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Serum Levels of Angiogenic Factors Distinguish Between Women with Preeclampsia and Normotensive Pregnant Women But Not Severity of Preeclampsia in an Obstetric Center in Turkey

Background This study aimed to compare serum levels of vascular endothelial growth factor (VEGF) and the VEGF receptors, VEGFR-1 and VEGFR-2, free placental growth factor (fPGF), endostatin, and serum pregnancy-associated plasma protein-A (PAPP-A) levels in women with mild and severe preeclampsia and healthy pregnant women. Material/Methods A included patients diagnosed with mild preeclampsia (n=32), severe preeclampsia (n=32), and healthy pregnant women (n=24). Serum levels of VEGF-A, VEGFR-1, VEGFR-2, fPGF, endostatin, and PAPP-A levels were measured by enzyme-linked immunosorbent assay (ELISA). Results In women with mild and severe preeclampsia, the gestation age at birth and birth weight were found to be significantly lower than the control group (p<0.001). Serum levels of endostatin, VEGFR-1, and VEGF-A levels were significantly increased in pregnant women with preeclampsia compared with healthy pregnant women (p<0.001). Serum levels of PAPP-A, VEGFR-2, and fPGF were significantly higher in healthy pregnant women when compared with women with preeclampsia (p=0.024, p<0.001, and p<0.001, respectively), but there were no significant differences between women with mild and severe preeclampsia. Conclusions Reduced serum levels of the angiogenic factors PAPP-A, VEGFR-2, and fPGF distinguished between women with preeclampsia and normotensive pregnant women but did not significantly distinguish between mild and severe preeclampsia.


Background
Worldwide, preeclampsia affects between 2-8% of all women in pregnancy and is the main cause of maternal and perinatal mortality and morbidity [1,2]. Preeclampsia accounts for 17-24% of all maternal deaths in developing countries [3,4]. Preeclampsia is a complex clinical condition that is currently believed to have to main causes, inadequate trophoblast invasion and placentation and endothelial damage with impaired angiogenesis [1,2].
In the pathogenesis of preeclampsia, disrupted angiogenic balance and endothelial damage are considered to be associated with the development of clinical symptoms and signs of preeclampsia [3,4]. Vascular endothelial growth factor (VEGF), vascular endothelial growth factor receptor (VEGFR), and placental growth factor (PGF) have recently been studied in abnormal angiogenesis and angiogenic balance in preeclampsia [5,6]. The mean plasma and serum levels of soluble VEGFR-1 and soluble fms-like tyrosine kinase-1 (sFlt-1), which are anti-angiogenic in the placenta of patients with preeclampsia have previously been shown to be higher than in healthy pregnant women. Increased sFlt-1 in preeclampsia binds to VEGF and PGF molecules in the circulation and antagonizes their effects [7,8].
Therefore, this study aimed to compare the serum levels of vascular endothelial growth factor (VEGF) and the VEGF receptors, VEGFR-1 and VEGFR-2, free placental growth factor (fPGF), endostatin, and serum pregnancy-associated plasma protein-A (PAPP-A) levels in women with mild and severe preeclampsia and healthy pregnant women.

Material and Methods
Patients A randomized case-control study included all patients who were diagnosed with mild and severe preeclampsia in the Adnan Menderes University Clinic of Obstetrics and Gynecology and who filled the informed consent forms. A total of 88 patients were included in the study. Thirty-two women were diagnosed with mild preeclampsia, 32 women were diagnosed with severe preeclampsia. Also, 24 healthy pregnant women were included with similar age, and body mass index (BMI). The study protocol was approved by the Ethics Committee of Adnan Menderes University (2017/1203). This study was performed in accordance with the Helsinki Declaration and good clinical practice.

Diagnosis of preeclampsia
The diagnosis of preeclampsia was based on the blood pressure of ³140\90 mmHg and 300 mg/L of protein (at least +1 on dipstick) in spot urine testing found with two measurements made at least at 6-hourly intervals [9]. The criteria for severe preeclampsia were a blood pressure of ³160\110 mmHg or oliguria (400 ml/24 hr), impairment of renal function tests, visual impairment, symptoms of cerebral dysfunction, convulsion, headache, epigastric pain, nausea, vomiting, elevation in serum creatinine, thrombocytopenia (<100.000×10 3 /mm 3 ), abnormal peripheral blood smear findings, impaired liver function, pulmonary edema, and cyanosis.

