Association between the Presence of Autoantibodies against Adrenoreceptors and Severe Pre-Eclampsia: A Pilot Study

Background Pre-eclampsia is the leading cause of maternal and neonatal morbidity and mortality with incompletely understood etiopathogenesis. The purpose of the current study is to determine whether there is a relationship between the presence of autoantibodies against β1, β2 and α1 adrenoreceptors and severe pre-eclampsia. Methodology/Principal Findings Synthetic peptides corresponding to amino acid sequences of the second extracellular loops of β1, β2 and α1 adrenoreceptors were synthesized as antigens to test 34 patients with severe pre-eclampsia, 36 normal pregnancy women and 40 non-pregnant controls for the presence of autoantibodies using enzyme-linked immunosorbent assay. The respective frequencies of autoantibodies against β1, β2 and α1 adrenoreceptors were 50.0% (17/34), 52.9% (18/34) and 55.9% (19/34) in patients with severe pre-eclampsia, 19.4% (7/36) (p = 0.011), 19.4% (7/36) (p = 0.006) and 17.6% (6/36) (p = 0.001) in normal pregnancy women and 10% (4/40), 7.5% (3/40) and 10% (4/40) (p<0.001) in non-pregnant controls. Titers of these autoantibodies were also significantly increased in patients with severe pre-eclampsia. By logistic regression analysis, the presence of these three autoantibodies significantly increased the risk of neonatal death (odds ratio, 13.5; 95% confidence interval, 1.3–141.3; p = 0.030) and long-term neonatal hospitalization (odds ratio, 5.0; 95% confidence interval, 1.3–19.1; p = 0.018). The risk of hypertension and fetal distress were also associated with the presence of these three autoantibodies. Conclusions/Significance This novel pilot study demonstrated for the first time that the presence of autoantibodies against β1, β2 and α1 adrenoreceptors are increased in patients with severe pre-eclampsia. Pregnant women who are positive for the three autoantibodies are at increased risks of neonatal mortality and morbidity. We posit that these autoantibodies may be involved in the pathogenesis of severe pre-eclampsia.


Introduction
Pre-eclampsia is a serious hypertensive disorder during pregnancy that affects 3%-5% of pregnancies, and remains the leading cause of maternal and neonatal mortalities and morbidities in the world [1,2]. It is a multi-systemic disease with features such as hypertension and proteinuria [3]. In serious cases, termination of pregnancy is the only available option to prevent further health deterioration of the fetus and mother [4]. To date, the factors and mechanisms involved in the pathogenesis of pre-eclampsia remain poorly understood.
Studies have described the role of autoantibodies against a 1 adrenoreceptor (anti-a 1 -AR) in primary and malignant hypertension [5,6]. Previously, we demonstrated that the presence of autoantibodies against b 1 , b 2 , and a 1 adrenoreceptors (anti-b 1 , b 2 , and a 1 -ARs), which bind to the second extracellular loop of the receptors, are highly prevalent in hypertensive heart disease and may participate in its pathogenesis [7,8]. In recent years, evidence has accumulated that suggests that autoimmunity participates in the pathogenesis of pre-eclampsia. Recently, numerous studies have shown that pre-eclamptic women possess autoantibodies against angiotension II type 1 receptor, which bind to and activate the receptor, consequently provoking biological responses relevant to the pathogenesis of pre-eclampsia [9][10][11][12][13]. The aim of this study was to investigate differences in the frequencies of anti-b 1 , b 2 , and a 1 -ARs among patients with severe pre-eclampsia, compared to normal pregnancy women and non-pregnant controls. The second aim was to investigate the relationship between the presence of anti-b 1 , b 2 , and a 1 -ARs and perinatal mortality and morbidity. We used synthetic peptides corresponding to amino acid sequences of the second extracellular loop of the human b 1 , b 2 , and a 1 ARs, to test sera from patients with severe pre-eclampsia, normal pregnancy women, and non-pregnant controls.

Results
Study subjects were enrolled from May 2011 to November 2011. Clinical characteristic of women from three study groups are shown in Table 1.

