Pregnancy Hypertension: An International Journal of Women’s Cardiovascular Health 1 (2011) 206–212
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Original Article
Interleukin-18 concentrations in pregnancies complicated by preeclampsia with and without IUGR: A comparison with normotensive pregnant women with isolated IUGR and healthy pregnant women Marzena Laskowska a,⇑, Katarzyna Laskowska b, Jan Oleszczuk a a b
Department of Obstetrics and Perinatology, Medical University of Lublin, 20-950 Lublin, ul. Jaczewskiego 8, Poland Department of Gastroenterology, Medical University of Lublin, Poland, 20-950 Lublin, ul. Jaczewskiego 8, Poland
a r t i c l e
i n f o
Article history: Received 27 March 2011 Received in revised form 1 June 2011 Accepted 7 July 2011 Available online 23 July 2011 Keywords: Interleukin-18 Severe preeclampsia IUGR
a b s t r a c t Objective: The aim of present study was to assess the maternal serum levels and clinical significance of interleukin-18 (IL-18) in pregnancies complicated by preeclampsia and/or intrauterine growth restriction (IUGR). Patients and methods: The study was carried out on 30 patients with pregnancy complicated by severe preeclampsia (15 patients with IUGR and 15 with appropriate-for-gestational-age weight fetuses), 11 normotensive pregnant patients with pregnancy complicated by isolated IUGR and 32 healthy normotensive women with uncomplicated pregnancies. The interleukin-18 levels were determined using an ELISA assay. Results: Decreased levels of maternal serum IL-18 in preeclamptic patients with and without IUGR were observed. Contrary to the preeclamptic women, no difference was found in the maternal serum levels of IL-18 in normotensive patients with pregnancies complicated by isolated fetal growth restriction. These levels were the same as observed in the healthy controls. The mean values of maternal serum IL-18 were 219.118 ± 180.079 pg/mL in the PRE group, 438.170 ± 229.657 pg/mL in the group of women with isolated IUGR, and 457.053 ± 528.142 pg/mL in the control group. The levels of maternal serum IL-18 were similar in both study preeclamptic subgroups. The mean values of IL-18 were 204.823 ± 188.171 pg/mL in the group PI and 233.414 ± 176.995 pg/mL in the P group. Conclusions: Our findings suggest that decreased levels of IL-18 in maternal serum play a significant role in etiology and pathogenesis of preeclampsia. But normotensive pregnancies complicated by isolated IUGR are not associated with the altered interleukin 18 levels in maternal serum. Ó 2011 International Society for the Study of Hypertension in Pregnancy Published by Elsevier B.V. All rights reserved.
1. Introduction Preeclampsia is a major complication in human pregnancy, affecting 5–10% pregnant women. Severe preeclampsia is one of the common maternal conditions associated with intrauterine fetal growth restriction [1]. But it is impor⇑ Corresponding author. Tel./fax: +48 81 7244841. E-mail address: [email protected] (M. Laskowska).
tant that in many pregnancies complicated by intrauterine fetal growth restriction there is an idiopathic placental failure with an abnormal placental development and suboptimal fetal nutrition and oxygenation without maternal hypertension or preeclampsia [2,3]. Despite recent progress in the immunobiology of preeclampsia its etiology remains unknown. There are many theories regarding the pathogenesis of this disorder: impaired trophoblast invasion and abnormal
2210-7789/$ - see front matter Ó 2011 International Society for the Study of Hypertension in Pregnancy Published by Elsevier B.V. All rights reserved. doi:10.1016/j.preghy.2011.07.003
M. Laskowska et al. / Pregnancy Hypertension: An International Journal of Women’s Cardiovascular Health 1 (2011) 206–212
immunological adaptation to pregnancy, oxidative stress, vascular endothelial dysfunction, defective angiogenesis processes, dietary deficiencies and genetic predisposition [4,5] and an abnormal maternal systemic inflammatory response [6–8]. It was suggested that preeclampsia is a syndrome caused by an excessive systemic inflammatory response to pregnancy [9] and dysfunction of maternal endothelial cells that represent the pathological scheme of pregnancy induced hypertension [10–13]. Altered expression of cytokines was reported in pregnancies complicated by severe preeclampsia, but their role in the pathogenesis of this pregnancy specific disorder remains controversial [14]. The mechanisms by which Th-1 type cytokines induce preeclampsia have not been fully explained, but it has been observed that inflammatory cytokines induce apoptosis of trophoblast and may inhibit angiogenesis [15]. Seol et al. [16] suggested that interleukin-18 (IL-18) may be associated with activated macrophages and acute atherosclerosis in the preeclamptic placenta. IL-18 participates in both innate and acquired immunity and plays an important role in the T-cell-helper type 1 response, primarily by its ability to induce IFN-c production in T cells and natural killer (NK) cells [17,18] and is a cofactor for Th1 development [15,17–21] IL-18 also plays a crucial role in controlling the effect of NK cells on spiral artery remodeling mice and in humans [22]. Furthermore, it was also observed that IL-18 induces gene expression and synthesis of TNF-a, IL-1, Fas Ligand and several chemokines. Increased concentrations of IL18 were found in patients with hepatic dysfunction, after hepatectomy [23], in inflammatory liver diseases [24], and in patients with acute graft-versus-host disease after bone marrow transplantation [25]. IL-18 is a proinflammatory cytokine capable of promoting atherosclerotic plaque formation and is mainly synthesized by macrophages and monocytes [26]. This process is observed in pregnancies complicated by preeclampsia. On the other hand, it is poorly understood the process of the pathological development of the placenta and the mechanism that lead in intrauterine growth restriction of the fetus (IUGR). The research to explain the role of the various cytokines in pregnancy complicated by intrauterine fetal growth restriction is rather scarce. The aim of present study was firstly to estimate serum interleukin 18 levels in preeclamptic pregnancies with and without IUGR and compare these levels with healthy pregnant women with proper fetal growth and secondly, to compare maternal serum IL-18 in pregnancies complicated by intrauterine fetal growth restriction in the course of preeclampsia with their levels in normotensive pregnancies complicated by IUGR alone. 2. Material and methods 2.1. Patient information This study was approved by the local institutional Ethics Committee. Patients with severe preeclampsia were recruited when they were admitted to the Department of Obstetrics and