Inclusion and exclusion criteria
All pregnant women with a singleton pregnancy, who were over 18 years of age, >20 weeks' gestation, and who signed an informed consent form were included in the study. Women with multiple pregnancies, with known systemic diseases, pregnant women with hypertension before pregnancy, or with diabetes, and fetal abnormalities, and with cardiac and renal diseases, and urinary tract infections, were excluded from the study.

Obstetric findings
The demographic information (age, parity, height, and weight) and routine laboratory tests of all pregnant women were recorded. The uterine artery resistance at birth, mode of delivery, birth weights, and 1 st -minute and 5 th -minute APGAR scores of all newborns were recorded. The details of the newborns who were admitted to the newborn intensive care unit (NICU) were recorded. The verbal and written consents of all pregnant women who agreed to participate in the study were recorded. Local Ethics Committee approval was obtained for this study.
Sampling of maternal blood and enzyme-linked immunosorbent assay (ELISA) of serum samples Blood samples were taken from all patients during labor from the antecubital region of the forearm and were sampled in two sterile vacutainer tubes after the area was wiped with 70% alcohol. Serum samples were divided and placed in three Eppendorf tubes. All samples were centrifuged at 3,000×g for 20 minutes at 4°C and were stored at -85°C.

Measurement of serum levels of VEGF-A
Serum VEGF-A levels were measured using a commercial human ELISA kit (Elabscience Biotechnology Co., Ltd., Wuhan, China) (Cat. No. E-EL-H0111). The test results were determined with an ELx800 absorbance microplate reader (Biokit, Barcelona, Spain) using a standard curve. The kit detection range was 031.25-2000 pg/ml. The kit performance characteristics were: sensitivity, 18.75 pg/ml, and coefficient of variation (CV%), <10.

Measurement of serum levels of VEGFR1/FLT1
Serum levels of VEGFR1/FLT1 were determined using commercial human ELISA kit (Elabscience Biotechnology Co., Ltd Building 4, Room 401, Guandong Science and Technology Industry Park, Wuhan, China) (Catalogue No. E-EL-H1087). The test results were determined with an ELx800 absorbance microplate reader (Biokit, Barcelona, Spain) using a standard curve. The kit detection range was125-8000 pg/ml. The kit performance characteristics were sensitivity, 75 pg/ml and coefficient of variation (CV%), <10.
Measurement of serum levels of vascular endothelial growth factor receptor 2 (VEGFR2) or KDR Serum VEGFR2/KDR levels were determined using a commercial human ELISA kit (Elabscience Biotechnology Co., Ltd, Wuhan, China) (Catalogue No: E-EL-H1603). The test results were calculated with an ELx800 absorbance microplate reader (Biokit, Barcelona, Spain) using a standard curve. The kit detection range was 78.13-5000 pg/ml. The kit performance characteristics were sensitivity, 46.88 pg/ml and coefficient of variation (CV%), <10.

Measurement of serum levels of pregnancy-associated plasma protein-A (PAPP-A)
Serum PAPP-A levels determined via commercial human ELISA kit (Elabscience Biotechnology Co., Ltd., Wuhan, China) (Catalogue No: E-EL-H0145). The test results were calculated with an ELx800 absorbance microplate reader (Biokit, Barcelona, Spain) using a standard curve. The kit detection range was 0.78-50 ng/ml. The kit performance characteristics were sensitivity, 0.47 ng/ml, and the coefficient of variation (CV%), <10.

Measurement of human endostatin
In serum samples, human endostatin levels were determined using a commercial human ELISA kit (Elabscience Biotechnology Co., Ltd., Wuhan, China) (Catalogue No: E-EL-H0063). The test results were calculated with an ELx800 absorbance microplate reader (Biokit, Barcelona, Spain) using a standard curve. The kit detection range was 0.16-10 ng/ml. The kit performance characteristics were sensitivity, 0.1ng/ml, and the coefficient of variation (CV%), <10.

