Angiogenic Effect of Pravastatin Alone and with Sera from Healthy and Complicated Pregnancies Studied by in vitro Vasculogenesis/Angiogenesis Assay.

OBJECTIVE: To determine the direct effect of pravastatin on angiogenesis and to study the interaction between pravastatin and maternal sera from women with early- or late-onset pre-eclampsia (PE), intrauterine growth restriction, or healthy pregnancy. METHODS: We collected 5 maternal serum samples from each group. The effect of pravastatin on angiogenesis was assessed with and without maternal sera by quantifying tubule formation in a human-based in vitro assay. Pravastatin was added at 20, 1,000, and 8,000 ng/mL concentrations. Concentrations of angiogenic and inflammatory biomarkers in serum and in test medium after supplementation of serum alone and with pravastatin (1,000 ng/mL) were measured. RESULTS: Therapeutic concentration of pravastatin (20 ng/mL) did not have significant direct effect on angiogenesis, but the highest concentrations inhibited angiogenesis. Pravastatin did not change the levels of biomarkers in the test media. There were no changes in angiogenesis when therapeutic dose of pravastatin was added with maternal sera, but there was a trend to wide individual variation towards enhanced angiogenesis, particularly in the early-onset PE group. CONCLUSIONS: At therapeutic concentration, pravastatin alone or with maternal sera has no significant effect on angiogenesis, but at high concentrations the effect seems to be anti-angiogenic estimated by in vitro assay.

Angiogenic capacity in pre-eclampsia and uncomplicated pregnancy estimated by assay of angiogenic proteins and an in vitro vasculogenesis/angiogenesis test.

OBJECTIVE: The purpose of the study was to determine the angiogenic capacity of sera in early and late pregnancy and in umbilical blood serum after childbirth, and to define how angiogenic properties assessed in a functional in vitro test are related to individual angiogenic proteins in six women with pre-eclampsia and in six healthy pregnant controls. METHODS: Maternal first and third trimester serum samples, and umbilical blood samples after childbirth, were tested in an in vitro human adipose stromal cell-human umbilical vein endothelial cell (hASC-HUVEC) vasculogenesis/angiogenesis assay. The angiogenic properties of the samples were measured by quantifying tubule formation. Concentrations of total placental growth factor (PlGF), total vascular endothelial growth factor (VEGF), soluble fms-like tyrosine kinase 1 (sFlt-1) and soluble endoglin (sEng) were determined by immunoassay. RESULTS: First-trimester maternal sera of both groups had a stimulatory effect on angiogenesis in vitro and levels of angiogenic proteins did not differ between the groups. Third-trimester maternal sera in the pre-eclampsia group had an inhibitory effect on tubule formation, while those from normal pregnancies remained stimulatory. Compared with the first trimester there was a significant change in the concentrations of angiogenic proteins toward an anti-angiogenic state in pre-eclampsia. Umbilical blood serum exhibited strong anti-angiogenic effects without a significant difference between groups. CONCLUSIONS: Third-trimester serum of pre-eclamptic patients is anti-angiogenic. This phenomenon is not yet present in the first trimester. Umbilical blood serum shows inhibitory effects on angiogenesis after normal as well as pre-eclamptic pregnancy.

Novel Biomarkers: Soluble Urokinase-Type Plasminogen Activator Receptor and Procalcitonin- and Histological Chorioamnionitis after Preterm Premature Rupture of Membranes.