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Perinatology at the Medical University Hospital in Lublin. A total of 30 preeclamptic women were recruited in this study (the PRE group). In the group of preeclamptic women, there were 15 patients with preeclampsia complicated by intrauterine growth restriction (the PI group) and 15 preeclamptic patients with appropriate-for-gestational-age weight infants (the P group). Diagnosis of severe preeclampsia is defined as blood pressure >160/110 mmHg on at least two occasions 6 h apart with proteinuria >2 g in a 24-h urinary protein excretion, and when hypertension and proteinuria were associated with one or more of the following clinical manifestations: presence of persistent headache and visual disturbances, or epigastric pain and HELLP syndrome (hemolysis, elevated AST, ALT and LDH, thrombocytopenia). Preeclamptic patients were admitted to the Hospital because of the symptoms of the disease and without signs of labor. None of the pregnant patients with preeclampsia were affected by chronic hypertension, renal disorders and/or proteinuria before pregnancy and all were normotensive before 20th week of gestation. The control group consisted of 32 healthy normotensive pregnant patients with singleton uncomplicated pregnancies, without any renal, cardiac and vascular diseases and with normal laboratory tests and with appropriate-for-gestational-age weight infants (the C group). The study was carried out on also 11 normotensive pregnant patients with intrauterine growth restricted fetuses (the IUGR group). Fetal biometry was based on the non-invasive ultrasound method, and comprised of the estimation of the gestational age in early gestation and the diagnosis of fetal intrauterine growth restriction by monitoring fetal growth later in the second or third trimester of pregnancy. Intrauterine growth restricted fetuses (IUGR) were classified as such according to ultrasound measurements when the weight of the fetus was lower than expected in relation to the gestational age, as determined by the standard curves characteristic of the Polish population, when the fetus was below the 5th percentile for gestational age. Additionally, IUGR pregnancies were characterized by at least one disturbed placental function and abnormal ultrasound examination [27,28]. The diagnosis was confirmed by the infant’s weight at birth. All arterial blood pressure measurements in the control group and in the group of patients with isolated fetal intrauterine growth restriction were normal and did not exceed 135/85 mm Hg. None of the patients from any of these groups suffered from proteinuria. 2.2. Sample collection and cytokine assay A venous blood sample was drawn at the time of admission and collected by venipuncture from each preeclamptic patient and from each woman from the control group and placed in sterile tubes. It was centrifuged for 15 min at 500g. Serum was collected and stored at 70 °C until assayed. Interleukin-18 levels were measured in the maternal serum samples using commercially available enzyme-linked immunosorbent assay (ELISA) system kit. The human IL-18 immunoassay kits were
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Table 1 Analysis of obtained results. Data
Preeclamptic patients the PRE group (n = 30)
Control group the C group (n = 32)
p value
Gravidity Parity Maternal age (years) Maternal weight (kg) Maternal height (cm) Maternal BMI (kg/m2) Age of pregnancy (weeks) Systolic blood pressure (mmHg) Diastolic blood pressure (mmHg) IL-18 levels in maternal serum (pg/ml)
1.333 ± 0.702 1.292 ± 0.690 28.944 ± 4.717 86.269 ± 15.212 165.118 ± 3.407 31.972 ± 5.332 33.443 ± 4.504 167.500 ± 16.178 113.375 ± 20.832 219.1183 ± 180.079
1.303 ± 0.586 1.242 ± 0.435 29.761 ± 4.279 78.933 ± 15.633 165.158 ± 5.786 31.909 ± 14.264 38.469 ± 1.538 115.545 ± 14.407 72.424 ± 9.747 457.053 ± 528.142
p = 0.859860 p = 0.322997 p = 0.526282 p = 0.176242 p = 0.980172 p = 0.987244 p = 0.000001* p < 0.000001* p < 0.000001* p = 0.022507*
Data presented as a mean ± SD. PRE – women with pregnancies complicated by severe preeclampsia. C – the control group - healthy normotensive pregnant women. * statistical significance (p < 0.05).
IL 18 in maternal serum (pg/ml) 700
600
IL 18 (pg/ml)
500
400
300
200
100 C
PRE
Mean Mean +/- SEM Mean +/- 1,96* SEM
groups Fig. 1. Maternal serum IL-18 in pregnancies complicated by preeclampsia and in the control subjects.
obtained from Bender MedSystems (Vienna, Austria, Europe). Assay procedures were followed according to the manufacturer’s instructions. The standard was serially diluted and used to generate a standard curve for each ELISA assay. The assay was conducted according to manufacturer’s instruction. The sensitivities for IL-18 were 9.2 pg/ml. Intra-assay coefficient of variation was 6.5%. 2.3. Statistical analysis Data were presented in mean and standard deviation (SD). The Student t-test was conducted to evaluate the differences of means and the Mann–Whitney test was conducted to evaluate the differences of the medians. Statistical package STATISTICA 8.0 was used for data analysis. Probability level of p < 0.05 was considered significant.
3. Results There were no statistically significant differences in gravidity, parity, maternal age, maternal weight, height and BMI in patient profiles between both preeclamptic and control groups. Lower BMI was observed in the group of pregnant patients with pregnancy complicated by isolated IUGR. But the levels of IL-18 were not associated with parity or BMI. Creatinine and urea levels were normal in all patients. Lower gestational age was found in both preeclamptic groups and in the group of normotensive women with isolated IUGR. The mean age of gestation was 36.931 ± 1.990 weeks in the IUGR group, 33.753 ± 5.067 weeks in the P group, 33.262 ± 3.925 weeks in the PI group, and 38.462 ± 1.538 weeks in the control group. Systolic and diastolic blood pressure and mean arterial blood pressure were higher in the study group of
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Maternal serum levels of Interleukin 18 in studied groups of pregnant women 1200
IL-18 in maternal serum (pg/ml)
1000
800
600
400
200
0
-200 P
PI
C
iugr
Mean Mean +/- SEM Mean +/- SD
groups Fig. 2. IL-18 levels in maternal serum in studied groups of pregnant women.
