Maternal plasma concentrations of the soluble tumor necrosis factor receptor 2 are increased prior to the diagnosis of preeclampsia

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OBSTETRICS

Maternal plasma concentrations of the soluble tumor necrosis factor receptor 2 are increased prior to the diagnosis of preeclampsia Baha Sibai, MD; Roberto Romero, MD; Mark A. Klebanoff, MD; Madeline Murguia Rice, PhD; Steve Caritis, MD; Marshall D. Lindheimer, MD; J. Peter Van Dorsten, MD; Mark Landon, MD; Menachem Miodovnik, MD; Mitchell Dombrowski, MD; Paul Meis, MD; for the Eunice Kennedy Shriver National Institute of Child Health and Human Development Maternal-Fetal Medicine Units Network OBJECTIVE: Soluble receptor levels of tumor necrosis factor (sTNF-

R)-1 and -2 are increased during preeclampsia. We postulated the increase preceded overt disease. STUDY DESIGN: Archived plasma from the Eunice Kennedy Shriver National Institute of Child Health and Human Development aspirin to prevent preeclampsia in high risk women trial were used to measure serial sTNF-R1 and sTNF-R2 (enrollment, 24-28 week’s gestation) in 986 women (577 also sampled at 34-38 weeks). RESULTS: Preeclampsia incidence was 21.2%. sTNF-R2 levels were

higher at enrollment (P ⫽ .02) and weeks 24-28 (P ⫽ .01) in women who eventually developed preeclampsia. The magnitude of increase

from baseline of both receptors was significantly greater in women who developed preeclampsia in the future. Women with week 24-28 sTNF-R2 levels in the highest quartile had significantly increased odds to develop preeclampsia (P ⫽ .03 vs quartile 1). This association was observed in the placebo but not the aspirin arm (P ⱕ .002). Sensitivities and positive predictive values were low. CONCLUSION: sTNF-R2 levels are elevated prior to overt preeclamp-

sia, suggesting a pathogenetic role for these proinflammatory cytokines. Key words: prediction, preeclampsia, tumor necrosis factor

Cite this article as: Sibai B, Romero R, Klebanoff MA, et al. Maternal plasma concentrations of the soluble tumor necrosis factor receptor 2 are increased prior to the diagnosis of preeclampsia. Am J Obstet Gynecol 2009;200:630.e1-630.e8.

umor necrosis factor-␣ (TNF-␣) is a 17 kDa polypeptide proinflammatory pleiotropic cytokine, produced by many cells in response to a wide range of stimuli such as microbial products (eg, bacterial endotoxin), viruses, and immune complexes.1 This cytokine can

T

induce endothelial cell activation,2-4 production of tissue factor,5,6 and hypertrigliceridemia,7 as well as induce/potentiate oxidative stress.8,9 TNF-␣ exerts its effects by interacting with 2 receptors, which have distinct biological effects: the 55 kDa TNF recep-

From the Departments of Obstetrics and Gynecology at the University of Tennessee, Memphis, TN (Dr Sibai); University of Cincinnati, Cincinnati, OH (Drs Sibai and Miodovnik); Eunice Kennedy Shriver National Institute of Child Health and Human Development, Bethesda, MD (Drs Romero and Klebanoff); The George Washington University Biostatistics Center, Washington, DC (Dr Rice); University of Pittsburgh, Pittsburgh, PA (Dr Caritis); University of Chicago, Chicago, IL (Dr Lindheimer); Medical University of South Carolina, Charleston, SC (Dr Van Dorsten); Ohio State University, Columbus, OH (Dr Landon); Wayne State University, Detroit, MI (Dr Dombrowski); and Wake Forest University, Winston-Salem, NC (Dr Meis). Received Aug. 18, 2008; revised Oct. 21, 2008; accepted Jan. 16, 2009. Reprints: Baha M. Sibai, MD, Department of Obstetrics and Gynecology, University of Cincinnati College of Medicine, 231 Albert Sabin Way, Cincinnati, OH 45267-0526. This study was supported in part by the Division of Intramural Research of the National Institutes of Health and Grants from the Eunice Kennedy Shriver National Institute of Child Health and Human Development (HD19897, HD36801, HD21410, HD21414, HD21434, HD27860, HD27861, HD27869, HD27883, HD27889, HD27905, HD27915, and HD27917) . 0002-9378/$36.00 • © 2009 Mosby, Inc. All rights reserved. • doi: 10.1016/j.ajog.2009.01.033