Maternal Clinical Characteristics
Maternal hospital stay was significantly longer in the severe preeclampsia group than in the normal pregnancy group (8.961.1 days versus 4.560.5 days, p,0.001). Complications of pregnancy, such as placental abruption, placenta remnants and postpartum hemorrhage, were significantly higher in the severe pre-eclampsia group than in the normal pregnancy group (7/34 versus 0/36, p = 0.004).
Birth weight in the severe pre-eclampsia group was significantly lower than in the normal pregnancy group (1807.36137.7 g versus 3279.2665.9 g, p,0.001). Similarly, placental weight was also reduced in the severe pre-eclampsia group (507.9639.6 g versus 649.6620.1 g, p = 0.002, Figure 1).
Neonatal Apgar score (includes assessment of the appearance of skin color, pulse, grimace, activity, and respiration) was used to classify or grade newborn infants [14]. The Apgar scores were significantly lower in the severe pre-eclampsia group than in the normal pregnancy group at one minute (7.760.5 versus 9.560.1, p,0.001), five minutes (8.460.5 versus 9.960.06, p,0.001), and ten minutes (8.460.6 versus 10.060.03, p,0.001) ( Figure 2). Neonatal asphyxia was defined according to the Apgar score at one minute: mild was between 4 and 7 and severe was between 0 and 3. The incidence of neonatal asphyxia was significantly higher in the severe pre-eclampsia group than in the normal pregnancy group (17.7% versus 0%, p = 0.001).
There were different outcomes for the 34 neonates in the severe pre-eclampsia group: 11.8% (4/34) of infants died; 23.5% (8/34) of infants were transferred to the BaYi Children's Hospital, affiliated to the military general hospital of Beijing PLA; 41.2% (14/34) of infants were transferred to the pediatric department of our hospital; 23.5% (8/34) did not require further treatment. The mean hospital stay for the neonates was 28.464.6 days.
Positive sera from the severe pre-eclampsia group contained different kinds of autoantibodies. Twelve patients were positive for a single autoantibody, four patients were positive for two kinds of autoantibody, and 11 patients were positive for all the three kinds of autoantibody. A significant correlation was found between antib 1 -AR and anti-a 1 -AR (r s = 0.4, p = 0.02), anti-b 1 -AR and anti-b 2 -AR (r s = 0.6, p,0.001). In the severe pre-eclampsia group, 76.5% (13/17) of the 17 women with anti-b 1 -AR, were also positive for anti-a 1 -AR, and 82.4% (14/17) had anti-b 2 -AR.

The Association of Autoantibody and Clinical Outcomes
Univariate logistic regression assessed the association of autoantibody with hypertension ($160 mmHg systolic or $110 mmHg diastolic), pregnancy complications, fetal growth restriction, fetal distress, preterm birth, low birth weight, neonatal long-term hospitalization (more than two weeks), and perinatal death. The presence of one or two kinds of autoantibody against b 1 , b 2 , and a 1 ARs had no statistical relationship with any complication. In contrast, the risks for both maternal and perinatal complications were closely associated with the presence of the three autoantibodies. Positivity for anti-b 1 , b 2 , and a 1 -ARs was associated with hypertension (OR, 6.5; 95% CI, 1.6-25.7; p = 0.008), pregnancy complication (OR, 4.1; 95%CI, 1.0-16.0;

Main Findings
In this study, we demonstrated for the first time that positivity for anti-b 1 , b 2 , and a 1 -ARs was associated with severe preeclampsia. The frequencies and titers of anti-b 1 , b 2 , and a 1 -ARs were significantly higher in women with severe pre-eclamptic, when compared to normal pregnancy women and non-pregnant healthy controls. The presence of the three autoantibodies was associated with both adverse maternal and perinatal clinical outcomes including hypertension, pregnancy complications, fetal growth restriction, fetal distress, preterm birth, low birth weight, perinatal death, and long-term hospitalization of neonates.

Immune Mechanisms in Pre-eclampsia
The pathogenesis of pre-eclampsia remains obscure, but is likely multifactorial involving abnormal placentation, reduced placental perfusion, endothelial cell dysfunction, and systemic vasospasm [15]. An immune mechanism has long been postulated in the pathogenesis of pre-eclampsia. Immune maladaptation may impair invasion of spiral arteries by endovascular cytotrophoblast cells [16]. Studies have suggested that repeated exposure to sperm from a particular male partner prior to pregnancy promotes immune tolerance and reduces the risk of defective trophoblast invasion [17]. Autoantibodies, such as anticardiolipin and anti-b 2glycoprotein-1 antibody, have been detected in pre-eclampsia patients [18]. From the first report that described the presence of autoantibody against angiotensin II type 1 receptor in preeclampsia patients [9], researchers have gained a greater understanding on the pathogenic mechanisms underlying preeclampsia which implicate the immune system.