preeclamptic pregnant women than in the control group. These differences were statistically significant (p < 0.0000001). The mean systolic blood pressure values were 167.500 ± 16.178 mmHg in the group of preeclamptic patients and 115.545 ± 14.407 mmHg in the control group. The mean diastolic blood pressure values were 113.375 ± 20.832 mmHg in women with pregnancy complicated by preeclampsia and 72.424 ± 9.747 mmHg in the healthy controls. Systolic and diastolic blood pressure and mean arterial blood pressure were also higher in both study subgroups of preeclamptic pregnant women than in the control group and in the pregnant patients with isolated growth restricted fetus. These differences were statistically significant. The mean systolic blood pressure values were 109.020 ± 16.195 mmHg in the group of women with pregnancy complicated by isolated intrauterine growth restricted fetuses (the IUGR group), 172.833 ± 16.618 mmHg in the group of preeclamptic pregnant patients with IUGR (the PI group), 161.533 ± 13.279 mmHg in the P group and 115.545 ± 14.407 mmHg in the control group. The mean diastolic blood pressure values were 70.8000 ± 6.987 mmHg in the group of women with pregnancy complicated by isolated IUGR, 110.500 ± 9.545 mmHg in women with pregnancy complicated by preeclampsia with IUGR (the PI group), 106.000 ± 27.162 mmHg in preeclamptic patients without IUGR (the P group).and 72.424 ± 9.747 mmHg in the healthy controls. The preeclamptic women revealed lower levels of maternal serum interleukin-18 levels than the healthy normotensive pregnant women from the control group. The mean values were 219.118 ± 180.079 pg/mL in the PRE group compared with 457.053 ± 528.142 pg/mL in
the control group. This difference was statistically significant (p = 0.022507⁄). Data presented in Table 1 and Figure 1. When the preeclamptic women were subdivided into preeclampsia with normal intrauterine fetal growth (group P) and preeclampsia complicated by intrauterine growth restriction (group PI), the levels of maternal serum IL-18 were similar in both study subgroups (p = 0.671459). These levels of IL-18 in maternal serum in these both preeclamptic subgroups were lower than levels observed in normotensive pregnant women with isolated IUGR (p = 0.15777⁄ for group PI and p = 0.026602⁄ for group P respectively) and than in healthy normotensive controls with normal intrauterine fetal growth and uncomplicated course of pregnancy (p = 0.080463 for group PI and p = 0.118710 for group P respectively). Contrary to both preeclamptic groups of women, no difference was found in the maternal serum levels of IL-18 in normotensive patients with pregnancies complicated by isolated fetal growth restriction. These levels were the same as observed in the healthy controls. (see Fig. 1). The mean values were 204.823 ± 188.171 pg/mL in the PI group, 233.414 ± 176.995 pg/mL in the P group, 438.170 ± 229.657 g/ml in the IUGR group and 457.053 ± 528.142 pg/mL in the control group of pregnant patients with normal fetal growth (the C group) Fig. 2. Table 2 shows the clinical characteristics of patients with IUGR in the course of preeclampsia and normotensive patients with pregnancies complicated by IUGR in comparison with healthy controls. Table 3 shows the clinical characteristics of patients with fetal growth restriction with and without preeclampsia and preeclamptic pregnancies with normal fetal growth.
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4. Discussion Redman and Sargent [13] observed that preeclampsia is associated with the activation of innate immunity. It seems possible that immune disturbances and the abnormal activation of the process of inflammation may be involved in the etiology of preeclampsia or/and intrauterine growth restricted fetus. During normal pregnancy the balance between Th1 cells, which are involved in cellular immunity and Th2 cells, which are involved in humoral immunity, is shifted to a Th2 predominant condition. These Th1 and Th2 cells which cooperate with Treg cells, Th3 and Tr1 cells play the essential role in the maternal tolerance to paternal antigens and maintenance of pregnancy [29–34]. Interleukin-18 may also play roles in terminal differentiation of cells and protection of cells from stress-induced cell death [35–37]. It was also suggested that IL-18 is expressed at sites of chronic inflammation and that this interleukin can up-regulate cyclooxygenase, inducible nitric oxide synthase and adhesion molecules [38–40]. It is noteworthy that intravascular excessive inflammatory response is thought to be the cause of clinical symptoms and signs of preeclampsia [9,40]. The roles of IL-18 in pregnancies complicated by intrauterine fetal growth restriction with and without preeclampsia are yet to be elucidated. It seems that increased levels of interleukin-18 may be expected in preeclamptic patients. However, our study revealed a lower maternal serum concentration of interleukin-18 in pregnancies complicated by severe preeclampsia with and without IUGR than in healthy controls. These levels in both preeclamptic groups of women were also lower than in normotensive pregnancies complicated by isolated IUGR. Our findings suggest that decreased levels of IL-18 in the maternal serum of preeclamptic women may play a role in pathogenesis of preeclampsia with and without IUGR. Contrary to both preeclamptic groups of women, no difference was found in the maternal serum levels of IL-18 in normoten-