630.e1

American Journal of Obstetrics & Gynecology JUNE 2009

tor (TNF-R)-1 induces apoptosis, and the 75-kDa TNF-R2 induces proliferation through the activation of the transcription factor, nuclear factor ␬B.10 Shedding of the soluble receptors of TNF-␣ from the cell membranes plays a role in the regulation of TNF-␣’s biological functions by decreasing its availability as a ligand.11,12 Moreover, plasma or serum concentrations of soluble TNF-Rs (sTNF-Rs) are considered markers for excessive TNF biological activity because these receptors have longer half-lives than the ligand. Most, but not all, studies13-24 report circulating maternal TNF-␣, TNF-R1, and TNF-R2 concentrations as elevated during overt preeclampsia. We could locate only 2 studies reporting elevations in TNF-R1 concentrations,19,24 and none evaluating sTNF-R2, prior to clinical manifestations of the disease. Our study was designed to test whether elevations in circulating sTNF-R1 and sTNF-R2 do, in fact, occur during preg-

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www.AJOG.org nancy prior to the diagnosis of preeclampsia. If so, these cytokines would be implicated further in a plausible explanation of the cause of this disease or its phenotypes.

M ATERIALS AND M ETHODS Subjects The Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD) Maternal Fetal Medicine Network conducted a placebocontrolled randomized trial in 2539 pregnant women, enrolled between 13 and 26 weeks’ gestation, at risk for the development of preeclampsia to test the effectiveness of low dose aspirin (60 mg/ day) in preventing this serious hypertensive disorder of pregnancy.25 Randomized women had conditions that placed them into 1 of the following risk groups: pregestational insulin-treated diabetes mellitus, chronic hypertension, multifetal gestation, and preeclampsia during a previous pregnancy. The criteria for subject selection, enrollment, study protocol, and results have been reported elsewhere.25 Sample inclusion for this biomarker study required collection of serial samples (enrollment and 24-28 weeks’ gestation); samples collected prior to the date of hospital admission for delivery; and among those diagnosed with preeclampsia, samples collected prior to the date of diagnosis of preeclampsia. Of the 2539 randomized women, 986 were eligible for this biomarker study and had both an enrollment sample and a sample at 24-28 weeks’ gestation, and 577 also had a third sample collected at 34-38 weeks’ gestation. Collection of serial samples from only 39% of the trial population reflected the fact that collection of maternal blood to establish a repository for future secondary studies was initiated after the trial was well underway. Of the 986 women with samples from enrollment and 24-28 weeks’ gestation, 285 delivered before the date for the third sample collection at 34-38 weeks’ gestation. The original study was approved by the institutional review board (IRB) of each participating center in the network. Informed consent

was obtained from all subjects enrolled in the trial including collection of blood samples. Analysis of data from the stored blood samples was considered exempt from review by the IRB.

Blood samples During the NICHD trial, blood samples were obtained at enrollment, 24-28 weeks’ gestation, and 34-38 weeks’ gestation.25 The blood was collected into EDTA-containing tubes, centrifuged, and the plasma fraction aliquotted and stored at -70°C for future studies. TNF soluble receptor assays The sTNF-R1 and sTNF-R2 concentrations were determined using commercially available enzyme-linked immunoassay (R&D Systems, Minneapolis, MN). The samples were analyzed in a blinded fashion. The sensitivity of the assays for sTNF-R1 and sTNF-R2 were 5.2 and 4.5 pg/ml, respectively (intraassay coefficients of variation: 3.1% and 2.0%, respectively; interassay coefficients of variation: 2.8% and 2.7%, respectively). The effects of freezing the samples for longer than 10 years on these receptors are unknown. Outcome variables The primary outcome variable was preeclampsia, defined in women with neither hypertension nor proteinuria at baseline as the development of hypertension plus 1 of the following: proteinuria, thrombocytopenia, or pulmonary edema. Hypertension was defined as a systolic and/or diastolic blood pressure of 140 mm Hg or greater and/or 90 mm Hg or greater, respectively, on 2 occasions at least 4 hours apart. Abnormal protein excretion was defined as 300 mg/ day or greater or 2 dipstick determinations of 2⫹ or greater (ⱖ 100 mg/dL), recorded at least 4 hours apart, with no evidence of urinary tract infection. Thrombocytopenia was defined as a platelet count of less than 100,000/mL3. The diagnosis of preeclampsia in women who were proteinuric but normotensive at enrollment required the development of thrombocytopenia; a serum aspartate aminotransferase concentration of 70 U/L or greater; or hypertension accompanied by severe headaches, epigastric