Fetal inflammatory response syndrome or infection after preterm premature rupture of membranes (PPROM) increases neonatal morbidity in preterm deliveries. Biochemical markers from the amniotic fluid (AF) have been used to evaluate possible intra-amniotic infection during the asymptomatic phase after PPROM. This study aimed to describe whether soluble urokinase-type plasminogen activator receptor (suPAR) or procalcitonin (PCT) from AF or maternal sera could reveal fetal inflammatory response or infection after PPROM. AF and maternal serum samples were collected weekly after PPROM (23+ 0 - 34+ 6 gestational weeks) until delivery from twenty women and two women with possible chorioamnionitis with intact membranes. Levels of suPAR, PCT, interleukin-6 (IL-6), glucose, lactate dehydrogenase (LDH), and bacterial PCR were determined from AF and suPAR and PCT and IL-6 from maternal sera. Fetal infection or inflammation response were determined by the histology of the placenta after delivery. AF glucose was significantly lower and AF LDH higher in the fetal site histologic chorioamnionitis (HCA) group, while AF suPAR concentrations tended to be higher in this group. AF suPAR correlated significantly with AF glucose and LDH. Based on receiver operating characteristic (ROC) analysis, AF glucose had the best predictability for fetal site histological chorioamnionitis. The findings of AF PCT were insignificant considering HCA. AF glucose had the highest accuracy in predicting fetal site histologic chorioamnionitis. AF suPAR may be a promising marker; however, our findings were limited by a small study population.

Therapeutic doses of metformin do not have impact on angiogenesis in presence of sera from pre-eclamptic, IUGR and healthy pregnancies.

Recent evidence suggests that metformin may prevent pre-eclampsia by reverting the angiogenic imbalance in maternal sera. In this study, we investigated effect of metformin on angiogenesis by quantifying tubule formation in a human-based in vitro test with co-culture of human adipose stromal cell (hASC) and human umbilical vein endothelial cell (HUVEC). A total of 20 pregnant women were recruited in the study. Serum samples were obtained from women with early- and late-onset pre-eclampsia and from women with pregnancies complicated by intrauterine growth restriction (IUGR) without pre-eclampsia (N = 5 in each of the three groups). Serum samples from women with healthy pregnancies served as controls (N = 5). The direct effect of metformin on angiogenesis was first assessed without maternal sera. Secondly, we investigated the impact of metformin on angiogenesis in the present of maternal sera. Metformin was used at 5, 50 and 600 µg/ml concentrations. Angiogenic and inflammatory biomarkers in maternal sera were analyzed by immunoassays. When the direct effect of metformin was studied, the two lowest concentrations of metformin did not affect tubule formation (angiogenesis), but the highest concentration inhibited angiogenesis. When metformin was supplemented at therapeutic concentrations of 5 and 50 µg/ml along with serum samples, there was no change in tubule formation in comparison to maternal sera alone. However, strong inhibitory effect on tubule formation was observed in all groups with the highest, non-therapeutic (600 µg/ml), concentration of metformin.

Strong inhibitory effect of pre-eclampsia serum on angiogenesis detected in vitro by human cell-based angiogenesis tests.

OBJECTIVE: To explore in vitro angiogenic properties of maternal and umbilical cord blood sera from women with symptomatic pre-eclampsia in comparison with sera from women with normotensive pregnancies. STUDY DESIGN: Maternal and umbilical blood serum samples were collected from eleven primiparous women with pre-eclampsia and ten healthy gestational-age-matched primiparous controls. The samples were tested for tubule formation in two different types of in vitro angiogenesis tests. The first test (fibroblast-HUVEC) showed effects on angiogenesis and the second test (hASC-HUVEC), in addition to angiogenesis, also showed effects on vasculogenesis. The pro-angiogenic and inhibitory properties of the samples were microscopically quantified after immunostaining tubular structures, using markers for von Willebrand factor (vWf) and collagen IV. RESULTS: Serum samples from pre-eclamptic women inhibited tubule formation in both models, while those from normal pregnancy didn't. Umbilical blood samples were inhibitory both after pre-eclampsia and normal pregnancy. In the fibroblast-HUVEC model the inhibition was stronger after preeclampsia pregnancy, and the difference between groups was statistically significant. In the pre-eclampsia group a correlation between the inhibitory effect of umbilical blood and birth weight adjusted to gestational age was found. No clear correlation between sera from pregnant women and corresponding umbilical sera was found. CONCLUSION: The strong inhibitory effect of maternal serum samples on tubule formation reflects the anti-angiogenic state that is present in pre-eclampsia.

Decreased free water clearance is associated with worse respiratory outcomes in premature infants.