sive patients with pregnancies complicated by isolated fetal growth restriction. These levels were the same as observed in the healthy controls. The lack of such association with normotensive pregnancies complicated by isolated intrauterine fetal growth restriction is consistent with the idea that different pathophysiological mechanisms are involved in impairment of intrauterine fetal growth in pregnancies complicated by IUGR with and without preeclampsia. In our study the age of pregnancy was lower in preeclamptic patients and in normotensive pregnancies with isolated IUGR than in the control subjects. But it was presented by Adams et al. [41] that levels of IL-18 were not associated with gestational age and its levels in blood do not change substantially in the normal course of pregnancy. Similar results were presented by Roland et al. [42] who observed significantly lower concentrations of IL-18 in preeclamptic samples than in controls. These authors suggested that IL-18 was potentially linked to oxidative stress in preeclampsia, since its levels correlated with the concentration of the powerful antioxidant CoQ10. But different results of increased interleukin-18 serum levels in pregnancies complicated by preeclampsia were presented by Huang et al. [14]. Adams et al. [41] observed similar concentrations of IL18 in preeclamptic women and in control subjects. But lower levels of this cytokine were observed in preeclamptic women who were previously treated with bethamethasone or dihydralazine. Redman et al. [9] suggested that elevated levels of IL-18 in normal pregnancies and their excessive higher levels in pregnancies complicated by severe preeclampsia might contribute to the inflammatory response. Sakai et al. [40] observed elevated secretion of IL-18 by peripheral blood mononuclear cells (PBMC) in normal pregnant women and suggested that increased numbers of peripheral blood monocytes might contribute to this elevated IL-18 secretion. But these authors presented similar levels in both preeclamptic and healthy normotensive
Table 2 Characteristics of patients with pregnancy complicated by IUGR with and without preeclampsia. Data
Preeclamptic patients with IUGR the PI group (n = 15)
Statistical significance PI/IUGR p value
Normotensive patients with IUGR alone the IUGR group (n = 11)
Statistical significance IUGR/C p value
Control group the C group (n = 32)
Gravidity Parity Maternal age (years) Maternal weight (kg) Maternal height (cm) Maternal BMI (kg/m2) Age of pregnancy (weeks) Systolic blood pressure (mmHg) Diastolic blood pressure (mmHg) IL-18 levels in maternal serum (pg/ml)
1.00 ± 0.00 1.00 ± 0.00 30.068 ± 4.267 84.722 ± 14.988 164.778 ± 3.667 30.385 ± 4.746 33.262 ± 3.925 172.833 ± 16.618 110.500 ± 9.545 204.823 ± 188.171
p = 0.0602236 p = 0.042894⁄ p = 0.088505 p = .0031693 p = 0.854543 p = 0.040086* p = 0.000001* p = 0.000033* p = 0.000113* p = 0.069376
1.458 ± 0.771 1.396 ± 0.644 28.540 ± 4.400 69.171 ± 10.804 164.000 ± 5.292 25.852 ± 4.271 36.931 ± 1,990 109.020 ± 16.195 70.800 ± 6.987 438.170 ± 229.657
p = 0.480482 p = 0.446125 p = 0.0958003 p = 0.237141 p = 0.857979 p = 0.302298 p < 0.000001* p = 0.257057 p = 0.480482 p = 0.738081
1.303 ± 0.586 1.242 ± 0.435 29.761 ± 4.279 78.933 ± 15.633 165.158 ± 5.786 31.909 ± 14.264 38.469 ± 1.538 115.545 ± 14.407 72.424 ± 9.747 457.053 ± 528.142
Data presented as a mean ± SD. PI – preeclamptic women with pregnancies complicated by IUGR. IUGR – normotensive pregnant women with IUGR alone. C – the control group - healthy normotensive pregnant women. * Statistical significance (p < 0.05).
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Table 3 Comparison of the preeclamptic patients with and without IUGR and normotensive women with isolated IUGR. Data
Preeclamptic patients with IUGR the PI group (n = 15)
Statistical significance PI/P p value
Normotensive patients with IUGR alone the IUGR group (n = 11)
Statistical significance IUGR/P p value
Preeclamptic patients without IUGR the P group (n = 15)
Gravidity Parity Maternal age (years) Maternal weight (kg) Maternal height (cm) Maternal BMI (kg/m2) Age of gestation (weeks) Systolic blood pressure (mmHg) Diastolic blood pressure (mmHg) IL-18 levels in maternal serum (pg/ml)
1.00 ± 0.00 1.00 ± 0.00 30.068 ± 4.267 84.722 ± 14.988 164.778 ± 3.667 30.385 ± 4.746 33.262 ± 3.925 172.833 ± 16.618 110.500 ± 9.545 204.823 ± 188.171
p = 0.280019 p = 0.334108 p = 0.291616 p = 0.084022 p = 0.079362 p = 0.123220 0.673363 p = 0.040889* p = 0.440270 p = 0.671459
1.458 ± 0.771 1.396 ± 0.644 28.540 ± 4.400 69.171 ± 10.804 164.00 ± 5.292 25.852 ± 4.271 36.931 ± 1.990 109.020 ± 16.195 70.800 ± 6.987 438.170 ± 229.657
p = 0.174304 p = 0.308268 p = 0.718465 p = 0.000014* p = 0.113238 p = 0.000022* 0.000205* p < 0.000001* p < 0.000001* p = 0.026602*
1.750 ± 1.076 1.571 ± 0.826 28.045 ± 6.288 92.379 ± 19.739 167.000 ± 6.656 33.308 ± 5.302 33.753 ± 5.067 161.533 ± 13.279 106.000 ± 27.162 233.414 ± 176.995
Data presented as a mean ± SD. PI – preeclamptic women with pregnancies complicated by IUGR. IUGR – normotensive pregnant women with IUGR alone. P – preeclamptic women without IUGR. * Statistical significance (p < 0.05).
pregnant women [40]. They suggest that elevated Il-18 secretion alone is not sufficient to explain the excessive inflammatory response, but Th1/Th2 ratios were negatively correlated with the ratios of these cytokines, e.g. IL-18 and IL-12 [40]. In normal pregnancy monocytes could produce IL-18 but not IL-12, which results in depressed IL-12 and enhanced IL-18 secretion by non-stimulated PBMC [40]. This elevated IL-18 and depressed IL-12 secretion in normal pregnancy could induce Th2 dominant immune responses that enable fetal survival as a successful allograft [39]. But in preeclamptic pregnancies under oxidative stress condition the excessive stimulated monocytes could produce both IL-18 and IL-12 [40]. This elevated production of both IL-18 and IL-12 in pregnancies complicated by severe preeclampsia result in a Th1 predominant response [40]. Furthermore, IL-18 acts as an epigenetic regulator on chromatin and activates anti-apoptotic signal pathways [37,43]. Interleukin-18 increases the inflammatory process by stimulating the production of tumor necrosis factor, IL1beta and IL-6. IL-18 is also involved in cell-mediated immunity and its actions are synergistic with IL-12 and IL-15 [44]. 5. Conclusions Pregnancies complicated by severe preeclampsia are associated with the decreased levels of interleukin 18 in maternal serum. Normotensive pregnancies complicated by isolated IUGR without preeclampsia are not associated with the abnormal levels of IL-18 in maternal serum. The lack of such association in pregnancies complicated by isolated IUGR may suggest different pathomechanisms inducing impairment of fetal growth in pregnancies complicated by IUGR with and without preeclampsia. Further studies are needed to clarify these aspects and explain the role of IL-18 in preeclamptic pregnancies with and without IUGR in order to improve the management and therapeutic strategies for preeclamptic patients.
