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Does endothelial cell activation occur with intrauterine growth restriction?
BJOG: an International Journal of Obstetrics and Gynaecology July 2002, Vol. 109, pp. 836 – 839
SHORT COMMUNICATION
Does endothelial cell activation occur with intrauterine growth restriction? M.R. Johnsona,*, N. Anim-Nyamea, P. Johnsonb, S.R. Soorannaa, P.J. Steera It is possible that in fetal growth restriction without pre-eclampsia endothelial cell activation does not occur. This might be either because there is no release of ‘factor X’ or because of maternal resistance to its effects. To test this hypothesis, we took blood samples from 26 women with pre-eclampsia (without fetal growth restriction), 13 women with fetal growth restriction (without pre-eclampsia) and 24 normal pregnant controls, and measured the circulating levels of three markers of endothelial cell activation (soluble VCAM, ICAM and E-selectin) and three cytokines [tumour necrosis factor-a (TNF-a), interleukin-6 (IL-6) and -8 (IL-8)]. The levels of the markers of endothelial cell activation were raised in both pre-eclampsia and fetal growth restriction pregnancies compared with controls; however, the levels of TNF-a, IL-6 and IL-8 were significantly raised in pregnancies complicated by pre-eclampsia, but not in fetal growth restriction, compared with controls. These data show that endothelial cell activation is common to both pre-eclampsia and fetal growth restriction, but that the circulating levels of cytokines are elevated only in pre-eclampsia. Thus, it seems likely that endothelial cell activation is a consequence of a failure of trophoblast invasion and that a further step is required, possibly involving cytokine release, for the expression of the full clinical picture of pre-eclampsia. Introduction Pre-eclampsia and fetal growth restriction have both been attributed to a failure of trophoblast invasion which leads to underperfusion of the uteroplacental bed1. However, why one pregnancy with a failure of trophoblast invasion is complicated by pre-eclampsia alone, another with fetal growth restriction alone and yet another with both, is not known. In pre-eclampsia, it is suggested that underperfusion of the placenta is associated with the release of ‘factor X’ (variously suggested to be cytokines, oxygen free radicals or placental membrane fragments), which either directly or indirectly activates the maternal endothelium and gives rise to the clinical picture of pre-eclampsia. There are data supporting an aetiological role for cytokines in the pathogenesis of pre-eclampsia2. In most studies, maternal circulating levels of the cytokines tumour necrosis factor-a (TNF-a), interleukin-1 (IL-1) receptor antagonist and interleukin-6 (IL-6) are increased in preeclampsia2. The levels of IL-6 are correlated with the levels of VCAM-1 (a marker of endothelial cell activation) in
a
Department of Maternal Fetal Medicine, Imperial College School of Medicine, Chelsea and Westminster Hospital, London, UK b Queen Charlotte’s and Chelsea Hospital, London, UK * Correspondence: Dr M. R. Johnson, Department of Maternal Fetal Medicine, Imperial College School of Medicine, Chelsea and Westminster Hospital, 369 Fulham Road, London, SW10 9NH, UK. D RCOG 2002 BJOG: an International Journal of Obstetrics and Gynaecology PII: S 1 4 7 0 - 0 3 2 8 ( 0 2 ) 0 1 0 4 5 - 5
pre-eclampsia, and IL-6 and TNF-a are both related to disease severity3. In relation to oxygen free radicals, giving anti-oxidant vitamins has been shown to reduce the occurrence of pre-eclampsia in a high risk population4. The release of syncytiotrophoblast membrane fragments is increased in pre-eclampsia; preparations of syncytiotrophoblast membrane fragments inhibit endothelial cell proliferation and increase the release of markers of endothelial cell activation in vitro5. The presumption has been that cytokines, oxygen free radicals and syncytiotrophoblast membrane fragments are involved in mediating the endothelial activation which is thought to be responsible for the characteristic multisystem manifestations of pre-eclampsia. This is supported by the increased circulating levels of the markers of endothelial cell activation, including sVCAM-1, sE-selectin and Von Willebrand factor, which have been reported in women with pre-eclampsia6 – 10. In contrast to the well documented increases of cytokines in pre-eclampsia, there are few data concerning cytokine levels in pregnancies complicated by fetal growth restriction alone. Amniotic fluid levels of TNF-a and IL-10 are elevated11,12, however, placental TNF-a mRNA levels are reported to be unaltered in fetal growth restriction13. Maternal circulating levels of TNF-a, IL-6 and IL-8 are unaltered in fetal growth restriction11, but those of IL-18 are increased14. Similarly, there are limited data with regards to adhesion molecule levels in fetal growth restriction. Placental adhesion molecule mRNA levels and immunoreactivity are similar in fetal growth restriction and in uncomplicated pregnancies15. Extra villous trophoblasts are reported to express more ICAM-3 and less VCAM in fetal www.bjog-elsevier.com