Autoantibodies and Pre-eclampsia
While our results need to be confirmed by larger studies, there are biologically plausible mechanisms by which anti-b 1 , b 2 , and a 1 -ARs may lead to severe pre-eclampsia. The b 1 and b 2 adrenoreceptors in the human heart couple to the G protein Gs, which activates adenylyl cyclase. Stimulation of both receptor subtypes increases the intracellular level of cAMP, which leads to phosphorylation of target proteins [19]. a 1 adrenoreceptor couple predominantly via Gq, resulting in hydrolysis of membrane phospholipids via phospholipase C b, to yield the second messengers inositol triphosphate and diacylglycerol. This leads to muscle contraction through mobilization of intracellular Ca 2+ [20] and activation of protein kinase C. Frequencies and titers of antib 1 , b 2 , and a 1 -ARs were significantly increased in the severe preeclampsia group than in the normal pregnancy women and nonpregnant controls. Therefore we posit that there may be a relationship between the presence of anti-b 1 , b 2 , and a 1 -ARs and the development of severe pre-eclampsia. Alternatively, it is plausible that severe pre-eclampsia triggers the production of anti-b 1 , b 2 , and a 1 -ARs. Further studies are needed to delineate these two possible pathways. Studies have shown that the risk of long-term sequelae such as chronic hypertension, ischemic heart disease, stroke, and venous thromboembolism are significantly increased in pre-eclamptic women [21,22]. In this study, we were able to collect blood samples from six of the 34 patients with severe pre-eclampsia at the end of puerperium without scheduled follow-up. Three of the six samples were positive for anti-b 1 , b 2 , and a 1 -ARs at similar titers to levels at recruitment. This is but distinct from what has been observed for autoantibodies against angiotension II type 1 receptor which have been shown to subside after pregnancy termination [9]. We infer that the presence of autoantibody might  be related to high risk for cardiovascular sequelae, but this hypothesis remains to be further explored.
Three kinds of autoantibodies closely related to each other were detected in the severe pre-eclampsia group. Approximately 32.4% (11/34) of severe pre-eclamptic women had all three kinds of autoantibodies. This indicates that the autoimmune response in severe pre-eclamptic patients is multi-faceted.

Conclusion
This pilot study has demonstrated for the first time that the presence of anti-b 1 , b 2 , and a 1 -ARs were prevalent in a cohort of severe pre-eclamptic women. Risks of both maternal and perinatal complications were significantly increased when all the three kinds of autoantibody studied were present. We posit that autoantibodies against adrenoreceptors may be involved in the pathogenesis of severe pre-eclampsia. Further studies are needed to confirm these findings and dissect the underlying mechanisms for this novel observation.

Ethics Statement
The research protocol was conducted in accordance with the guidelines of the World Medical Association's Declaration of Helsinki and was performed following approval from the Medical Ethics Committee (12-S-63) of Capital Medical University Beijing Chao-Yang Hospital. All pregnant women were included in the study during the prepartum or early intrapartum period and provided written informed consent before inclusion in the study.

Subjects
Patients who were admitted to Beijing Chao-Yang Hospital were managed by the obstetrics faculty of the Capital Medical University. Thirty-four patients were diagnosed with severe preeclampsia based on the criteria set by the National High Blood Pressure Education Program Working Group report [23]. The criteria include increased blood pressure ($160 mmHg systolic or $110 mmHg diastolic on two occasions at least 6 hours apart) after 20 weeks of gestation in women with previously normal blood pressure or proteinuria of $2 g/24 h. Patients with severe preeclampsia and normal pregnancy women were approached for the study during the antepartum or early intrapartum period. We selected two comparison groups: 36 apparently healthy pregnant women and without hypertension or proteinuria (normal pregnancy group) and 40 healthy non-pregnant women (non-pregnant group). Exclusion criteria for all groups were diabetes mellitus, vasculitis, renal disease and autoimmune disease. Blood samples were collected from antecubital vein at recruitment using tubes containing EDTA, and centrifuged at 20006g for 10 minutes at 4uC within 2 h of the collection. Serum samples were stored at 270uC. We were able to collect blood samples from six patients with severe pre-eclampsia at the end of puerperium. Placentas were collected from study subjects and weighed.
We collected clinical data from mother and infants/neonates. In this study, low birth weight was defined as body weight less than 2500 g at birth. Gestational age less than 37 weeks was considered as preterm birth. Fetal growth restriction was defined as the failure to reach the predetermined growth potential. This was operationally defined as sonographic estimated fetal weight below the 10th percentile for their gestational age. A fetal heart rate $160 bpm or #110 bpm, evaluated by electronic fetal monitoring, or the third degree of meconium-stained amniotic fluid was considered evidence of fetal distress.