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Pregnancy Hypertension: An International Journal of Women’s Cardiovascular Health journal homepage: www.elsevier.com/locate/preghy
Original Article
Interleukin-18 concentrations in pregnancies complicated by preeclampsia with and without IUGR: A comparison with normotensive pregnant women with isolated IUGR and healthy pregnant women Marzena Laskowska a,⇑, Katarzyna Laskowska b, Jan Oleszczuk a a b
Department of Obstetrics and Perinatology, Medical University of Lublin, 20-950 Lublin, ul. Jaczewskiego 8, Poland Department of Gastroenterology, Medical University of Lublin, Poland, 20-950 Lublin, ul. Jaczewskiego 8, Poland
a r t i c l e
i n f o
Article history: Received 27 March 2011 Received in revised form 1 June 2011 Accepted 7 July 2011 Available online 23 July 2011 Keywords: Interleukin-18 Severe preeclampsia IUGR
a b s t r a c t Objective: The aim of present study was to assess the maternal serum levels and clinical significance of interleukin-18 (IL-18) in pregnancies complicated by preeclampsia and/or intrauterine growth restriction (IUGR). Patients and methods: The study was carried out on 30 patients with pregnancy complicated by severe preeclampsia (15 patients with IUGR and 15 with appropriate-for-gestational-age weight fetuses), 11 normotensive pregnant patients with pregnancy complicated by isolated IUGR and 32 healthy normotensive women with uncomplicated pregnancies. The interleukin-18 levels were determined using an ELISA assay. Results: Decreased levels of maternal serum IL-18 in preeclamptic patients with and without IUGR were observed. Contrary to the preeclamptic women, no difference was found in the maternal serum levels of IL-18 in normotensive patients with pregnancies complicated by isolated fetal growth restriction. These levels were the same as observed in the healthy controls. The mean values of maternal serum IL-18 were 219.118 ± 180.079 pg/mL in the PRE group, 438.170 ± 229.657 pg/mL in the group of women with isolated IUGR, and 457.053 ± 528.142 pg/mL in the control group. The levels of maternal serum IL-18 were similar in both study preeclamptic subgroups. The mean values of IL-18 were 204.823 ± 188.171 pg/mL in the group PI and 233.414 ± 176.995 pg/mL in the P group. Conclusions: Our findings suggest that decreased levels of IL-18 in maternal serum play a significant role in etiology and pathogenesis of preeclampsia. But normotensive pregnancies complicated by isolated IUGR are not associated with the altered interleukin 18 levels in maternal serum. Ó 2011 International Society for the Study of Hypertension in Pregnancy Published by Elsevier B.V. All rights reserved.
1. Introduction Preeclampsia is a major complication in human pregnancy, affecting 5–10% pregnant women. Severe preeclampsia is one of the common maternal conditions associated with intrauterine fetal growth restriction [1]. But it is impor⇑ Corresponding author. Tel./fax: +48 81 7244841. E-mail address: [email protected] (M. Laskowska).
tant that in many pregnancies complicated by intrauterine fetal growth restriction there is an idiopathic placental failure with an abnormal placental development and suboptimal fetal nutrition and oxygenation without maternal hypertension or preeclampsia [2,3]. Despite recent progress in the immunobiology of preeclampsia its etiology remains unknown. There are many theories regarding the pathogenesis of this disorder: impaired trophoblast invasion and abnormal
2210-7789/$ - see front matter Ó 2011 International Society for the Study of Hypertension in Pregnancy Published by Elsevier B.V. All rights reserved. doi:10.1016/j.preghy.2011.07.003
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immunological adaptation to pregnancy, oxidative stress, vascular endothelial dysfunction, defective angiogenesis processes, dietary deficiencies and genetic predisposition [4,5] and an abnormal maternal systemic inflammatory response [6–8]. It was suggested that preeclampsia is a syndrome caused by an excessive systemic inflammatory response to pregnancy [9] and dysfunction of maternal endothelial cells that represent the pathological scheme of pregnancy induced hypertension [10–13]. Altered expression of cytokines was reported in pregnancies complicated by severe preeclampsia, but their role in the pathogenesis of this pregnancy specific disorder remains controversial [14]. The mechanisms by which Th-1 type cytokines induce preeclampsia have not been fully explained, but it has been observed that inflammatory cytokines induce apoptosis of trophoblast and may inhibit angiogenesis [15]. Seol et al. [16] suggested that interleukin-18 (IL-18) may be associated with activated macrophages and acute atherosclerosis in the preeclamptic placenta. IL-18 participates in both innate and acquired immunity and plays an important role in the T-cell-helper type 1 response, primarily by its ability to induce IFN-c production in T cells and natural killer (NK) cells [17,18] and is a cofactor for Th1 development [15,17–21] IL-18 also plays a crucial role in controlling the effect of NK cells on spiral artery remodeling mice and in humans [22]. Furthermore, it was also observed that IL-18 induces gene expression and synthesis of TNF-a, IL-1, Fas Ligand and several chemokines. Increased concentrations of IL18 were found in patients with hepatic dysfunction, after hepatectomy [23], in inflammatory liver diseases [24], and in patients with acute graft-versus-host disease after bone marrow transplantation [25]. IL-18 is a proinflammatory cytokine capable of promoting atherosclerotic plaque formation and is mainly synthesized by macrophages and monocytes [26]. This process is observed in pregnancies complicated by preeclampsia. On the other hand, it is poorly understood the process of the pathological development of the placenta and the mechanism that lead in intrauterine growth restriction of the fetus (IUGR). The research to explain the role of the various cytokines in pregnancy complicated by intrauterine fetal growth restriction is rather scarce. The aim of present study was firstly to estimate serum interleukin 18 levels in preeclamptic pregnancies with and without IUGR and compare these levels with healthy pregnant women with proper fetal growth and secondly, to compare maternal serum IL-18 in pregnancies complicated by intrauterine fetal growth restriction in the course of preeclampsia with their levels in normotensive pregnancies complicated by IUGR alone. 2. Material and methods 2.1. Patient information This study was approved by the local institutional Ethics Committee. Patients with severe preeclampsia were recruited when they were admitted to the Department of Obstetrics and