SHORT COMMUNICATION
growth restriction than normal16. Thus, the available data regarding cytokines and adhesion molecule behaviour in fetal growth restriction are incomplete and of uncertain significance. Previous studies investigating the pathogenesis of preeclampsia have usually compared cases with pre-eclampsia to normal controls. Many women with early-onset preeclampsia also have babies which exhibit fetal growth restriction17 but many cases of fetal growth restriction are not associated with pre-eclampsia18. In this study, we have compared women with pre-eclampsia with two further populations, one in which the outcome of the pregnancy was complicated by fetal growth restriction alone and the other in which the outcome of the pregnancy was normal. This approach allows us to test the hypothesis that in fetal growth restriction without pre-eclampsia, endothelial cell activation does not occur. We chose to measure the circulating levels of three markers of endothelial cell activation, VCAM, ICAM and E-selectin and of three cytokines previously reported to be elevated in pre-eclampsia, TNF-a, IL-6 and IL-8.
837
below the third centile for gestational age, in the presence of persistent notching of the uterine artery blood flow waveform on Doppler examination (initial examination performed between 24 and 28 weeks). Exclusion criteria were diabetes, chronic hypertension, known history of peripheral vascular disease and booking body mass index (BMI) less than 19 or more than 30. Blood samples were collected into lithium heparin tubes, spun immediately at 3000 rpm (4jC), separated into aliquots and the plasma stored at 70jC until assayed. Ethics committee approval for the study was obtained at both Chelsea and Westminster Hospital and Queen Charlotte’s Hospital sites. Documented informed consent was obtained from all patients involved in the study. TNF-a, IL-6 and IL-8 were measured using quantikine sandwich ELISA kits obtained from Diaclone Research, Cedex, France. Soluble VCAM, ICAM and E-selectin were measured using kits from R&D Systems, Minneapolis, MN55413. All samples were assayed in a single batch with a within-assay variation of <5%.
Statistical analysis As this was a pilot study to generate data for a grant application, we did not perform a power calculation. The data were not normally distributed and are presented as median (range). Initial comparisons were made with a Kruskall – Wallis test followed by a Mann –Whitney U test.
Methods Venous blood samples were obtained from 26 women with pre-eclampsia, 13 women with fetal growth restriction and 24 normal pregnant controls all in the third trimester of pregnancy. Pre-eclampsia was diagnosed when patients developed both hypertension and proteinuria and was confirmed retrospectively when both hypertension and proteinuria reversed after the pregnancy. Proteinuria was defined as more than 500 mg/L in a 24-hour urine specimen19. Hypertension was defined as an absolute blood pressure greater than 140 mmHg systolic or 90 mmHg diastolic taken twice, at least 6 hours apart. The first and fifth Korotkoff sounds were used to determine the systolic and diastolic components, respectively. Disease severity was assessed using blood pressure, urate levels and proteinuria. Fetal growth restriction was defined as evidence of reduced growth velocity on prenatal ultrasound (‘crossing centiles’) or fetal biometry below the third centile, confirmed by birthweight (Gairdner/Pearson revised 1985)
Results The clinical and demographic characteristics of the subjects are shown in Table 1. The groups were similar in maternal age, gestational age at delivery and pre-pregnancy BMI. Mean arterial blood pressure was significantly greater in the pre-eclampsia group than in either the fetal growth restriction or the normal control groups ( P < 0.0001 for both). Centile birthweight was lower in the fetal growth restriction group than in either pre-eclampsia or control groups ( P < 0.0001 for both). All the women were white, European, non-smokers and none had received antihypertensive treatment at the time of sampling.
