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Placenta growth factor is not an early marker for the development of severe preeclampsia
Placenta growth factor is not an early marker for the development of severe preeclampsia Jeffrey C. Livingston, MD,a Bassam Haddad, MD,a Laura A. Gorski, DO,a Paul Neblett, MD,a Robert A. Ahokas, PhD,a Risa Ramsey,a and Baha M. Sibai, MDb Memphis, Tennessee, and Cincinnati, Ohio OBJECTIVE: Our purpose was to determine whether plasma concentrations of placenta growth factor may be used as a marker for women who ultimately have severe preeclampsia. STUDY DESIGN: We performed a nested case-control study to compare plasma concentrations of placenta growth factor in women with severe preeclampsia with the concentrations in normotensive pregnant control subjects. Plasma samples were collected at <20 weeks’ gestation and again in the third trimester. Twenty-two women who ultimately had severe preeclampsia were matched for gestational age at delivery with 22 normotensive control subjects. Placenta growth factor concentrations were measured by a specific antigen capture enzyme-linked immunosorbent assay. Comparisons were made by using the Mann-Whitney U test for nonparametric data such as placenta growth factor concentrations. The Student t test was used for parametric data. RESULTS: A total of 880 pregnant women were screened. Severe preeclampsia developed in 22, for an incidence of 2.5%. As expected, women with severe preeclampsia had significantly higher systolic and diastolic blood pressures, and their infants had lower birth weights. Placental weights at delivery were similar between those with severe preeclampsia and control subjects (659 vs 699 g; P = .51). During the third trimester, the median placenta growth factor concentrations were significantly lower in women with severe preeclampsia than in normotensive control subjects (125 vs 449 pg/mL; P = .003). When samples drawn at <20 weeks’ gestation were compared, there was no difference between the group with severe preeclampsia and those who remained normotensive (98.8 vs 56.34 pg/mL; P = .15). CONCLUSION: During the third trimester, patients with severe preeclampsia have decreased maternal concentrations of placenta growth factor. This difference is not seen earlier in pregnancy. Lower concentrations of placenta growth factor may be a result of severe preeclampsia rather than a causal factor. Placenta growth factor is not a good marker for the subsequent development of severe preeclampsia. (Am J Obstet Gynecol 2001;184:1218-20.)
Key words: Preeclampsia, placenta growth factor, predictor
Preeclampsia is a multisystem disorder that remains one of the leading causes of maternal and fetal morbidity and death. Diagnosed in 5% to 10% of pregnancies, preeclampsia ranks as the number two cause of maternal mortality, second only to thromboembolic events of pregnancy.1 Despite considerable investigation, the exact pathogenesis of preeclampsia remains evasive. Placenta growth factor (PLGF) is a homologous protein member of the endothelial growth factor family. PLGF possesses potent angiogenic and mitogenic activity capable of inducing proliferation, migration, and activation of enFrom the Department of Obstetrics and Gynecology, University of Tennessee Health Sciences Center,a and the Department of Obstetrics and Gynecology, University of Cincinnati.b Received for publication June 7, 2000; revised August 2, 2000; accepted December 27, 2000. Reprint requests: Baha M. Sibai, MD, Professor and Chair, Department of Obstetrics and Gynecology, University of Cincinnati, 231 Bethesda Ave, PO Box 670526, Cincinnati, OH 45267. Copyright © 2001 by Mosby, Inc. 0002-9378/2001 $35.00 + 0 6/1/113877 doi:10.1067/mob.2001.113877
1218
dothelial cells.2, 3 Endothelial cell damage is a hallmark of preeclampsia. Recently, PLGF concentrations have been shown to be decreased in pregnancies at the time of diagnosis of preeclampsia.4-6 The site of primary expression and synthesis of PLGF is the placenta, specifically, the trophoblast.7 Reduced PLGF concentrations during development of the uteroplacental unit may result in decreased angiogenesis and decreased trophoblastic invasion into maternal spiral arteries. Decreased spiral artery invasion by the trophoblast early in pregnancy is considered a major abnormality in women with preeclampsia.8 We hypothesized that the concentration of PLGF should be lower before 20 weeks’ gestation in women in whom severe preeclampsia develops than in those who remain normotensive. Alteration in PLGF concentrations before clinically evident disease makes PLGF a potential marker for severe preeclampsia. The purpose of this study was to investigate whether circulating concentrations of PLGF are decreased early in pregnancy in women who subsequently have severe preeclampsia.