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Perinatology at the Medical University Hospital in Lublin. A total of 30 preeclamptic women were recruited in this study (the PRE group). In the group of preeclamptic women, there were 15 patients with preeclampsia complicated by intrauterine growth restriction (the PI group) and 15 preeclamptic patients with appropriate-for-gestational-age weight infants (the P group). Diagnosis of severe preeclampsia is defined as blood pressure >160/110 mmHg on at least two occasions 6 h apart with proteinuria >2 g in a 24-h urinary protein excretion, and when hypertension and proteinuria were associated with one or more of the following clinical manifestations: presence of persistent headache and visual disturbances, or epigastric pain and HELLP syndrome (hemolysis, elevated AST, ALT and LDH, thrombocytopenia). Preeclamptic patients were admitted to the Hospital because of the symptoms of the disease and without signs of labor. None of the pregnant patients with preeclampsia were affected by chronic hypertension, renal disorders and/or proteinuria before pregnancy and all were normotensive before 20th week of gestation. The control group consisted of 32 healthy normotensive pregnant patients with singleton uncomplicated pregnancies, without any renal, cardiac and vascular diseases and with normal laboratory tests and with appropriate-for-gestational-age weight infants (the C group). The study was carried out on also 11 normotensive pregnant patients with intrauterine growth restricted fetuses (the IUGR group). Fetal biometry was based on the non-invasive ultrasound method, and comprised of the estimation of the gestational age in early gestation and the diagnosis of fetal intrauterine growth restriction by monitoring fetal growth later in the second or third trimester of pregnancy. Intrauterine growth restricted fetuses (IUGR) were classified as such according to ultrasound measurements when the weight of the fetus was lower than expected in relation to the gestational age, as determined by the standard curves characteristic of the Polish population, when the fetus was below the 5th percentile for gestational age. Additionally, IUGR pregnancies were characterized by at least one disturbed placental function and abnormal ultrasound examination [27,28]. The diagnosis was confirmed by the infant’s weight at birth. All arterial blood pressure measurements in the control group and in the group of patients with isolated fetal intrauterine growth restriction were normal and did not exceed 135/85 mm Hg. None of the patients from any of these groups suffered from proteinuria. 2.2. Sample collection and cytokine assay A venous blood sample was drawn at the time of admission and collected by venipuncture from each preeclamptic patient and from each woman from the control group and placed in sterile tubes. It was centrifuged for 15 min at 500g. Serum was collected and stored at 70 °C until assayed. Interleukin-18 levels were measured in the maternal serum samples using commercially available enzyme-linked immunosorbent assay (ELISA) system kit. The human IL-18 immunoassay kits were
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Table 1 Analysis of obtained results. Data
Preeclamptic patients the PRE group (n = 30)
Control group the C group (n = 32)
p value
Gravidity Parity Maternal age (years) Maternal weight (kg) Maternal height (cm) Maternal BMI (kg/m2) Age of pregnancy (weeks) Systolic blood pressure (mmHg) Diastolic blood pressure (mmHg) IL-18 levels in maternal serum (pg/ml)
1.333 ± 0.702 1.292 ± 0.690 28.944 ± 4.717 86.269 ± 15.212 165.118 ± 3.407 31.972 ± 5.332 33.443 ± 4.504 167.500 ± 16.178 113.375 ± 20.832 219.1183 ± 180.079
1.303 ± 0.586 1.242 ± 0.435 29.761 ± 4.279 78.933 ± 15.633 165.158 ± 5.786 31.909 ± 14.264 38.469 ± 1.538 115.545 ± 14.407 72.424 ± 9.747 457.053 ± 528.142
p = 0.859860 p = 0.322997 p = 0.526282 p = 0.176242 p = 0.980172 p = 0.987244 p = 0.000001* p < 0.000001* p < 0.000001* p = 0.022507*
Data presented as a mean ± SD. PRE – women with pregnancies complicated by severe preeclampsia. C – the control group - healthy normotensive pregnant women. * statistical significance (p < 0.05).
IL 18 in maternal serum (pg/ml) 700
600
IL 18 (pg/ml)
500
400
300
200
100 C
PRE
Mean Mean +/- SEM Mean +/- 1,96* SEM
groups Fig. 1. Maternal serum IL-18 in pregnancies complicated by preeclampsia and in the control subjects.
obtained from Bender MedSystems (Vienna, Austria, Europe). Assay procedures were followed according to the manufacturer’s instructions. The standard was serially diluted and used to generate a standard curve for each ELISA assay. The assay was conducted according to manufacturer’s instruction. The sensitivities for IL-18 were 9.2 pg/ml. Intra-assay coefficient of variation was 6.5%. 2.3. Statistical analysis Data were presented in mean and standard deviation (SD). The Student t-test was conducted to evaluate the differences of means and the Mann–Whitney test was conducted to evaluate the differences of the medians. Statistical package STATISTICA 8.0 was used for data analysis. Probability level of p < 0.05 was considered significant.
3. Results There were no statistically significant differences in gravidity, parity, maternal age, maternal weight, height and BMI in patient profiles between both preeclamptic and control groups. Lower BMI was observed in the group of pregnant patients with pregnancy complicated by isolated IUGR. But the levels of IL-18 were not associated with parity or BMI. Creatinine and urea levels were normal in all patients. Lower gestational age was found in both preeclamptic groups and in the group of normotensive women with isolated IUGR. The mean age of gestation was 36.931 ± 1.990 weeks in the IUGR group, 33.753 ± 5.067 weeks in the P group, 33.262 ± 3.925 weeks in the PI group, and 38.462 ± 1.538 weeks in the control group. Systolic and diastolic blood pressure and mean arterial blood pressure were higher in the study group of
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Maternal serum levels of Interleukin 18 in studied groups of pregnant women 1200
IL-18 in maternal serum (pg/ml)
1000
800
600
400
200
0
-200 P
PI
C
iugr
Mean Mean +/- SEM Mean +/- SD
groups Fig. 2. IL-18 levels in maternal serum in studied groups of pregnant women.
