PMC:2952583 / 4271-12964 JSONTXT

{"project":"2_test","denotations":[{"id":"20948996-9494145-87413003","span":{"begin":250,"end":251},"obj":"9494145"},{"id":"20948996-9494145-87413004","span":{"begin":2659,"end":2660},"obj":"9494145"},{"id":"20948996-9494145-87413005","span":{"begin":5194,"end":5195},"obj":"9494145"},{"id":"T54605","span":{"begin":250,"end":251},"obj":"9494145"},{"id":"T46726","span":{"begin":2659,"end":2660},"obj":"9494145"},{"id":"T11032","span":{"begin":5194,"end":5195},"obj":"9494145"}],"text":"Methods\n\nStudy Population\nWe performed a secondary analysis of serum samples obtained during the Maternal-Fetal Medicine Units Network multicenter randomized controlled trial of low-dose aspirin for the prevention of preeclampsia in high-risk women [4]. This study, conducted between May 1991 and June 1995, investigated four groups of high-risk women: those with pre-gestational insulin treated diabetes mellitus, chronic hypertension, multifetal gestation, and those with a history of preeclampsia in a previous pregnancy. At enrollment, subjects were offered participation in an ancillary study that collected blood samples at three time points (study entry, 24–28 and 34–38 weeks' gestation). The ancillary study started after the original trial was in progress and only a subset of trial subjects participated. Subjects were included if they had at least a study entry sample available for analysis.\nThe diagnosis of chronic hypertension required documentation of antihypertensive-drug therapy by medical records or a blood pressure while sitting of greater than or equal to 140/90 mmHg taken on two occasions at least four hours apart, either before pregnancy or during pregnancy, but prior to study entry and before 20 weeks' gestation. Multifetal gestation was documented by ultrasound examination before enrollment. Previous preeclampsia was defined as new-onset proteinuric hypertension as determined by medical records or, in the absence of a record, an oral history of preeclampsia that resulted in delivery before the 37th gestational week. At the screening visit, all women underwent urinary-protein testing by dipstick. If the test was 1+ or greater, a 24-hour urine sample was collected; women with values of 300mg of protein per 24 hours were considered to have proteinuria. Women with multi-fetal gestations were ineligible for the study if they also had diabetes mellitus, chronic hypertension, or proteinuria as defined above, as were women with a history of preeclampsia and current proteinuria. Women with both diabetes and hypertension were included in the diabetes group.\nInstitutional Review Board approval for the use of these stored samples was obtained at both the University of Pittsburgh and George Washington University. Subjects provided written informed consent prior to enrolling in the initial study. Laboratory assessment was completed at Magee-Womens Research Institute, and data was subsequently linked and statistical assessment completed at the George Washington University Biostatistics Center.\n\nDiagnosis of Preeclampsia\nThe diagnosis of preeclampsia for each of these high-risk groups has been described previously [4]. In brief, preeclampsia was defined in normotensive women with normal urinary protein excretion at baseline as the development of hypertension plus one of the following: proteinuria, thrombocytopenia, or pulmonary edema. Hypertension was defined as a systolic and/or diastolic blood pressure equal to or greater than 140 mmHg and 90 mmHg respectively, on two occasions at least four hours apart and within the same period (antepartum, intrapartum, postpartum). Proteinuria was defined as excretion of ≥ 300mg of protein in a 24-hour urine collection, or two dipstick-test results of ≥ 2+ (100 mg per deciliter), the values recorded at least 4 hours apart, with no evidence of urinary tract infection. Thrombocytopenia was defined as a platelet count of less than 100,000 per cubic millimeter. In women who had normal blood pressure but proteinuria at baseline, the diagnosis of preeclampsia required thrombocytopenia, or a serum aspartate aminotransferase concentration of ≥ 70 U per liter, or hypertension and either severe headaches, epigastric pain, or worsening proteinuria (either five times the baseline value or twice baseline if the baseline value exceeded 5 g per 24 hours). In women who had hypertension but no proteinuria at baseline, a diagnosis of preeclampsia required the development of proteinuria or thrombocytopenia. A woman was deemed to have preeclampsia if she had an eclamptic convulsion or the HELLP syndrome, defined as hemolysis (serum total bilirubin concentration of ≥1.2 mg per deciliter (20 mmol per liter), a serum lactate dehydrogenase concentration of ≥ 600 U per liter, or hemolytic anemia as determined by the presence of schistocytes on a peripheral smear), elevated serum concentration of aspartate aminotransferase (≥ 70 U per liter), and thrombocytopenia. The records of all the women with apparent preeclampsia, worsening hypertension, new-onset proteinuria, or proteinuria at baseline (≥ 1+) were reviewed independently by three physicians who had to agree unanimously on the validity of the designated outcomes, a policy designed to ensure diagnostic consistency.