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pain, or a sudden increase in proteinuria (to a ⱖ 5-fold increase over baseline or, if baseline values exceeded 5 g/day, to ⱖ 2-fold increase). In nonproteinuric hypertensive women, the diagnosis of preeclampsia required the development of proteinuria or thrombocytopenia. In the women both hypertensive and proteinuric at baseline, at least 1 of the following criteria were required to diagnose preeclampsia: thrombocytopenia; an elevated serum concentration of aspartate aminotransferase (ⱖ 70 U/L); or acceleration of the hypertension (defined by 2 diastolic readings of 110 mm Hg or greater taken 4 hours apart during the week preceding delivery) combined with either exacerbation of proteinuria (see aforementioned criteria), severe headaches, or epigastric pain.25 Other criteria for a diagnosis of preeclampsia were an eclamptic convulsion and manifestation of the HELLP syndrome, the latter defined as hemolysis (serum total bilirubin concentration, ⱖ 1.2 mg/dL [20 ␮mol/L], elevated serum aspartate aminotransferase concentrations (ⱖ 70 U/L), lactate dehydrogenase concentration of 600 U/ L or greater, or evidence of microangiopathic hemolytic anemia (presence of schistocytes in the peripheral smear), and thrombocytopenia. Consistency of diagnosis was further controlled by a committee comprised of 3 physicians unaware of the treatment group assignments who reviewed the records of women with new or accelerating hypertension, new-onset proteinuria, or a baseline dipstick protein recording of 1 or greater. Unanimous concurrence on the diagnosis was achieved.

Statistical analysis Statistical analysis was conducted using SAS software, version 8 (SAS Institute, Cary, NC). Continuous variables were compared using the Wilcoxon rank sum test. Categorical variables were analyzed using the ␹2 or Fisher’s exact test, where appropriate. Spearman correlation coefficients were estimated to summarize the association between the continuous biomarker variables. Logistic regression models included sTNF-R1 and sTNF-R2 from all 3 sam-

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TABLE 1

Characteristics of the study population Characteristics

Patients with samples collected at enrollment and 24-28 wks’ gestation (n ⴝ 986)

Patients with a sample also collected at 34-38 wks’ gestation (n ⴝ 577)

Patients diagnosed with preeclampsia (n ⴝ 209)

BASELINE RISK GROUP Diabetes

187 (19.0%)

119 (20.6%)

46 (22.0%)

Chronic hypertension

303 (30.7%)

184 (31.9%)

75 (35.9%)

Multiple gestation

237 (24.0%)

100 (17.3%)

39 (18.7%)

Previous history of preeclampsia

259 (26.3%)

174 (30.2%)

49 (23.4%)

Maternal age, y (mean and SD)

26.5 (6.3)

26.6 (6.3)

27.3 (6.5)

279 (28.3%)

164 (28.4%)

69 (33.0%)

29.3 (8.5)

29.6 (8.6)

30.7 (9.2)

White

338 (34.3%)

215 (37.3%)

70 (33.5%)

African American

561 (56.9%)

309 (53.6%)

125 (59.8%)

87 (8.8%)

53 (9.2%)

14 (6.7%)

................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................

Nullipara

................................................................................................................................................................................................................................................................................................................................................................................

Body mass index (mean and SD)

................................................................................................................................................................................................................................................................................................................................................................................

RACE OR ETHNIC GROUPS ................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................

Hispanic/other

................................................................................................................................................................................................................................................................................................................................................................................

BLOOD PRESSURE (mm Hg) AT ENROLLMENT Systolic (mean and SD)

117 (15)

117 (15)

122 (15)

69 (11)

73 (12)

................................................................................................................................................................................................................................................................................................................................................................................

Diastolic (mean and SD)

69 (11)

................................................................................................................................................................................................................................................................................................................................................................................

Married

386 (39.2%)

231 (40.0%)

94 (45.0%)

747 (75.8%)

446 (77.3%)

167 (79.9%)

77 (7.8%)

49 (8.5%)

13 (6.2%)

162 (16.4%)

82 (14.2%)

29 (13.9%)

................................................................................................................................................................................................................................................................................................................................................................................

CIGARETTE SMOKING DURING PREGNANCY No

................................................................................................................................................................................................................................................................................................................................................................................

Discontinued during pregnancy

................................................................................................................................................................................................................................................................................................................................................................................

Yes

................................................................................................................................................................................................................................................................................................................................................................................

GESTATIONAL AGE AT RANDOMIZATION (WEEKS) Median

20

19

19

Range

(13-27)

(13-27)

(13-27)

2944 (791)

3216 (635)

2886 (844)

................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................