Statistical analysis
SPSS version 25.0 (IBM Corporation, Armonk, NY, USA) was used to analyze the data and variables. The conformity of the data with a normal distribution was evaluated with the Shapiro-Wilk test, and the variance homogeneity with the Levene test. One-way analysis of variance (ANOVA) was determined using the Jonckheere-Terpstra Test. The Kruskal-Wallis test was used to compare the groups according to the quantitative data and Dunn's test. Tukey's test was used for the post hoc analysis. One-way ANOVA was used to compare multiple independent groups according to quantitative data, and the Games-Howell test was used for the post hoc analysis. The Wilcoxon signed rank test was used with the Monte Carlo simulation method for the comparison of the two repetitive measurements of the dependant quantitative variables. The Pearson-Fisher-Freeman-Holton test was used with the Monte Carlo simulation method in the comparison of the categorical variables, and the column rates were compared and expressed according to the Benjamini-Hochberg corrected p-value results. The quantitative variables were shown as the mean ± standard deviation (SD) and median (minimum/maximum) and the categorical variables as n (%) in the tables. The variables were analyzed at 95% confidence interval (CI), and the p-value <0.05 was accepted as significant.

Demographic findings
There were no significant differences between the pregnant women with preeclampsia and without preeclampsia in terms of age, body mass index (BMI), parity, aborted and live birth numbers (p>0.05). In pregnant women diagnosed with preeclampsia, the arterial blood pressures (systolic and diastolic), umbilical artery pulsatility index (UAPI), and protein and creatinine levels in spot urine testing were significantly higher than in healthy pregnant women (p<0.001, p=0.037, and p=0.010, respectively) ( Table 1). The gestational age and birth weights were significantly lower in pregnant women diagnosed with preeclampsia (mild and severe) compared with the control group (p<0.001) ( Table 1).

Obstetric findings
The APGAR scores at 1 minute and 5 minutes were lower in patients diagnosed with preeclampsia compared with healthy pregnant women (p<0.001), and there was no significant difference between pregnant women diagnosed with mild and severe preeclampsia (p>0.05) ( Almost 50% of the healthy pregnant women had a cesarean section, but the cesarean section rate was significantly higher in pregnant women with severe preeclampsia (81.3%) compared with women with mild preeclampsia and healthy pregnant women (Table 2). Births with intrauterine growth restriction (IUGR) were found in half of the pregnant women diagnosed with preeclampsia (p<0.001), but there was no difference between the rates of birth with IUGR in the pregnant women diagnosed with mild and severe preeclampsia (p>0.05).

Laboratory results
There was no significant difference between pregnant women in terms of blood urea nitrogen (BUN), creatinine, hemoglobin (Hb), white blood cell (WBC) count, platelets (PLT), aspartate transaminase (AST) and alanine transaminase (ALT) results (p>0.05). The uric acid levels for pregnant women diagnosed with mild and severe preeclampsia were significantly higher than the control group (p<0.001) ( Table 3).
The levels of endostatin, vascular endothelial growth factor-A (VEGF-A), and the VEGF receptor-1) were significantly higher in pregnant women diagnosed with preeclampsia compared with healthy pregnant women (p<0.001). However, the pregnancy-associated plasma protein-A (PAPP-A), VEGFR-2, and free placental growth factor (fPGF) levels were higher in healthy pregnant women (p=0.024, p<0.001, and p<0.001, respectively). There was no significant difference between women with mild and severe preeclampsia in terms of these biomarkers (p>0.05) ( Table 3).