Table 1. Clinical data for the three groups of women. Data are expressed as mean (standard deviation), except centile birthweight which are expressed as median [range]. P < 0.05 is considered significant (ANOVA or Kruskall – Wallis). Gestation refers to timing of blood sampling. MABP ¼ mean arterial blood pressure; BMI ¼ body mass index. Normal Subject number Age (years) BMI (kg/m2) Gestation (weeks) Centile birthweight MABP (mmHg)
31.1 19.9 37.4 60 82.6
24 (4.0) (0.02) (2.0) [2 – 99] (2.6)
D RCOG 2002 Br J Obstet Gynaecol 109, pp. 836 – 839
Fetal growth restriction
27.1 23.6 35.5 2.4 91
13 (2.3) (2.3) (4) [0.5 – 5] (8.9)
Pre-eclampsia
P
26 30.6 (7.1) 25.0 (4.4) 36.1 (3.2) 50 [2 – 96] 105.6 (3.5)
0.49 0.34 0.32 <0.0001 <0.0001
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Table 2. The circulating levels of cytokines and markers of endothelial activation in women with a normal outcome of pregnancy, fetal growth restriction (FGR) and pre-eclampsia (PET). Data are expressed as median and range. P < 0.05 is considered significant [Kruskall – Wallis (KW) followed by Mann – Whitney U test]. Control ICAM (Ag/L) VCAM (Ag/L) E-selectin (Ag/L) TNF-a (ng/L) IL-6 (ng/L) IL-8 (ng/L)
186 168 27.5 35.4 15.6 51.4
[89 – 353] [59 – 267] [2.1 – 69.1] [7.8 – 91.9] [10.3 – 38.5] [18.5 – 702.6]
PET 348 340 51.3 176.9 22.6 513.1
[133 – 658] [111 – 527] [26.2 – 158.5] [14.3 – 1064] [10.6 – 376.7] [94.5 – 1829]
FGR 332 401 37.7 40.5 13.2 48.3
[171 – 500] [171 – 758] [25.2 – 91.2] [19 – 208.7] [9.8 – 57.9] [21.4 – 78.2]
The Kruskall – Wallis tests showed significant differences for each analyte ( P ¼ 0.043 –0.0001). The levels of the markers of endothelial cell activation, VCAM, ICAM and E-selectin, were raised in both fetal growth restriction and pre-eclampsia samples compared with normal controls ( P ¼ 0.034 – 0.0001, Table 2) but were not significantly different from one another ( P ¼ 0.07, 0.13 and 0.1, respectively). TNF-a, IL-6 and IL-8 were higher in pregnancies complicated by pre-eclampsia than in either fetal growth restriction ( P ¼ 0.005, 0.0001 and 0.028, respectively) or control ( P ¼ 0.0001, 0.0001 and 0.1, respectively, Table 2). The levels in fetal growth restriction were not significantly different from controls ( P ¼ 0.26, 0.18, 0.86, respectively). There were no associations between the markers of disease severity in pre-eclampsia and any of the analytes.