OBJECTIVE: The goal was to elucidate predictors of decreased free water clearance (DFWC) in very low birth weight (VLBW) infants. We hypothesized that DFWC and fluid retention are linked to the severity of pulmonary problems and prolonged respiratory support, especially to nCPAP treatment. METHODS: The investigation was carried out at Tampere University Hospital between 2001 and 2006. The study population comprised 74 VLBW infants born at 29.21 (24.57-34.14) weeks of gestation. Median birth weight was 1175 (575-1490) grams. We measured plasma and urine osmolality and 24-hour urine volume to calculate free water clearance (FWC) for each infant. If FWC was less than 30 ml/kg/day the infant was classified as having DFWC. RESULTS: There were 38 (51.4%) infants with DFWC in the study population. The median duration of the observed DFT period was 14 (4-44) days. The gestational age at birth was lower for DFWC infants compared to infants with normal FWC (NFWC), 28.29 (24.57-32.86) vs. 30.00 (25.57-34.14) weeks (p = 0.001). DFWC infants also needed longer ventilator treatment, 2 (0-23) vs. 0.50 (0-23) days (p = 0.046), nCPAP treatment 30 (0-100) vs. 3 (0-41) days (p<0.0001) and longer oxygen supplementation 47 (0-163) vs. 22 (0-74) days (p = 0.011) than NFWC infants. All values presented here are medians with ranges. CONCLUSIONS: DFWC appears to be frequently connected with exacerbation and prolongation of pulmonary problems in VLBW infants. Cautious fluid administration seems to be indicated in VLBW infants with prolonged respiratory problems and DFWC.

Predictors of delayed first voiding in newborn.

AIM: Delay>24 h of age in neonates' first voiding attracts attention, although the phenomenon is usually benign. Earlier studies indicate that stress increases the infant's arginine vasopressin (AVP) and aldosterone secretion during birth. Our aim was to seek predictors of delayed first voiding and indirect evidence of AVP effect behind this phenomenon. METHODS: The study population comprised 20 normal-term newborns whose first voiding was delayed>24 h of age (cases), and 19 age-matched control infants who voided for the first time at <24 h of age (controls). The first urine was collected and osmolality (U-Osm) and sodium content (U-Na) measured. RESULTS: The median of U-osm in cases was 432.50 (284-519) and in controls 337.50 (169-497) mOsm/L (p=0.005), and U-Na 21.50 (9-241) and 40.00 (13-226) mmol/L (p=0.001), respectively. Cases were more frequently born to primiparous mothers than controls (70% vs. 21%, p=0.004). Duration of labour was longer in cases than controls, first stage 10.5 h (3.92-20.50 h) versus 5.7 h (1.17-16.00 h) (p=0.045) and second stage 0.42 h (0.08-1.25 h) versus 0.17 h (0.08-0.92 h) (p=0.015). All seven (35%) abnormal cardiotocographies were recorded with cases (p=0.008). CONCLUSIONS: Delayed voiding appears to be related to a prolonged and stressful birth. Laboratory findings in the first urine suggest increased AVP and aldosterone secretion in such cases.

Predictors of AVP and TSH levels and the timing of first voiding in the newborn.

To evaluate obstetric predictors of umbilical cord plasma AVP levels, serum TSH levels and the timing of first voiding, 87 singleton term newborns were divided into three groups: group A, vaginal delivery (n = 30); group B, cesarean section (CS) during labor (n = 26); and group C, elective CS (n = 31). The AVP concentration was 120 (0.7-2170) ng/L in group A, 1.8 (0.01-183) ng/L in group B, and 0.8 (0.01-30) ng/L in group C (p < 0.001). In group A, the TSH concentration was 10.20 (3.5-30.80) mU/L; in group B, 5.40 (2.10-43.00) mU/L; and in group C, 5.30 (2.90-11.00) mU/L (p = 0.001). Duration of labor had a positive correlation with AVP (p < 0.001) and TSH (p = 0.001) concentrations. The timing of first voiding had a positive correlation with gestational age (p = 0.003), volume of additional feeding before first voiding (p < 0.001), and umbilical AVP concentration (p = 0.023). The AVP and TSH concentrations are associated with mode of delivery and duration of labor and AVP levels also with the timing of first voiding in the newborn.