preeclamptic pregnant women than in the control group. These differences were statistically significant (p < 0.0000001). The mean systolic blood pressure values were 167.500 ± 16.178 mmHg in the group of preeclamptic patients and 115.545 ± 14.407 mmHg in the control group. The mean diastolic blood pressure values were 113.375 ± 20.832 mmHg in women with pregnancy complicated by preeclampsia and 72.424 ± 9.747 mmHg in the healthy controls. Systolic and diastolic blood pressure and mean arterial blood pressure were also higher in both study subgroups of preeclamptic pregnant women than in the control group and in the pregnant patients with isolated growth restricted fetus. These differences were statistically significant. The mean systolic blood pressure values were 109.020 ± 16.195 mmHg in the group of women with pregnancy complicated by isolated intrauterine growth restricted fetuses (the IUGR group), 172.833 ± 16.618 mmHg in the group of preeclamptic pregnant patients with IUGR (the PI group), 161.533 ± 13.279 mmHg in the P group and 115.545 ± 14.407 mmHg in the control group. The mean diastolic blood pressure values were 70.8000 ± 6.987 mmHg in the group of women with pregnancy complicated by isolated IUGR, 110.500 ± 9.545 mmHg in women with pregnancy complicated by preeclampsia with IUGR (the PI group), 106.000 ± 27.162 mmHg in preeclamptic patients without IUGR (the P group).and 72.424 ± 9.747 mmHg in the healthy controls. The preeclamptic women revealed lower levels of maternal serum interleukin-18 levels than the healthy normotensive pregnant women from the control group. The mean values were 219.118 ± 180.079 pg/mL in the PRE group compared with 457.053 ± 528.142 pg/mL in
the control group. This difference was statistically significant (p = 0.022507⁄). Data presented in Table 1 and Figure 1. When the preeclamptic women were subdivided into preeclampsia with normal intrauterine fetal growth (group P) and preeclampsia complicated by intrauterine growth restriction (group PI), the levels of maternal serum IL-18 were similar in both study subgroups (p = 0.671459). These levels of IL-18 in maternal serum in these both preeclamptic subgroups were lower than levels observed in normotensive pregnant women with isolated IUGR (p = 0.15777⁄ for group PI and p = 0.026602⁄ for group P respectively) and than in healthy normotensive controls with normal intrauterine fetal growth and uncomplicated course of pregnancy (p = 0.080463 for group PI and p = 0.118710 for group P respectively). Contrary to both preeclamptic groups of women, no difference was found in the maternal serum levels of IL-18 in normotensive patients with pregnancies complicated by isolated fetal growth restriction. These levels were the same as observed in the healthy controls. (see Fig. 1). The mean values were 204.823 ± 188.171 pg/mL in the PI group, 233.414 ± 176.995 pg/mL in the P group, 438.170 ± 229.657 g/ml in the IUGR group and 457.053 ± 528.142 pg/mL in the control group of pregnant patients with normal fetal growth (the C group) Fig. 2. Table 2 shows the clinical characteristics of patients with IUGR in the course of preeclampsia and normotensive patients with pregnancies complicated by IUGR in comparison with healthy controls. Table 3 shows the clinical characteristics of patients with fetal growth restriction with and without preeclampsia and preeclamptic pregnancies with normal fetal growth.
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4. Discussion Redman and Sargent [13] observed that preeclampsia is associated with the activation of innate immunity. It seems possible that immune disturbances and the abnormal activation of the process of inflammation may be involved in the etiology of preeclampsia or/and intrauterine growth restricted fetus. During normal pregnancy the balance between Th1 cells, which are involved in cellular immunity and Th2 cells, which are involved in humoral immunity, is shifted to a Th2 predominant condition. These Th1 and Th2 cells which cooperate with Treg cells, Th3 and Tr1 cells play the essential role in the maternal tolerance to paternal antigens and maintenance of pregnancy [29–34]. Interleukin-18 may also play roles in terminal differentiation of cells and protection of cells from stress-induced cell death [35–37]. It was also suggested that IL-18 is expressed at sites of chronic inflammation and that this interleukin can up-regulate cyclooxygenase, inducible nitric oxide synthase and adhesion molecules [38–40]. It is noteworthy that intravascular excessive inflammatory response is thought to be the cause of clinical symptoms and signs of preeclampsia [9,40]. The roles of IL-18 in pregnancies complicated by intrauterine fetal growth restriction with and without preeclampsia are yet to be elucidated. It seems that increased levels of interleukin-18 may be expected in preeclamptic patients. However, our study revealed a lower maternal serum concentration of interleukin-18 in pregnancies complicated by severe preeclampsia with and without IUGR than in healthy controls. These levels in both preeclamptic groups of women were also lower than in normotensive pregnancies complicated by isolated IUGR. Our findings suggest that decreased levels of IL-18 in the maternal serum of preeclamptic women may play a role in pathogenesis of preeclampsia with and without IUGR. Contrary to both preeclamptic groups of women, no difference was found in the maternal serum levels of IL-18 in normoten-