Discussion These data show that endothelial cell activation is not specific to pre-eclampsia, but that it occurs to a similar degree in fetal growth restriction. Thus, it seems likely that endothelial cell activation is associated with a failure of adequate trophoblast invasion and that a further step is required for the expression of the full clinical picture of pre-eclampsia, which, given their higher levels in preeclampsia in this study, may involve cytokine release. Endothelial cell activation is thought both to be an essential step in the development of pre-eclampsia and, in pregnancy, to occur only in association with pre-eclampsia. These data show that endothelial cell activation is present to a similar degree in fetal growth restriction as well as preeclampsia. The fact that it occurs in both suggests that it is a feature of impaired trophoblast invasion, which is also common to both conditions, although less consistent in mild cases of fetal growth restriction20. The mechanism responsible for the endothelial cell activation is uncertain, but may include the release of syncytiotrophoblast membrane fragments or oxidative stress. The similarity in the circulating cytokine levels in control and fetal growth restriction pregnancies suggests that cytokines are not involved in the process of endothelial cell activation in fetal growth restriction. The elevated levels of VCAM,
KW test
Control vs PET
Control vs FGR
PET vs FGR
0.0001 0.0001 0.0002 0.0001 0.042 0.0001
0.0001 0.0001 0.0001 0.0001 0.09 0.0001
0.0002 0.0001 0.034 0.26 0.15 0.86
0.13 0.07 0.1 0.005 0.028 0.0001
ICAM and E-selectin in pre-eclampsia found in this study are in agreement with most previous data. Where inconsistencies exist in the literature these may reflect differences in disease severity or assay used8 – 10,21,22. Our results also show that the presence of endothelial cell activation itself does not result in the clinical picture of pre-eclampsia, and we suggest that another step is required. This is consistent with previous studies that found no relationship between circulating adhesion molecules and the vascular reactivity of omental vessels23. Taken with our results, it seems likely that an additional mechanism is responsible for the increase in vascular reactivity seen in pre-eclampsia. Given the higher levels of cytokines in the pre-eclampsia group in this study, it is possible that they are responsible as has been suggested previously3,24. These data suggest that in studies of pre-eclampsia, the presence of fetal growth restriction should be considered and controlled for. We have demonstrated that endothelial cell activation is common to both pre-eclampsia and fetal growth restriction, but that raised cytokine levels are found only in pre-eclampsia. We plan further studies to establish whether the degree of oxidative stress or the release of membrane fragments by the placenta is greater in preeclampsia than in fetal growth restriction alone, and thus, whether they may have a role in the pathogenesis of the clinical syndrome of pre-eclampsia.
References 1. Lyall F, Bulmer JN, Kelly H, Duffie E, Robson SC. Human trophoblast invasion and spiral artery transformation: the role of nitric oxide. Am J Pathol 1999;154:1105 – 1114. 2. Conrad KP, Benyo DF. Placental cytokines and the pathogenesis of preeclampsia. Am J Reprod Immunol 1997;37:240 – 249. 3. Greer IA, Lyall F, Perera T, Boswell F, Macara LM. Increased concentrations of cytokines interleukin-6 and interleukin-1 receptor antagonist in plasma of women with preeclampsia: a mechanism for endothelial dysfunction? Obstet Gynecol 1994;84:937 – 940. 4. Chappell LC, Seed PT, Briley AL, et al. Effect of antioxidants on the occurrence of pre-eclampsia in women at increased risk: a randomised trial. Lancet 1999;354:810 – 816. 5. Redman CW, Sargent IL. Placental debris, oxidative stress and pre-eclampsia. Placenta 2000;21:597 – 602. 6. Lyall F, Greer IA, Boswell F, Macara LM, Walker JJ, Kingdom JCP.
D RCOG 2002 Br J Obstet Gynaecol 109, pp. 836 – 839
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The cell adhesion molecule, VCAM-1 is selectively elevated in serum in pre-eclampsia: does this indicate the mechanism of leucocyte activation? Br J Obstet Gynaecol 1994;101:485 – 487. Heyl W, Handt S, Reister F, et al. Elevated soluble adhesion molecules in women with pre-eclampsia. Do cytokines like tumour necrosis factor-alpha and interleukin-1beta cause endothelial activation. Eur J Obstet Gynecol Reprod Biol 1999;86:35 – 41. Heyl W, Handt S, Reister F, Gehlen J, Mittermayer C, Rath W. The role of soluble adhesion molecules in evaluating endothelial cell activation in preeclampsia. Am J Obstet Gynecol 1999;180:68 – 72. Budak E, Madazli R, Aksu MF, et al. Vascular cell adhesion molecule-1 (VCAM-1) and leukocyte activation in pre-eclampsia and eclampsia. Int J Gynaecol Obstet 1998;63:115 – 121. Wang Y, Adair CD, Coe L, Weeks JW, Lewis DF, Alexander JS. Activation of endothelial cells in preeclampsia: increased neutrophilendothelial adhesion correlates with up-regulation of adhesion molecule P-selectin in human umbilical vein endothelial cells isolated from preeclampsia. J Soc Gynecol Invest 1998;5:237 – 243. Ida A, Tsuji Y, Muranaka J, et al. IL-18 in pregnancy; the elevation of IL-18 in maternal peripheral blood during labour and complicated pregnancies. J Reprod Immunol 2000;47:65 – 74. Stallmach T, Hebisch G, Joller-Jemelka HI, Orban P, Schwaller J, Engelmann M. Cytokine production and visualized effects in the fetomaternal unit. Quantitative and topographic data on cytokines during intrauterine disease. Lab Invest 1995;73:384 – 392. Heinig J, Wilhelm S, Muller H, Briese V, Bittorf T, Brock J. Determination of cytokine mRNA-expression in term human placenta of patients with gestational hypertension, intrauterine growth retardation and gestational diabetes mellitus using polymerase chain reaction. Zentralbl Gynaekol 2000;122:413 – 418. Heyborne KD, McGregor JA, Henry G, Witkin SS, Abrams JS. Interleukin-10 in amniotic fluid at midtrimester: immune activation and suppression in relation to fetal growth. Am J Obstet Gynecol 1994; 171:55 – 59.