Livingston et al 1219
Volume 184, Number 6 Am J Obstet Gynecol
Table I. Pregnancy characteristics
Gestational age at delivery (wk) Mean ± SD Range Systolic blood pressure (mm Hg, mean ± SD) Diastolic blood pressure (mm Hg, mean ± SD) Birth weight (g, mean ± SD) Placental weight (g, mean ± SD)
Severe preeclampsia
Control subjects
38.3 ± 2.3 33.5-41.4 160.0 ± 14.3 106.5 ± 9.9 2889 ± 603 659 ± 216
39.2 ± 1.6 35.6-42.0 134.5 ± 12.5 84.8 ± 6.4 3284 ± 501 699 ± 160
Statistical significance
P = .09 P < .0001 P < .0001 P = .02 P = .51
Table II. Growth factor concentrations
Early pregnancy Gestational age (wk, mean ± SD and range) PLGF (pg/mL, median and range) Late pregnancy Gestational age (wk, mean ± SD and range) PLGF (pg/mL, median and range)
Severe preeclampsia
Control subjects
Statistical significance
15.0 ± 3.3 (10.9-19.7) 98.8 (29.3-540.6)
15.1 ± 3.3 (10.4-19.6) 56.3 (11.4-298.8)
P = .89 P = .15
37.3 ± 1.6 (35.1-40.9) 125.2 (64-657)
37.0 ± 1.5 (34.3-40.9) 449 (101-1101)
P = .50 P = .003
Material and methods The Institutional Review Board at the University of Tennessee, Memphis, approved this study. We performed a nested, case-control analysis to compare plasma concentrations of PLGF in women with severe preeclampsia with the concentrations in normotensive pregnant control subjects. Reuvekamp et al6 demonstrated a decrease in PLGF concentrations in women with preeclampsia in the third trimester (54.19 ± 32.5 pg/mL vs 497.95 ± 340.51 pg/mL). On the basis of this difference, we calculated that a sample size of 11 patients in each arm would be needed, with β = .2.4 Because we had no previous data on early pregnancy concentrations of PLGF in women with preeclampsia, we doubled the sample size. The inclusion criterion for entry was intrauterine pregnancy at <20 weeks without major fetal anomalies. The exclusion criteria included maternal chronic hypertension, multifetal gestation, known preexisting microvascular disease, and fetal growth restriction, defined as birth weight <10th percentile for gestational age.9 Pregnancies with intrauterine growth restriction were excluded because differences in placental weights may introduce a confounding bias. Severe preeclampsia was defined as systolic blood pressure >160 mm Hg or diastolic blood pressure >110 mm Hg on two occasions 6 hours apart in the presence of new-onset proteinuria. Two women with eclampsia were also included in the group with severe preeclampsia. Maternal blood samples were collected at <20 weeks’ gestation into standard collection tubes (with ethylenediaminetetraacetic acid) and centrifuged, at room temperature, at 5000 rpm for 5 minutes. A second maternal sample was obtained and processed at the time of diagnosis of severe preeclampsia or, for the control group, at admission for delivery. Both plasma aliquots were stored at –70°C until assayed. Concentrations of plasma PLGF
were then assessed by specific antigen-capture enzymelinked immunosorbent assay (R&D Systems, Inc, Minneapolis, Minn). Concentrations of PLGF in the plasma samples were calculated from a standard curve generated with doubling dilutions of recombinant human PLGF. The minimum detectable level of PLGF by this assay is 5 pg/mL. A 5% cross-reactivity occurs with this assay and PLGF–vascular endothelial growth factor heterodimers. Control subjects were selected from the 880 women who enrolled but remained normotensive. Patients were matched for the time of gestation at which blood samples were obtained. The same patients were used for both early and late plasma samples. The Mann-Whitney U test was used to compare PLGF concentrations and other nonparametric continuous variables. Patient characteristics were compared by the Mann-Whitney U test or the Student t test, where appropriate. A P value of < .05 was considered to be significant. Results A total of 880 nonconsecutive patients were entered into the study. Severe preeclampsia developed in 22, for a prevalence of 2.5%. Pregnancy characteristics are noted in Table I. In comparison with normotensive pregnant control subjects, women with severe preeclampsia had significantly higher blood pressures, and their infants had lower birth weights. In addition, there were no differences in maternal age, parity, weight, or ethnicity (data not shown). Table II summarizes PLGF concentrations and gestational time of sampling in both groups. PLGF concentrations in the third trimester were significantly lower in women with preeclampsia than in women with normotensive pregnancies. There was no decrease in PLGF