Birthweight, g (mean and SD)

................................................................................................................................................................................................................................................................................................................................................................................

SD, standard deviation. Sibai. TNF-R2 and preeclampsia. Am J Obstet Gynecol 2009.

ples, categorized into quartiles. Logistic regression models also examined change per 10 unit increase per day in sTNF-R1 and sTNF-R2 from enrollment to 24-28 weeks’ gestation and from enrollment to 34-38 weeks’ gestation. Multivariable logistic regression was used to adjust for assigned treatment group and potential confounding maternal clinical characteristics. Stratified analyses and models with interaction terms examined whether associations varied by baseline risk group, singleton or twin gestation, smoking status, or treatment group (aspirin or placebo). Analyses were also conducted to determine the sensitivity, specificity, and pos630.e3

itive and negative predictive values for the 75th percentile of sTNF-R1 and sTNF-R2. A nominal 2-tailed P value of ⬍ .05 was considered significant. No adjustments were made for multiple comparisons.

R ESULTS Plasma sTNF-R concentrations were measured in 986 of the 2539 women (39%) enrolled in the randomized clinical trial. Table 1 describes the characteristics of all enrolled women and those with preeclampsia. The baseline characteristics of the women included in this analysis were similar to those who did

American Journal of Obstetrics & Gynecology JUNE 2009

not participate in this biomarker study (data not shown). The incidence of preeclampsia in this biomarker study was 21.2% (209/986), which was similar to that of the trial as a whole (19.4%). A significant correlation was found between plasma concentrations of sTNF-R1 and sTNF-R2 (enrollment: r ⫽ 0.84; P ⬍ .001; 24-28 weeks’ gestation: r ⫽ 0.82; P ⬍ .001; and 34-38 weeks’ gestation: r ⫽ 0.80; P ⬍ .001). Table 2 shows the plasma concentrations of sTNF-R1 and sTNF-R2, generally increasing with advanced gestational age in both those with and without preeclampsia. The median plasma concentrations of sTNF-R2,

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TABLE 2

sTNF-R1 and sTNF-R2, and preeclampsia Enrollment Preeclampsia (n ⴝ 209)

No preeclampsia (n ⴝ 777)

24-28 wks’ gestation

34-38 wks’ gestation

Preeclampsia (n ⴝ 209)

No preeclampsia (n ⴝ 777)

Preeclampsia (n ⴝ 100)

No preeclampsia (n ⴝ 477)

1045.7

1007.7

1175.7

1143.0

sTNF-R1 Median (pg/mL)

933.8

914.0

Range

448.50-3239.3

359.0-5060.1

422.1-4071.4

313.0-4758.0

463.4-3192.5

300.0-3062.5

Interquartile range

775.4-1138.2

758.6-1080.2

853.9-1318.1

845.3-1206.4

1007.0-1419.5

997.1-1402.7

................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................

P value (vs no PE)

.26

.10

.41

................................................................................................................................................................................................................................................................................................................................................................................

sTNF-R2 Median (pg/mL)

2672.6

2530.6

2873.9

2668.0

933.6-6889.6

1021.5-13000.0

1237.5-11100.0

Interquartile range

2229.2-3218.3

2127.3-3051.0

2323.7-3510.0

P value (vs no PE)

.02

2873.8

2757.4

................................................................................................................................................................................................................................................................................................................................................................................

Range

786.9-11740.0

1187.5-5883.2

863.0-6996.0

2403.6-3583.0

2336.0-3380.7

................................................................................................................................................................................................................................................................................................................................................................................

2219.9-3268.5

................................................................................................................................................................................................................................................................................................................................................................................

.01

.12

................................................................................................................................................................................................................................................................................................................................................................................

PE, preeclampsia; TNF, tumor necrosis factor. Sibai. TNF-R2 and preeclampsia. Am J Obstet Gynecol 2009.

but not sTNF-R1, in the enrollment and 24-28 weeks’ gestation samples were significantly higher in patients who subsequently developed preeclampsia than in those who did not (Table 2). Values for samples collected at 34-38 weeks’ gestation demonstrated results similar to the 24-28 weeks’ gestation samples, but statistical significance was not reached. The data were then reanalyzed according to gestational age at onset of preeclampsia. Table 3 summarizes plasma concentrations of the 2 receptors at time of enrollment and at 24-28 and 34-38 week’s gestation in predicting preeclampsia developing at gestations of less than 34, 34-36, and ⱖ 37 weeks. The median plasma concentrations of sTNF-R1 and sTNF-R2 in the enrollment and 24-28 week’s gestation samples were significantly higher in patients who subsequently developed preeclampsia at less than 34 week’s gestation only. The association between plasma concentration of sTNF-R2, and the subsequent diagnosis of preeclampsia remained significant after adjusting for assigned treatment group and variables known to be associated with preeclampsia in this population: body mass index, black race, and chronic hypertension at baseline. Table 4 depicts these associations by quartiles of sTNF-R2. Women in the