Materials
Three peptides corresponding to the amino acid sequence of the second extracellular loop of human b 1 , b 2 , and a 1 ARs were synthesized by Genomed (Genomed Synthesis, Inc., CA, USA) and the sequences are shown in Table 3 [24][25][26]. The purity of the peptides, determined by high performance liquid chromatography (HPLC) using a Vydac C-18 column, was above 95% as shown in Figure S1. The molecular weight of peptides was analyzed by mass spectrometry as shown in Figure S2. Nunc microtiter plates were purchased from Kastrup, Denmark. Tween-20, thimerosal, and ABTS were obtained from Sigma, St. Louis, MO, USA. Fetal bovine serum, biotinylated goat anti-human IgG (H+L), and horseradish peroxidase-streptavidin were bought from Zhongshan Golden Bridge Biotech, Beijing, China. The microplate reader was purchased from Molecular Devices Corp, Menlo Park, CA.

ELISA Protocol
Samples were classified as positive or negative based upon the presence or absence of anti-b 1 -AR, anti-b 2 -AR, and anti-a 1 -AR. An ELISA protocol, previously described by Fu et al [27], was used to screen for the presence of the autoantibodies. Briefly, 50 mL of peptide (5 mg/L) in 100 mmol Na 2 CO 3 solution (pH = 11) was coated on microtiter plates overnight at 4uC. The wells were saturated with PMT (16 PBS, 1 mL/L Tween-20, and 0.1 g/L thimerosal (PBS-T) supplemented with 100 mL/L fetal bovine serum) for 1 hour at 37uC. Then 50 mL of serum diluted from 1:20 to 1:160, positive control and negative control were added to the wells for 1 hour at 37uC. After washing the wells with PBS-T for three times, affinity-purified biotinylated goat antihuman IgG (H+L) (1:500 dilution in PMT) was added for 1 hour at 37uC. Following another round of washing three times, the bound biotinylated antibody was detected by incubating the microtiter plate for 1 hour at 37uC with horseradish peroxidasestreptavidin (1:500 dilution in PMT). This was followed by three times washing in PBS, the addition of 2.5 mmol/L H 2 O 2, and then of 2 mmol/L ABTS in citrate buffer solution. Absorbance (A) was measured after 30 minutes at 405 nm in a microplate reader.

Data Analysis
Quantitative data were expressed as the mean 6 SD. Positivity was defined as the ratio of (sample Ablank A)/(negative control A -blank A) $2.1. Antibody titer was reported as geometric mean. Table 3. Amino acid sequences of human b 1 , b 2 , and a 1 -adrenoreceptors. Data were analyzed using SPSS 16.0. (SPSS, Chicago, Illinois, USA) Fisher's exact test and unpaired t tests were used to determine the significance in differences between groups. The correlation with autoantibodies was tested using the Spearman correlation coefficient. Association between the presence of autoantibodies and categorical outcomes was assessed using univariable logistic regression analysis. Tests with p,0.05 were considered statistical significant. Figure S1 Purity of the peptides. The purity of the synthesized peptides corresponding to the amino acid sequence of the second extracellular loop of human b 1 , b 2 , and a 1 adrenoreceptors, determined by HPLC, was 96.66%, 96.21% and 95.34%.

Supporting Information
(TIF) Figure S2 Molecular weight of peptides. Molecular weight of peptides corresponding to the amino acid sequence of the second extracellular loop of human b 1 , b 2 , and a 1 adrenoreceptors was analyzed by mass spectrometry and the molecular weight was 3484.9, 3237.5 and 2872.2. (TIF)