1220 Livingston et al
concentrations between the group with severe preeclampsia and the control group when samples were obtained at <20 weeks’ gestation. In fact, there was a trend toward an increase in PLGF in early pregnancy. Comment Abnormal placentation is one of the initial events of severe preeclampsia. The main feature of abnormal placentation is the inadequate trophoblastic invasion of the maternal spiral arterioles. Recent evidence has shown that the presence and function of vascular endothelial growth factor and PLGF are critical during this cytotrophoblastic invasion.10 Our study is the first to evaluate PLGF concentrations early in pregnancy in women who subsequently had severe preeclampsia. Our findings of decreased PLGF at term (but not early in pregnancy) do not support the hypothesis that a reduction in growth factors is responsible for abnormal placentation. Rather, our findings suggest that lower PLGF concentrations seen later in pregnancy may be a result of earlier abnormal placentation. Moreover, because PLGF is known to be down-regulated in hypoxic environments,7 normal PLGF levels in early pregnancy do not support a hypoxiainitiated pathogenesis of preeclampsia. Our sample size was large enough to confirm previously published findings of decreased PLGF at the diagnosis of preeclampsia.4, 6 Our findings do not support a trend of decreased PLGF concentrations early in pregnancy. One limitation of our study is the fact that we evaluated circulating maternal plasma concentrations of PLGF rather than studying PLGF in its local uteroplacental environment. Growth factors are known to work locally, and circulating concentrations may not be representative of values in the uteroplacental circulation. Our
May 2001 Am J Obstet Gynecol
assay measured only the free PLGF 149 isoform, and we cannot comment on total PLGF concentrations. In conclusion, PLGF concentrations are reduced in pregnancies once severe preeclampsia becomes clinically evident. PLGF concentrations were not altered earlier in pregnancy, which suggests that PLGF is not a good marker for the development of subsequent severe preeclampsia. REFERENCES
1. Sibai BM. Hypertension in pregnancy. Obstet Gynecol Clin North Am 1992;19:15-32. 2. Cao Y, Chen A, An SSA, Ji RW, Davidson D, Llinas M. Kringle 5 of plasminogen is a novel inhibitor of endothelial cell growth. J Biol Chem 1997;272:22924-8. 3. Park JE, Chen HH, Winer J, Houck KA, Ferrara N. Placenta growth factor: potentiation of vascular endothelial growth factor bioactivity, in vitro and in vivo, and high affinity binding to Flt-1 but not to Flk-1/KDR. J Biol Chem 1994;269:25646-54. 4. Livingston JC, Chin R, Haddad B, McKinney ET, Ahokas R, Sibai BM. Reductions of vascular endothelial growth factor and placental growth factor concentrations in severe preeclampsia. Am J Obstet Gynecol 2000;183:1554-7. 5. Torry DS, Wang HS, Wang TH, Caudle MR, Torry RJ. Preeclampsia is associated with reduced plasma levels of placenta growth factor. Am J Obstet Gynecol 1998;179:1539-44. 6. Reuvekamp A, Velsing-Aarts FV, Poulina IEJ, Capello JJ, Duits AJ. Selective deficit of angiogenic growth factors characterizes pregnancies complicated by pre-eclampsia. BJOG 1999;106:1019-22. 7. Shore VH, Wang TH, Wang CL, Torry RJ, Caudle MR, Torry DS. Vascular endothelial growth factor and their receptors in isolated human trophoblast. Placenta 1997;18:657-65. 8. Kong TY, DeWolf F, Robertson WB, Brosens I. Inadequate maternal vascular response to placentation in pregnancies complicated by preeclampsia and by small-for-gestational age infants. BJOG 1986;93:1049-59. 9. Lubchenco LO, Hansman C, Boyd E. Intrauterine growth in length and head circumference as estimated from live births at gestational ages 26 to 42 weeks. Pediatrics 1966;37:403-8. 10. Vuoral P, Hatva E, Lymboussaki A, et al. Expression of vascular endothelial growth factor and placental growth factor in human placenta. Biol Reprod 1997;56:489-94.