Discussion
In a healthy pregnancy, the placental cytotrophoblast cells invade the uterine wall and high-resistance uterine spiral arteries and arterioles form a low-resistance vascular bed [10]. This angiogenic remodeling is abnormal in women with preeclampsia, and placental ischemia develops [11]. In placental development, vascular endothelial growth factor (VEGF) and the VEGF receptors, VEGFR-1 and VEGFR-2, and free placental growth factor (fPGF), which are members of VEGF family, have been studied and are believed to be involved in angiogenic balance in placentation [12][13][14].
This study aimed to measure serum levels of factors involved in the development and regulation of the placental vasculature in maternal serum obtained at birth. Women with mild and severe preeclampsia were compared with healthy pregnant women. The findings were consistent with those reported by previous studies [15,16]. Serum levels of endostatin, VEGFR-1, and VEGF-A were significantly increased in women with preeclampsia, whereas free placental growth factor (fPGF) and serum pregnancy-associated plasma protein-A (PAPP-A) levels were significantly higher in healthy pregnant women [15,16].
VEGF binds to its receptors, VEGFR-1 (sFlt1) or VEGFR-2, whereas fPGF binds specifically to the sFlt-1 receptor [7]. As a result of placental dysfunction, a significant increase occurs in the production of sVEGFR-1 (sFlt-1), or soluble VEGFR-1. Previous studies have shown that sFlt-1 was significantly increased in the development of preeclampsia and bound to VEGF and fPGF in the circulation, reducing the free levels of these molecules in the plasma and created an anti-angiogenic effect [5,7,17].
Geva et al. reported that women with preeclampsia who gave birth to infants with intrauterine growth restriction (IUGR), had increased serum levels of VEGF-A, VEGFR-1, and sVEGFR-1 levels, but there was no change in the fPGF, VEGFR-2, and (neuropilin-1) NRP-1 levels [16]. The increase in VEGF, VEGFR-1, and sVEGFR-1 levels in pregnant women with preeclampsia were considered to be associated with placental hypoxia [16,17]. Bahlman et al. showed that the diagnostic sensitivity of the combined use of the sFlt-1/PGF ratio and uterine artery pulsatility index (UAPI) was 74% for the diagnosis of preeclampsia, and the sensitivity was 83.7%, and specificity was 68.1% when the cut-off value was taken as 5424 pg/ml for the sFlt-1 [18]. High sFlt-1 levels developed before the clinical symptoms and the sFlt-1 levels were associated with the time of onset of clinically evident preeclampsia, and partly with the severity of preeclampsia [18]. It was previously shown that sFlt-1 levels were  6928 higher in early-onset cases of preeclampsia [15]. Also, in a recent animal experimental study, in a model of hypertension associated with proteinuria, impaired renal function, thrombocytopenia, abnormal liver function and epigastric pain, with hemolysis-elevated liver enzymes-low platelet count (HELLP) syndrome was induced following VEGF-associated reduction in endothelial nitric oxide synthase (eNOS) production [19].
Soluble placental growth factor (sPGF) is a proangiogenic protein secreted from the placenta, which peaks in the 30 th gestational week and levels then reduce until the end of pregnancy [6]. Also, sPGFplays a role in angiogenesis, vasculogenesis, and embryogenesis during pregnancy [6]. Reduction in free PGF (fPGF) is found in the serum in late pregnancy due to binding of fPGF to sFlt-1 [6]. However, previous studies have shown that there was no change, or there was an increase in the fPGF levels in placental tissues in preeclampsia [15,20]. Previous studies have shown that low levels of fPGF in pregnant women diagnosed with preeclampsia were secondary to the binding of the fPGF molecules by sFlt-1 that were increased in the circulation secondary to dysfunction of the placenta [20,21]. In the present study, serum levels of fPGF were found to be higher in healthy pregnant women, which is in keeping with previously published findings.
PAPP-A is a glycoprotein secreted in the placenta, and it has been used for more than three decades as an important Max -maximum; A -significant compared with the control group; B -significant compared with the mild preeclampsia group; C -significant compared with the severe preeclampsia group; WBC -white blood cells; VEGF -vascular endothelial growth factor; sVEGFR -soluble vascular endothelial growth factor receptor; PAPP-A -pregnancy-associated plasma protein-A; fPGF -free placental growth factor.