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15. Lyall F, Greer IA, Boswell F, Young A, Macara LM, Jeffers MD. Expression of cell adhesion molecules in placentae from pregnancies complicated by pre-eclampsia and intrauterine growth retardation. Placenta 1995;16:579 – 587. 16. Zygmunt M, Boving B, Weinhard J, Munstedt K, Bohle RM, Lang U. Expression of cell adhesion molecules in the extravillous trophoblast is altered in IUGR. Am J Reprod Immunol 1997;38:295 – 301. 17. Long PA, Abell DA, Beischer NA. Fetal growth retardation and pre-eclampsia. Br J Obstet Gynaecol 1980;87:13 – 18. 18. von Dadelszen P, Watson RW, Noorwali F, et al. Maternal neutrophil apoptosis in normal pregnancy, preeclampsia, and normotensive intrauterine growth restriction. Am J Obstet Gynecol 1999;181:408 – 414. 19. Davey DA, MacGillivray I. The classification and definition of the hypertensive disorders of pregnancy. Am J Obstet Gynecol 1988; 158:892 – 898. 20. Gerretsen G, Huisjes HJ, Elema JD. Morphological changes of the spiral arteries in the placental bed in relation to pre-eclampsia and fetal growth retardation. Br J Obstet Gynaecol 1981;88:876 – 881. 21. Hayman R, Brockelsby J, Kenny L, Baker P. Preeclampsia: the endothelium, circulating factor(s) and vascular endothelial growth factor. J Soc Gynecol Invest 1999;6:3 – 10. 22. Haller H, Ziegler EM, Houth V, et al. Endothelial adhesion molecules and leukocyte integrins in preeclamptic patients. Hypertension 1997; 29:291 – 296. 23. Lyall F, Hayman RG, Ashworth JR, Duffie E, Baker PN. Relationship of cell adhesion molecule expression to endothelium-dependent relaxation in normal pregnancy and pregnancies complicated with preeclampsia or fetal growth restriction. J Soc Gynecol Invest 1999; 6:196 – 201. 24. Kupferminc MJ, Peaceman AM, Aderka D, Wallach D, Socol ML. Soluble tumor necrosis factor receptors and interleukin-6 levels in patients with severe preeclampsia. Obstet Gynecol 1996;88:420 – 427. Accepted 17 January 2002
SHORT COMMUNICATION
Does endothelial cell activation occur with intrauterine growth restriction? M.R. Johnsona,*, N. Anim-Nyamea, P. Johnsonb, S.R. Soorannaa, P.J. Steera It is possible that in fetal growth restriction without pre-eclampsia endothelial cell activation does not occur. This might be either because there is no release of ‘factor X’ or because of maternal resistance to its effects. To test this hypothesis, we took blood samples from 26 women with pre-eclampsia (without fetal growth restriction), 13 women with fetal growth restriction (without pre-eclampsia) and 24 normal pregnant controls, and measured the circulating levels of three markers of endothelial cell activation (soluble VCAM, ICAM and E-selectin) and three cytokines [tumour necrosis factor-a (TNF-a), interleukin-6 (IL-6) and -8 (IL-8)]. The levels of the markers of endothelial cell activation were raised in both pre-eclampsia and fetal growth restriction pregnancies compared with controls; however, the levels of TNF-a, IL-6 and IL-8 were significantly raised in pregnancies complicated by pre-eclampsia, but not in fetal growth restriction, compared with controls. These data show that endothelial cell activation is common to both pre-eclampsia and fetal growth restriction, but that the circulating levels of cytokines are elevated only in pre-eclampsia. Thus, it seems likely that endothelial cell activation is a consequence of a failure of trophoblast invasion and that a further step is required, possibly involving cytokine release, for the expression of the full clinical picture of pre-eclampsia. Introduction Pre-eclampsia and fetal growth restriction have both been attributed to a failure of trophoblast invasion which leads to underperfusion of the uteroplacental bed1. However, why one pregnancy with a failure of trophoblast invasion is complicated by pre-eclampsia alone, another with fetal growth restriction alone and yet another with both, is not known. In pre-eclampsia, it is suggested that underperfusion of the placenta is associated with