Receive tables of contents by e-mail To receive the tables of contents by e-mail, sign up through our Web site at http://www.mosby.com/ajog. Choose E-mail Notification. Simply type your e-mail address in the box and click the Subscribe button. Alternatively, you may send an e-mail message to [email protected]. Leave the subject line blank, and type the following as the body of your message: Subscribe ajog_toc. You will receive an e-mail to confirm that you have been added to the mailing list. Note that table of contents e-mails will be sent out when a new issue is posted to the Web site.
Key words: Preeclampsia, placenta growth factor, predictor
Preeclampsia is a multisystem disorder that remains one of the leading causes of maternal and fetal morbidity and death. Diagnosed in 5% to 10% of pregnancies, preeclampsia ranks as the number two cause of maternal mortality, second only to thromboembolic events of pregnancy.1 Despite considerable investigation, the exact pathogenesis of preeclampsia remains evasive. Placenta growth factor (PLGF) is a homologous protein member of the endothelial growth factor family. PLGF possesses potent angiogenic and mitogenic activity capable of inducing proliferation, migration, and activation of enFrom the Department of Obstetrics and Gynecology, University of Tennessee Health Sciences Center,a and the Department of Obstetrics and Gynecology, University of Cincinnati.b Received for publication June 7, 2000; revised August 2, 2000; accepted December 27, 2000. Reprint requests: Baha M. Sibai, MD, Professor and Chair, Department of Obstetrics and Gynecology, University of Cincinnati, 231 Bethesda Ave, PO Box 670526, Cincinnati, OH 45267. Copyright © 2001 by Mosby, Inc. 0002-9378/2001 $35.00 + 0 6/1/113877 doi:10.1067/mob.2001.113877
1218
dothelial cells.2, 3 Endothelial cell damage is a hallmark of preeclampsia. Recently, PLGF concentrations have been shown to be decreased in pregnancies at the time of diagnosis of preeclampsia.4-6 The site of primary expression and synthesis of PLGF is the placenta, specifically, the trophoblast.7 Reduced PLGF concentrations during development of the uteroplacental unit may result in decreased angiogenesis and decreased trophoblastic invasion into maternal spiral arteries. Decreased spiral artery invasion by the trophoblast early in pregnancy is considered a major abnormality in women with preeclampsia.8 We hypothesized that the concentration of PLGF should be lower before 20 weeks’ gestation in women in whom severe preeclampsia develops than in those who remain normotensive. Alteration in PLGF concentrations before clinically evident disease makes PLGF a potential marker for severe preeclampsia. The purpose of this study was to investigate whether circulating concentrations of PLGF are decreased early in pregnancy in women who subsequently have severe preeclampsia.
