples obtained from all the women. MAIN OUTCOMES MEASURES: Lipid analysis, including cholesterol to phospholipids ratio, distribution of phospholipid classes and fatty acid composition of total phospholipids in erythrocyte ghosts. RESULTS: The cholesterol/phospholipid ratio was significantly higher in the women with pregnancy induced hypertension compared with the normotensive pregnant women (mean 1.24, SD 0.31, 95% CI 1.12 to 1.35 vs mean 0.90, SD 0.09, 95% CI 0.86 to 0.94; P less than 0.01). Normotensive non-pregnant erythrocyte membrane cholesterol/phospholipid ratio was 0.88 (SD 0.11, 95% CI 0.79 to 0.96). The percentage distribution of different phospholipid classes and fatty acid composition was similar in all the four groups. CONCLUSIONS: The increased cholesterol/phospholipid ratio of the erythrocyte membrane found in pregnancy-induced hypertension represents one factor involved in the pathophysiology of this condition and a possible biochemical marker of the disease.

Serum and salivary magnesium levels in migraine. Results in a group of juvenile patients.

In the last few years a fundamental role for magnesium in establishing the threshold for migraine attacks and involvement in the pathophysiologic mechanisms related to its onset has become evident. We measured serum and salivary magnesium levels in juvenile migraine patients (with and without aura) and in a group of healthy young individuals by atomic absorption spectrophotometry. Migraineurs were studied in migraine-free (interictal) periods and during attacks. In comparison with normal subjects, migraine patients had lower levels of serum and salivary magnesium interictally. Serum magnesium levels tended to be further reduced during attacks. With respect to the values of interictal periods we observed a reduction, not statistically significant, of salivary magnesium levels for both migraine groups. Serum, and to a lesser extent salivary magnesium level reduction, could be an expression, at the peripheral level, of reduced cerebral magnesium levels which would contribute, at least in part, to defining the threshold for migraine attacks.

Serum and salivary magnesium levels in migraine and tension-type headache. Results in a group of adult patients.

It has been suggested that magnesium plays a central role in different etiopathogenetic conditions involved in the onset of migraine. We measured, by atomic absorption spectrophotometry, serum and salivary magnesium levels in drug-free migraine patients with and without aura and in tension-type headache patients. Migraine sufferers with and without aura and tension-type headache had significantly lower levels of serum and salivary magnesium concentrations in the interical periods than a group of healthy young individuals. Serum magnesium levels tended to be further reduced during attacks in all patient groups studied. A statistically significant decrease in salivary magnesium levels was evident only for migraine patients with aura. Serum magnesium levels and to a lesser extent salivary magnesium levels might express indirectly the lowering of brain extracellular magnesium concentration which occurs in migraine patients both in the intererictal periods and ictally.

Abnormal platelet lipid membrane composition in pregnancy induced hypertension.

The cholesterol:phospholipid ratio (C/PL) was measured in platelet plasma membrane in patients with pregnancy-induced hypertension [with proteinuria (PE), and without proteinuria (PIH)] and in matched normotensive gestational controls (NT). The C/PL was raised in the platelet membrane from PE (1.52 +/- 0.50, 95% CI 1.13 to 1.90) and PIH (1.38 +/- 0.34, 95% CI 1.08 to 1.67) compared with that from NT controls (0.88 +/- 0.13, 95% CI 0.80 to 0.95) (p less than 0.01, ANOVA test). No correlation was found when C/PL was regressed against total serum cholesterol levels. The abnormality of lipid composition of the platelet plasma membrane could account for some of the changes in platelet function that have been described in PIH.

A comparative chemometric study of QSAR descriptors.

Traditional descriptors for Qsar models are compared with two alternative blocks obtained by computer chemistry tools: electronic densities and principal properties. All three sets show similar prediction abilities by PLS modelling.