the release of ‘factor X’ (variously suggested to be cytokines, oxygen free radicals or placental membrane fragments), which either directly or indirectly activates the maternal endothelium and gives rise to the clinical picture of pre-eclampsia. There are data supporting an aetiological role for cytokines in the pathogenesis of pre-eclampsia2. In most studies, maternal circulating levels of the cytokines tumour necrosis factor-a (TNF-a), interleukin-1 (IL-1) receptor antagonist and interleukin-6 (IL-6) are increased in preeclampsia2. The levels of IL-6 are correlated with the levels of VCAM-1 (a marker of endothelial cell activation) in
a
Department of Maternal Fetal Medicine, Imperial College School of Medicine, Chelsea and Westminster Hospital, London, UK b Queen Charlotte’s and Chelsea Hospital, London, UK * Correspondence: Dr M. R. Johnson, Department of Maternal Fetal Medicine, Imperial College School of Medicine, Chelsea and Westminster Hospital, 369 Fulham Road, London, SW10 9NH, UK. D RCOG 2002 BJOG: an International Journal of Obstetrics and Gynaecology PII: S 1 4 7 0 - 0 3 2 8 ( 0 2 ) 0 1 0 4 5 - 5
pre-eclampsia, and IL-6 and TNF-a are both related to disease severity3. In relation to oxygen free radicals, giving anti-oxidant vitamins has been shown to reduce the occurrence of pre-eclampsia in a high risk population4. The release of syncytiotrophoblast membrane fragments is increased in pre-eclampsia; preparations of syncytiotrophoblast membrane fragments inhibit endothelial cell proliferation and increase the release of markers of endothelial cell activation in vitro5. The presumption has been that cytokines, oxygen free radicals and syncytiotrophoblast membrane fragments are involved in mediating the endothelial activation which is thought to be responsible for the characteristic multisystem manifestations of pre-eclampsia. This is supported by the increased circulating levels of the markers of endothelial cell activation, including sVCAM-1, sE-selectin and Von Willebrand factor, which have been reported in women with pre-eclampsia6 – 10. In contrast to the well documented increases of cytokines in pre-eclampsia, there are few data concerning cytokine levels in pregnancies complicated by fetal growth restriction alone. Amniotic fluid levels of TNF-a and IL-10 are elevated11,12, however, placental TNF-a mRNA levels are reported to be unaltered in fetal growth restriction13. Maternal circulating levels of TNF-a, IL-6 and IL-8 are unaltered in fetal growth restriction11, but those of IL-18 are increased14. Similarly, there are limited data with regards to adhesion molecule levels in fetal growth restriction. Placental adhesion molecule mRNA levels and immunoreactivity are similar in fetal growth restriction and in uncomplicated pregnancies15. Extra villous trophoblasts are reported to express more ICAM-3 and less VCAM in fetal www.bjog-elsevier.com
SHORT COMMUNICATION
growth restriction than normal16. Thus, the available data regarding cytokines and adhesion molecule behaviour in fetal growth restriction are incomplete and of uncertain significance. Previous studies investigating the pathogenesis of preeclampsia have usually compared cases with pre-eclampsia to normal controls. Many women with early-onset preeclampsia also have babies which exhibit fetal growth restriction17 but many cases of fetal growth restriction are not associated with pre-eclampsia18. In this study, we have compared women with pre-eclampsia with two further populations, one in which the outcome of the pregnancy was complicated by fetal growth restriction alone and the other in which the outcome of the pregnancy was normal. This approach allows us to test the hypothesis that in fetal growth restriction without pre-eclampsia, endothelial cell activation does not occur. We chose to measure the circulating levels of three markers of endothelial cell activation, VCAM, ICAM and E-selectin and of three cytokines previously reported to be elevated in pre-eclampsia, TNF-a, IL-6 and IL-8.