\nThe clinical onset of preeclampsia was defined as the time at which a patient met the criteria for preeclampsia as described above rather than the time at which any single symptom first occurred.\n\nLaboratory methods\nSerum samples were collected and aliquoted as part of the Maternal-Fetal Medicine Units Network multicenter randomized controlled trial of low-dose aspirin for the prevention of preeclampsia in high-risk women [4]. Samples were collected between June 1992 and December 1995, aliquoted, frozen and stored at −80°C. Samples were thawed one time (74%) or thawed two times (26%), and the storage time and handling of samples was similar to that of other similar studies including the CPEP cohort. Serum samples were assayed concurrently for sFlt1, sEng and PlGF using commercially available immunoassay kits purchased from R\u0026D Systems (Minneapolis, MN). All kits utilized were from the same manufacturing lot for each specific analyte, a policy designed to minimize variability. ELISAs were validated by performing dilutional parallelism and spike-recovery tests. Correlations between the degree of sample dilution and measured analytes were linear (r2 \u003e0.99, all). Calculated recoveries of excess analytes were \u003e94% for all. Measurements of each angiogenic factor were performed in duplicate according to the manufacturer's protocol. All laboratory analyses were performed by personnel unaware of either pregnancy outcome or the high-risk group from which the sera were obtained. In general, samples were diluted 1 to 5 for sFlt1 and sEng, and 1 to 2 for PlGF in order for the samples to fall within the measurable range of the kit's standard curves. A minimal number of samples required re-analysis because the results were outside of the range of the standard curve. Of the total number of serum samples, 135 (5%) required re-assay for sFlt1, 29 (0.9%) for sEng, and 33 (1.2%) for PlGF. The inter-assay variability for each analyte was 10% for sFlt1, 11% for sEng and 7% for PlGF.\n\nStatistical Methods\nData from all high-risk groups were analyzed separately. Continuous variables were compared using the Wilcoxon rank-sum test and categorical variables using the chi-square test. Change in the concentration of a factor was calculated by taking the difference of the value at study entry and the value obtained between 24 and 28 weeks and dividing by the number of weeks between the two sample collections. Logistic regression was used to calculate odds ratios and the multivariable analysis included gestational age at sample collection, smoking, maternal age, race or ethnic group, body mass index and study treatment group (i.e., aspirin or placebo).\nData were also analyzed cross-sectionally, according to intervals of gestational age and the number of weeks before the onset of preeclampsia. In the intervals, when more than one sample existed per woman, the earliest sample was used. All available samples were used in the gestational age analysis while a matched (1∶1) pair analysis was performed to evaluate differences in relation to the number of weeks before the onset of preeclampsia. Specifically, in the paired comparisons all available samples from subjects who developed preeclampsia were distributed into time blocks according to when the sample was collected in relation to the clinical onset of preeclampsia for each subject (onset of clinically recognized preeclampsia is time 0). Then a separate sample from a control subject from the same high-risk group was matched within one week of the same gestational age in weeks and by treatment group to the sample from the preeclampsia subject. Data were log transformed and the difference was evaluated using the Wilcoxon Signed Rank test.\nAll P values are two-tailed and a value less than 0.05 was considered statistically significant. No adjustments were made for multiple comparisons. Analyses were performed with the use of the SAS software."}