Livingston et al 1219
Volume 184, Number 6 Am J Obstet Gynecol
Table I. Pregnancy characteristics
Gestational age at delivery (wk) Mean ± SD Range Systolic blood pressure (mm Hg, mean ± SD) Diastolic blood pressure (mm Hg, mean ± SD) Birth weight (g, mean ± SD) Placental weight (g, mean ± SD)
Severe preeclampsia
Control subjects
38.3 ± 2.3 33.5-41.4 160.0 ± 14.3 106.5 ± 9.9 2889 ± 603 659 ± 216
39.2 ± 1.6 35.6-42.0 134.5 ± 12.5 84.8 ± 6.4 3284 ± 501 699 ± 160
Statistical significance
P = .09 P < .0001 P < .0001 P = .02 P = .51
Table II. Growth factor concentrations
Early pregnancy Gestational age (wk, mean ± SD and range) PLGF (pg/mL, median and range) Late pregnancy Gestational age (wk, mean ± SD and range) PLGF (pg/mL, median and range)
Severe preeclampsia
Control subjects
Statistical significance
15.0 ± 3.3 (10.9-19.7) 98.8 (29.3-540.6)
15.1 ± 3.3 (10.4-19.6) 56.3 (11.4-298.8)
P = .89 P = .15
37.3 ± 1.6 (35.1-40.9) 125.2 (64-657)
37.0 ± 1.5 (34.3-40.9) 449 (101-1101)
P = .50 P = .003
Material and methods The Institutional Review Board at the University of Tennessee, Memphis, approved this study. We performed a nested, case-control analysis to compare plasma concentrations of PLGF in women with severe preeclampsia with the concentrations in normotensive pregnant control subjects. Reuvekamp et al6 demonstrated a decrease in PLGF concentrations in women with preeclampsia in the third trimester (54.19 ± 32.5 pg/mL vs 497.95 ± 340.51 pg/mL). On the basis of this difference, we calculated that a sample size of 11 patients in each arm would be needed, with β = .2.4 Because we had no previous data on early pregnancy concentrations of PLGF in women with preeclampsia, we doubled the sample size. The inclusion criterion for entry was intrauterine pregnancy at <20 weeks without major fetal anomalies. The exclusion criteria included maternal chronic hypertension, multifetal gestation, known preexisting microvascular disease, and fetal growth restriction, defined as birth weight <10th percentile for gestational age.9 Pregnancies with intrauterine growth restriction were excluded because differences in placental weights may introduce a confounding bias. Severe preeclampsia was defined as systolic blood pressure >160 mm Hg or diastolic blood pressure >110 mm Hg on two occasions 6 hours apart in the presence of new-onset proteinuria. Two women with eclampsia were also included in the group with severe preeclampsia. Maternal blood samples were collected at <20 weeks’ gestation into standard collection tubes (with ethylenediaminetetraacetic acid) and centrifuged, at room temperature, at 5000 rpm for 5 minutes. A second maternal sample was obtained and processed at the time of diagnosis of severe preeclampsia or, for the control group, at admission for delivery. Both plasma aliquots were stored at –70°C until assayed. Concentrations of plasma PLGF
were then assessed by specific antigen-capture enzymelinked immunosorbent assay (R&D Systems, Inc, Minneapolis, Minn). Concentrations of PLGF in the plasma samples were calculated from a standard curve generated with doubling dilutions of recombinant human PLGF. The minimum detectable level of PLGF by this assay is 5 pg/mL. A 5% cross-reactivity occurs with this assay and PLGF–vascular endothelial growth factor heterodimers. Control subjects were selected from the 880 women who enrolled but remained normotensive. Patients were matched for the time of gestation at which blood samples were obtained. The same patients were used for both early and late plasma samples. The Mann-Whitney U test was used to compare PLGF concentrations and other nonparametric continuous variables. Patient characteristics were compared by the Mann-Whitney U test or the Student t test, where appropriate. A P value of < .05 was considered to be significant. Results A total of 880 nonconsecutive patients were entered into the study. Severe preeclampsia developed in 22, for a prevalence of 2.5%. Pregnancy characteristics are noted in Table I. In comparison with normotensive pregnant control subjects, women with severe preeclampsia had significantly higher blood pressures, and their infants had lower birth weights. In addition, there were no differences in maternal age, parity, weight, or ethnicity (data not shown). Table II summarizes PLGF concentrations and gestational time of sampling in both groups. PLGF concentrations in the third trimester were significantly lower in women with preeclampsia than in women with normotensive pregnancies. There was no decrease in PLGF