biochemical indicator in pregnancy [21]. Maternal plasma concentrations of PAPP-A increase during pregnancy [22]. In the first trimester of the pregnancy, PAPP-A is used in combination with the gonadotropin, b-human chorionic gonadotropin (b-hCG), and nuchal translucency (NT) thickness for screening for trisomy 21, 13, and 18 [22]. Previously published studies have reported the measurement of PAPP-A levels at different stages of pregnancy and to predict pregnancy outcomes [23].
Reduced PAPP-A levels in the first trimester of pregnancy were found to be associated with an increased risk of preeclampsia, intrauterine growth restriction (IUGR), small for gestational age (SGA), and preterm birth [23]. Also, measurement of serum PAPP-A combined with the uterine artery pulsatility index (UAPI) is a strong predictive (70%) biochemical indicator for the diagnosis of preeclampsia [23]. Although previous studies showed that the plasma PAPP-A concentrations increased in pregnancies complicated by preeclampsia and HELLP, the association with the severity of preeclampsia could not be demonstrated [24][25][26][27]. Although there is no clear consensus about the use of measurement of PAPP-A level in the diagnosis and follow-up of preeclampsia, its combined use with other serum indicators has been recommended [28].
Previous studies have also shown that the serum levels of PAPP-A in women with preeclampsia in the second trimester were lower than those in healthy pregnant women [8,21,29]. However, serum levels of PAPP-A in women with preeclampsia towards the end of the third trimester reached a higher level compared with healthy pregnant women [21]. Decreased PAPP-A levels in the first trimester are believed to play an important role in the development of preeclampsia. In the present study, the levels of maternal serum PAPP-A obtained at birth were significantly increased in healthy pregnant women. The findings from previous studies supported these results. However, the PAPP-A levels obtained at birth were not associated with the severity of preeclampsia.
Endostatin is an active component of the collagen XVII and is an inhibitor of angiogenesis by blocking VEGF signaling via the VEGFR-2 receptor (or KDR). Endostatin production, together with other anti-angiogenic factors, triggers the development of preeclampsia. However, few previous studies have evaluated the effect of endostatin alone or in combination with other anti-angiogenic molecules on the development of preeclampsia. Endostatin levels have been shown to increase, particularly in the 34 th to 37 th weeks in pregnant women with preeclampsia [30]. In the present study, endostatin levels were significantly increased in pregnant women diagnosed with preeclampsia compared with healthy pregnant women, which was supported by previous studies.
Infants with APGAR scores (cardiac apex beat, ventilatory effort, color, muscular tonus, response to a stimulus, color) between 7-10, taken in the first and fifth minutes after delivery, are usually healthy infants who do not require further follow-up. However, previous studies have shown that preeclampsia resulted in low neonatal APGAR scores, intrauterine death, low birth weight, intrauterine growth restriction (IUGR), and increased admission to the neonatal intensive care unit (NICU) [31]. In the present study, infants with the lowest average APGAR score, birth weight, and most severe degrees of IUGR were from pregnant women with preeclampsia, and there was a need for admission to the NICU in 65% of these infants.
This study had several limitations. There was a small number of study participants, and the study was conducted at a single center, which could have introduced bias. There were differences between the number of weeks of gestation of the pregnant women included in the study, which resulted in difficulty in the comparison of the birth weights, laboratory results, and clinical findings in the newborns. Differences in the gestational age and birth weights may have been associated with the high rate of early cesarean section in pregnant women diagnosed with preeclampsia compared with healthy pregnant women.

Conclusions
This study aimed to compare serum levels of vascular endothelial growth factor (VEGF) and the VEGF receptors, VEGFR-1 and VEGFR-2, free placental growth factor (fPGF), endostatin, and serum pregnancy-associated plasma protein-A (PAPP-A) levels in women with mild and severe preeclampsia and healthy pregnant women. Reduced serum levels of the angiogenic factors PAPP-A, VEGFR-2, and fPGF distinguished between women with preeclampsia and normotensive pregnant women but did not significantly differentiate between mild and severe preeclampsia. These findings were from a study in a single center, and there is a need for large-scale multicenter studies controlled studied to further investigate the role of serum markers of angiogenesis in preeclampsia.