837
below the third centile for gestational age, in the presence of persistent notching of the uterine artery blood flow waveform on Doppler examination (initial examination performed between 24 and 28 weeks). Exclusion criteria were diabetes, chronic hypertension, known history of peripheral vascular disease and booking body mass index (BMI) less than 19 or more than 30. Blood samples were collected into lithium heparin tubes, spun immediately at 3000 rpm (4jC), separated into aliquots and the plasma stored at 70jC until assayed. Ethics committee approval for the study was obtained at both Chelsea and Westminster Hospital and Queen Charlotte’s Hospital sites. Documented informed consent was obtained from all patients involved in the study. TNF-a, IL-6 and IL-8 were measured using quantikine sandwich ELISA kits obtained from Diaclone Research, Cedex, France. Soluble VCAM, ICAM and E-selectin were measured using kits from R&D Systems, Minneapolis, MN55413. All samples were assayed in a single batch with a within-assay variation of <5%.
Statistical analysis As this was a pilot study to generate data for a grant application, we did not perform a power calculation. The data were not normally distributed and are presented as median (range). Initial comparisons were made with a Kruskall – Wallis test followed by a Mann –Whitney U test.
Methods Venous blood samples were obtained from 26 women with pre-eclampsia, 13 women with fetal growth restriction and 24 normal pregnant controls all in the third trimester of pregnancy. Pre-eclampsia was diagnosed when patients developed both hypertension and proteinuria and was confirmed retrospectively when both hypertension and proteinuria reversed after the pregnancy. Proteinuria was defined as more than 500 mg/L in a 24-hour urine specimen19. Hypertension was defined as an absolute blood pressure greater than 140 mmHg systolic or 90 mmHg diastolic taken twice, at least 6 hours apart. The first and fifth Korotkoff sounds were used to determine the systolic and diastolic components, respectively. Disease severity was assessed using blood pressure, urate levels and proteinuria. Fetal growth restriction was defined as evidence of reduced growth velocity on prenatal ultrasound (‘crossing centiles’) or fetal biometry below the third centile, confirmed by birthweight (Gairdner/Pearson revised 1985)
Results The clinical and demographic characteristics of the subjects are shown in Table 1. The groups were similar in maternal age, gestational age at delivery and pre-pregnancy BMI. Mean arterial blood pressure was significantly greater in the pre-eclampsia group than in either the fetal growth restriction or the normal control groups ( P < 0.0001 for both). Centile birthweight was lower in the fetal growth restriction group than in either pre-eclampsia or control groups ( P < 0.0001 for both). All the women were white, European, non-smokers and none had received antihypertensive treatment at the time of sampling.
Table 1. Clinical data for the three groups of women. Data are expressed as mean (standard deviation), except centile birthweight which are expressed as median [range]. P < 0.05 is considered significant (ANOVA or Kruskall – Wallis). Gestation refers to timing of blood sampling. MABP ¼ mean arterial blood pressure; BMI ¼ body mass index. Normal Subject number Age (years) BMI (kg/m2) Gestation (weeks) Centile birthweight MABP (mmHg)
31.1 19.9 37.4 60 82.6
24 (4.0) (0.02) (2.0) [2 – 99] (2.6)
D RCOG 2002 Br J Obstet Gynaecol 109, pp. 836 – 839
Fetal growth restriction
27.1 23.6 35.5 2.4 91
13 (2.3) (2.3) (4) [0.5 – 5] (8.9)
Pre-eclampsia
P
26 30.6 (7.1) 25.0 (4.4) 36.1 (3.2) 50 [2 – 96] 105.6 (3.5)
0.49 0.34 0.32 <0.0001 <0.0001
838
SHORT COMMUNICATION
Table 2. The circulating levels of cytokines and markers of endothelial activation in women with a normal outcome of pregnancy, fetal growth restriction (FGR) and pre-eclampsia (PET). Data are expressed as median and range. P < 0.05 is considered significant [Kruskall – Wallis (KW) followed by Mann – Whitney U test]. Control ICAM (Ag/L) VCAM (Ag/L) E-selectin (Ag/L) TNF-a (ng/L) IL-6 (ng/L) IL-8 (ng/L)
186 168 27.5 35.4 15.6 51.4
[89 – 353] [59 – 267] [2.1 – 69.1] [7.8 – 91.9] [10.3 – 38.5] [18.5 – 702.6]