1220 Livingston et al
concentrations between the group with severe preeclampsia and the control group when samples were obtained at <20 weeks’ gestation. In fact, there was a trend toward an increase in PLGF in early pregnancy. Comment Abnormal placentation is one of the initial events of severe preeclampsia. The main feature of abnormal placentation is the inadequate trophoblastic invasion of the maternal spiral arterioles. Recent evidence has shown that the presence and function of vascular endothelial growth factor and PLGF are critical during this cytotrophoblastic invasion.10 Our study is the first to evaluate PLGF concentrations early in pregnancy in women who subsequently had severe preeclampsia. Our findings of decreased PLGF at term (but not early in pregnancy) do not support the hypothesis that a reduction in growth factors is responsible for abnormal placentation. Rather, our findings suggest that lower PLGF concentrations seen later in pregnancy may be a result of earlier abnormal placentation. Moreover, because PLGF is known to be down-regulated in hypoxic environments,7 normal PLGF levels in early pregnancy do not support a hypoxiainitiated pathogenesis of preeclampsia. Our sample size was large enough to confirm previously published findings of decreased PLGF at the diagnosis of preeclampsia.4, 6 Our findings do not support a trend of decreased PLGF concentrations early in pregnancy. One limitation of our study is the fact that we evaluated circulating maternal plasma concentrations of PLGF rather than studying PLGF in its local uteroplacental environment. Growth factors are known to work locally, and circulating concentrations may not be representative of values in the uteroplacental circulation. Our
May 2001 Am J Obstet Gynecol
assay measured only the free PLGF 149 isoform, and we cannot comment on total PLGF concentrations. In conclusion, PLGF concentrations are reduced in pregnancies once severe preeclampsia becomes clinically evident. PLGF concentrations were not altered earlier in pregnancy, which suggests that PLGF is not a good marker for the development of subsequent severe preeclampsia. REFERENCES
1. Sibai BM. Hypertension in pregnancy. Obstet Gynecol Clin North Am 1992;19:15-32. 2. Cao Y, Chen A, An SSA, Ji RW, Davidson D, Llinas M. Kringle 5 of plasminogen is a novel inhibitor of endothelial cell growth. J Biol Chem 1997;272:22924-8. 3. Park JE, Chen HH, Winer J, Houck KA, Ferrara N. Placenta growth factor: potentiation of vascular endothelial growth factor bioactivity, in vitro and in vivo, and high affinity binding to Flt-1 but not to Flk-1/KDR. J Biol Chem 1994;269:25646-54. 4. Livingston JC, Chin R, Haddad B, McKinney ET, Ahokas R, Sibai BM. Reductions of vascular endothelial growth factor and placental growth factor concentrations in severe preeclampsia. Am J Obstet Gynecol 2000;183:1554-7. 5. Torry DS, Wang HS, Wang TH, Caudle MR, Torry RJ. Preeclampsia is associated with reduced plasma levels of placenta growth factor. Am J Obstet Gynecol 1998;179:1539-44. 6. Reuvekamp A, Velsing-Aarts FV, Poulina IEJ, Capello JJ, Duits AJ. Selective deficit of angiogenic growth factors characterizes pregnancies complicated by pre-eclampsia. BJOG 1999;106:1019-22. 7. Shore VH, Wang TH, Wang CL, Torry RJ, Caudle MR, Torry DS. Vascular endothelial growth factor and their receptors in isolated human trophoblast. Placenta 1997;18:657-65. 8. Kong TY, DeWolf F, Robertson WB, Brosens I. Inadequate maternal vascular response to placentation in pregnancies complicated by preeclampsia and by small-for-gestational age infants. BJOG 1986;93:1049-59. 9. Lubchenco LO, Hansman C, Boyd E. Intrauterine growth in length and head circumference as estimated from live births at gestational ages 26 to 42 weeks. Pediatrics 1966;37:403-8. 10. Vuoral P, Hatva E, Lymboussaki A, et al. Expression of vascular endothelial growth factor and placental growth factor in human placenta. Biol Reprod 1997;56:489-94.
Receive tables of contents by e-mail To receive the tables of contents by e-mail, sign up through our Web site at http://www.mosby.com/ajog. Choose E-mail Notification. Simply type your e-mail address in the box and click the Subscribe button. Alternatively, you may send an e-mail message to [email protected]. Leave the subject line blank, and type the following as the body of your message: Subscribe ajog_toc. You will receive an e-mail to confirm that you have been added to the mailing list. Note that table of contents e-mails will be sent out when a new issue is posted to the Web site.