Materials and Methods

Our study was designed using a case-controlled approach. In all, 24 preeclamptic patients and 30 healthy pregnant women with uncomplicated pregnancies were involved in the study. The study participants were enrolled in the First Department of Obstetrics and Gynecology at the Semmelweis University, Budapest, Hungary. All women were Caucasian and resided in the same geographic area in Hungary. The preeclamptic patients were consecutively selected, while the healthy pregnant women were matched to them on the basis of age and gestational age at blood collection. Exclusion criteria were multifetal gestation, chronic hypertension, diabetes mellitus, autoimmune disease, angiopathy, renal disorder, maternal or fetal infection, and fetal congenital anomaly. The women were fasting, none of them were in active labor, and none had rupture of membranes.

Preeclampsia was defined by increased blood pressure (≥140 mm Hg systolic or ≥90 mm Hg diastolic on ≥2 occasions at least 6 hours apart) that occurred >20 weeks of gestation in a woman with previously normal blood pressure, accompanied by proteinuria (≥0.3 g/24 h or ≥1+ on dipstick in the absence of urinary tract infection). Blood pressure returned to normal by 12 weeks postpartum in each preeclamptic study patient. Preeclampsia was regarded as severe if any of the following criteria was present: blood pressure ≥ 160 mm Hg systolic or ≥110 mm Hg diastolic, or proteinuria ≥ 5 g/24 h (or ≥3+ on dipstick). Early onset of preeclampsia was defined as onset of the disease <34 weeks of gestation (between 20-33 completed gestational weeks). Intrauterine growth restriction (IUGR) was diagnosed if the fetal birthweight was <10th percentile for gestational age and sex, based on Hungarian birthweight percentiles.

The study protocol was approved by the Regional and Institutional Committee of Science and Research Ethics of the Semmelweis University (institutional review board no. 188/2008), and written informed consent was obtained from each patient. The study was conducted in accordance with the Declaration of Helsinki.

Blood samples were obtained from an antecubital vein into heparinized tubes. Peripheral blood mononuclear cells (PBMC) were isolated by the standard Ficoll-Hypaque method. The aliquots of PBMC were stored in fetal calf serum containing 10% dimethyl sulfoxide at –80°C until the analyses.

Intracellular VEGF expression of unstimulated lymphocytes was determined with flow cytometric examination. After thawing, isolated mononuclear cells were washed twice with phosphate-buffered saline and their viability was assessed by trypan blue exclusion (consistently >90%). Nonspecific binding sites were blocked by incubation with 10% mouse serum for 10 minutes at room temperature. Cells were stained with fluorescein isothiocyanate–labeled antihuman CD3 and phycoerythrin (PE)-cyanine 5–labeled antihuman CD56 or fluorescein isothiocyanate–conjugated antihuman CD8 and PE-cyanine 5–conjugated antihuman CD4 mouse monoclonal antibodies (BD Pharmingen, San Diego, CA) for 15 minutes at room temperature in the dark and then washed with washing buffer. Red blood cells were lysed by incubation with 1.5 mL of 1X fluorescence-activated cell sorter (FACS) Lysing Solution (BD Biosciences, San Jose, CA) for 10 minutes at room temperature in the dark. Cells were centrifuged and the supernatant was removed. A total of 500 μL of 1X FACS Permeabilizing Solution (BD Biosciences) was added and the mixture was incubated for 10 minutes at room temperature in the dark. After washing twice with washing buffer, the permeabilized cells were stained with PE-labeled mouse antihuman VEGF monoclonal antibody (R&D Systems, Minneapolis, MN) for 30 minutes at room temperature in the dark. PE-conjugated, isotype-matched mouse immunoglobulin G _2A (BD Pharmingen) was used as a control for detecting nonspecific binding. After washing twice with washing buffer, the cells were resuspended in 1% paraformaldehyde in phosphate-buffered saline. Flow cytometric analysis was performed on a FACSCalibur flow cytometer and data were processed using CellQuest Pro software (BD Biosciences). A total of 50,000 cells were counted. A real-time gate was set around the viable lymphocytes based on their forward-/side-scatter profile. Contaminating monocytes and necrotic cells were excluded from the analysis.

The normality of continuous variables was assessed using the Shapiro-Wilk W test. As the continuous variables were not normally distributed, nonparametric statistical methods were applied. To compare continuous variables between 2 groups, the Mann-Whitney U test was used. The Fisher’s exact and Pearson χ ² tests were performed to compare categorical variables between groups. The Spearman rank order correlation was applied to calculate correlation coefficients. As the proportions of VEGF-positive cells showed skewed distributions, we performed analysis of covariance with logarithmically transformed data.

Statistical analyses were carried out using the following software: Statistica (version 8.0; StatSoft Inc, Tulsa, OK) and Statistical Package for the Social Sciences (version 15.0 for Windows; SPSS Inc, Chicago, IL). For all statistical analyses, P < .05 was considered statistically significant.

In the article, data are reported as median (25-75 percentile) for continuous variables and as number (percent) for categorical variables, if not otherwise specified.