PET 348 340 51.3 176.9 22.6 513.1
[133 – 658] [111 – 527] [26.2 – 158.5] [14.3 – 1064] [10.6 – 376.7] [94.5 – 1829]
FGR 332 401 37.7 40.5 13.2 48.3
[171 – 500] [171 – 758] [25.2 – 91.2] [19 – 208.7] [9.8 – 57.9] [21.4 – 78.2]
The Kruskall – Wallis tests showed significant differences for each analyte ( P ¼ 0.043 –0.0001). The levels of the markers of endothelial cell activation, VCAM, ICAM and E-selectin, were raised in both fetal growth restriction and pre-eclampsia samples compared with normal controls ( P ¼ 0.034 – 0.0001, Table 2) but were not significantly different from one another ( P ¼ 0.07, 0.13 and 0.1, respectively). TNF-a, IL-6 and IL-8 were higher in pregnancies complicated by pre-eclampsia than in either fetal growth restriction ( P ¼ 0.005, 0.0001 and 0.028, respectively) or control ( P ¼ 0.0001, 0.0001 and 0.1, respectively, Table 2). The levels in fetal growth restriction were not significantly different from controls ( P ¼ 0.26, 0.18, 0.86, respectively). There were no associations between the markers of disease severity in pre-eclampsia and any of the analytes.
Discussion These data show that endothelial cell activation is not specific to pre-eclampsia, but that it occurs to a similar degree in fetal growth restriction. Thus, it seems likely that endothelial cell activation is associated with a failure of adequate trophoblast invasion and that a further step is required for the expression of the full clinical picture of pre-eclampsia, which, given their higher levels in preeclampsia in this study, may involve cytokine release. Endothelial cell activation is thought both to be an essential step in the development of pre-eclampsia and, in pregnancy, to occur only in association with pre-eclampsia. These data show that endothelial cell activation is present to a similar degree in fetal growth restriction as well as preeclampsia. The fact that it occurs in both suggests that it is a feature of impaired trophoblast invasion, which is also common to both conditions, although less consistent in mild cases of fetal growth restriction20. The mechanism responsible for the endothelial cell activation is uncertain, but may include the release of syncytiotrophoblast membrane fragments or oxidative stress. The similarity in the circulating cytokine levels in control and fetal growth restriction pregnancies suggests that cytokines are not involved in the process of endothelial cell activation in fetal growth restriction. The elevated levels of VCAM,
KW test
Control vs PET
Control vs FGR
PET vs FGR
0.0001 0.0001 0.0002 0.0001 0.042 0.0001
0.0001 0.0001 0.0001 0.0001 0.09 0.0001
0.0002 0.0001 0.034 0.26 0.15 0.86
0.13 0.07 0.1 0.005 0.028 0.0001
ICAM and E-selectin in pre-eclampsia found in this study are in agreement with most previous data. Where inconsistencies exist in the literature these may reflect differences in disease severity or assay used8 – 10,21,22. Our results also show that the presence of endothelial cell activation itself does not result in the clinical picture of pre-eclampsia, and we suggest that another step is required. This is consistent with previous studies that found no relationship between circulating adhesion molecules and the vascular reactivity of omental vessels23. Taken with our results, it seems likely that an additional mechanism is responsible for the increase in vascular reactivity seen in pre-eclampsia. Given the higher levels of cytokines in the pre-eclampsia group in this study, it is possible that they are responsible as has been suggested previously3,24. These data suggest that in studies of pre-eclampsia, the presence of fetal growth restriction should be considered and controlled for. We have demonstrated that endothelial cell activation is common to both pre-eclampsia and fetal growth restriction, but that raised cytokine levels are found only in pre-eclampsia. We plan further studies to establish whether the degree of oxidative stress or the release of membrane fragments by the placenta is greater in preeclampsia than in fetal growth restriction alone, and thus, whether they may have a role in the pathogenesis of the clinical syndrome of pre-eclampsia.
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