sive patients with pregnancies complicated by isolated fetal growth restriction. These levels were the same as observed in the healthy controls. The lack of such association with normotensive pregnancies complicated by isolated intrauterine fetal growth restriction is consistent with the idea that different pathophysiological mechanisms are involved in impairment of intrauterine fetal growth in pregnancies complicated by IUGR with and without preeclampsia. In our study the age of pregnancy was lower in preeclamptic patients and in normotensive pregnancies with isolated IUGR than in the control subjects. But it was presented by Adams et al. [41] that levels of IL-18 were not associated with gestational age and its levels in blood do not change substantially in the normal course of pregnancy. Similar results were presented by Roland et al. [42] who observed significantly lower concentrations of IL-18 in preeclamptic samples than in controls. These authors suggested that IL-18 was potentially linked to oxidative stress in preeclampsia, since its levels correlated with the concentration of the powerful antioxidant CoQ10. But different results of increased interleukin-18 serum levels in pregnancies complicated by preeclampsia were presented by Huang et al. [14]. Adams et al. [41] observed similar concentrations of IL18 in preeclamptic women and in control subjects. But lower levels of this cytokine were observed in preeclamptic women who were previously treated with bethamethasone or dihydralazine. Redman et al. [9] suggested that elevated levels of IL-18 in normal pregnancies and their excessive higher levels in pregnancies complicated by severe preeclampsia might contribute to the inflammatory response. Sakai et al. [40] observed elevated secretion of IL-18 by peripheral blood mononuclear cells (PBMC) in normal pregnant women and suggested that increased numbers of peripheral blood monocytes might contribute to this elevated IL-18 secretion. But these authors presented similar levels in both preeclamptic and healthy normotensive
Table 2 Characteristics of patients with pregnancy complicated by IUGR with and without preeclampsia. Data
Preeclamptic patients with IUGR the PI group (n = 15)
Statistical significance PI/IUGR p value
Normotensive patients with IUGR alone the IUGR group (n = 11)
Statistical significance IUGR/C p value
Control group the C group (n = 32)
Gravidity Parity Maternal age (years) Maternal weight (kg) Maternal height (cm) Maternal BMI (kg/m2) Age of pregnancy (weeks) Systolic blood pressure (mmHg) Diastolic blood pressure (mmHg) IL-18 levels in maternal serum (pg/ml)
1.00 ± 0.00 1.00 ± 0.00 30.068 ± 4.267 84.722 ± 14.988 164.778 ± 3.667 30.385 ± 4.746 33.262 ± 3.925 172.833 ± 16.618 110.500 ± 9.545 204.823 ± 188.171
p = 0.0602236 p = 0.042894⁄ p = 0.088505 p = .0031693 p = 0.854543 p = 0.040086* p = 0.000001* p = 0.000033* p = 0.000113* p = 0.069376
1.458 ± 0.771 1.396 ± 0.644 28.540 ± 4.400 69.171 ± 10.804 164.000 ± 5.292 25.852 ± 4.271 36.931 ± 1,990 109.020 ± 16.195 70.800 ± 6.987 438.170 ± 229.657
p = 0.480482 p = 0.446125 p = 0.0958003 p = 0.237141 p = 0.857979 p = 0.302298 p < 0.000001* p = 0.257057 p = 0.480482 p = 0.738081
1.303 ± 0.586 1.242 ± 0.435 29.761 ± 4.279 78.933 ± 15.633 165.158 ± 5.786 31.909 ± 14.264 38.469 ± 1.538 115.545 ± 14.407 72.424 ± 9.747 457.053 ± 528.142
Data presented as a mean ± SD. PI – preeclamptic women with pregnancies complicated by IUGR. IUGR – normotensive pregnant women with IUGR alone. C – the control group - healthy normotensive pregnant women. * Statistical significance (p < 0.05).
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Table 3 Comparison of the preeclamptic patients with and without IUGR and normotensive women with isolated IUGR. Data
Preeclamptic patients with IUGR the PI group (n = 15)
Statistical significance PI/P p value
Normotensive patients with IUGR alone the IUGR group (n = 11)
Statistical significance IUGR/P p value
Preeclamptic patients without IUGR the P group (n = 15)
Gravidity Parity Maternal age (years) Maternal weight (kg) Maternal height (cm) Maternal BMI (kg/m2) Age of gestation (weeks) Systolic blood pressure (mmHg) Diastolic blood pressure (mmHg) IL-18 levels in maternal serum (pg/ml)
1.00 ± 0.00 1.00 ± 0.00 30.068 ± 4.267 84.722 ± 14.988 164.778 ± 3.667 30.385 ± 4.746 33.262 ± 3.925 172.833 ± 16.618 110.500 ± 9.545 204.823 ± 188.171
p = 0.280019 p = 0.334108 p = 0.291616 p = 0.084022 p = 0.079362 p = 0.123220 0.673363 p = 0.040889* p = 0.440270 p = 0.671459
1.458 ± 0.771 1.396 ± 0.644 28.540 ± 4.400 69.171 ± 10.804 164.00 ± 5.292 25.852 ± 4.271 36.931 ± 1.990 109.020 ± 16.195 70.800 ± 6.987 438.170 ± 229.657
p = 0.174304 p = 0.308268 p = 0.718465 p = 0.000014* p = 0.113238 p = 0.000022* 0.000205* p < 0.000001* p < 0.000001* p = 0.026602*
1.750 ± 1.076 1.571 ± 0.826 28.045 ± 6.288 92.379 ± 19.739 167.000 ± 6.656 33.308 ± 5.302 33.753 ± 5.067 161.533 ± 13.279 106.000 ± 27.162 233.414 ± 176.995
Data presented as a mean ± SD. PI – preeclamptic women with pregnancies complicated by IUGR. IUGR – normotensive pregnant women with IUGR alone. P – preeclamptic women without IUGR. * Statistical significance (p < 0.05).
pregnant women [40]. They suggest that elevated Il-18 secretion alone is not sufficient to explain the excessive inflammatory response, but Th1/Th2 ratios were negatively correlated with the ratios of these cytokines, e.g. IL-18 and IL-12 [40]. In normal pregnancy monocytes could produce IL-18 but not IL-12, which results in depressed IL-12 and enhanced IL-18 secretion by non-stimulated PBMC [40]. This elevated IL-18 and depressed IL-12 secretion in normal pregnancy could induce Th2 dominant immune responses that enable fetal survival as a successful allograft [39]. But in preeclamptic pregnancies under oxidative stress condition the excessive stimulated monocytes could produce both IL-18 and IL-12 [40]. This elevated production of both IL-18 and IL-12 in pregnancies complicated by severe preeclampsia result in a Th1 predominant response [40]. Furthermore, IL-18 acts as an epigenetic regulator on chromatin and activates anti-apoptotic signal pathways [37,43]. Interleukin-18 increases the inflammatory process by stimulating the production of tumor necrosis factor, IL1beta and IL-6. IL-18 is also involved in cell-mediated immunity and its actions are synergistic with IL-12 and IL-15 [44]. 5. Conclusions Pregnancies complicated by severe preeclampsia are associated with the decreased levels of interleukin 18 in maternal serum. Normotensive pregnancies complicated by isolated IUGR without preeclampsia are not associated with the abnormal levels of IL-18 in maternal serum. The lack of such association in pregnancies complicated by isolated IUGR may suggest different pathomechanisms inducing impairment of fetal growth in pregnancies complicated by IUGR with and without preeclampsia. Further studies are needed to clarify these aspects and explain the role of IL-18 in preeclamptic pregnancies with and without IUGR in order to improve the management and therapeutic strategies for preeclamptic patients.
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