fourth quartile of sTNF-R2 at 24-28 weeks’ gestation had a significantly increased adjusted odds of preeclampsia (odds ratio [OR], 1.63; 95% confidence interval [CI], 1.06-2.50; P ⫽ .03), compared with women in the first quartile, with a similar but nonsignificant trend at an earlier measured sTNF-R2 at enrollment (OR, 1.47; 95% CI, 0.95-2.28; P ⫽ .09) and at a later measured sTNF-R2 at 34-38 weeks’ gestation (OR, 1.62; 95% CI, 0.87-3.01; P ⫽ .13). The observed associations were consistent by baseline risk group, singleton or twin gestation, and smoking status. An interaction was observed between sTNF-R2 and treatment group (aspirin or placebo), which showed a significant association between sTNF-R2 and outcome among the patients assigned to placebo but not among those assigned to aspirin (Table 5). When using the 75th percentile of plasma concentrations of sTNF-R1 and sTNF-R2 as a cut point, the sensitivity and positive predictive values for elevated plasma concentrations were low (sTNF-R1 enrollment: sensitivity 23.9%, specificity 74.8%, positive predictive value 20.3%, negative predictive value 78.5%; sTNF-R1 24-28 weeks’ gestation: sensitivity 22.0%, specificity 74.3%, positive predictive value 18.7%, negative predictive value 78.0%; sTNF-R1 34-38

weeks’ gestation: sensitivity 30.0%, specificity 76.1%, positive predictive value 20.8%, negative predictive value 83.8%; sTNF-R2 enrollment: sensitivity 27.3%, specificity 75.7%, positive predictive value 23.2%, negative predictive value 79.5%; sTNF-R2 24-28 weeks’ gestation: sensitivity 25.8%, specificity 75.3%, positive predictive value 22.0%, negative predictive value 79.1%; sTNF-R2 34-38 weeks’ gestation: sensitivity 26.0%, specificity 75.3%, positive predictive value 18.1%, negative predictive value 82.9%). The diagnostic indices were higher among the women assigned to placebo, although the indices remained relatively low (data not shown). An increase in the plasma concentration of sTNF-R1 and TNF-R2 between the first and second sample was associated with an increased adjusted odds for preeclampsia (sTNF-R1: OR per 10 unit increase per day 1.33; 95% CI 1.07-1.66; P ⫽ .01; sTNF-R2: OR per 10 unit increase per day 1.08; 95% CI, 1.00-1.16; P ⫽ .04). A significant association was also observed for an increase in the plasma concentration of sTNF-R1, but not TNF-R2, between the first and third sample (sTNF-R1: OR per 10 unit increase per day 2.21; 95% CI, 1.06-4.61; P ⫽ .04; sTNF-R2: OR per 10 unit increase per day 1.17; 95% CI, 0.88-1.56; P ⫽ .28).

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TABLE 3

sTNF-R1 and sTNF-R2, by timing of preeclampsia diagnosis sTNF-R1 PE < 34 wks’ gestation (n ⴝ 36)

sTNF-R2 PE 34-36 wks’ gestation (n ⴝ 88)

PE > 37 wks’ gestation (n ⴝ 85)

No PE (n ⴝ 777)

PE < 34 wks’ gestation (n ⴝ 36)

PE 34-36 wks’ PE > 37 wks’ gestation gestation (n ⴝ 88) (n ⴝ 85)

No PE (n ⴝ 777)

2670.4

ENROLLMENT Median (pg/mL)

918.5

888.9

914.0

2948.5

2535.1

2530.6

Range

1088.7

507.8-2595.8

448.5-3239.4

490.2-2473.6

359.0-5060.1

1145.1-6411.6

933.6-6889.6

1073.4-5808.3

1021.5-13000.0

Interquartile range

848.8-1312.4

764.7-1071.2

775.4-1066.2

758.6-1080.2

2301.2-3806.9

2241.6-3096.9

2157.0-3081.1

2127.3-3051.0

P value (vs no PE)

.002

.006

.15

.48

................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................

.84

.79

N/A

N/A

................................................................................................................................................................................................................................................................................................................................................................................

24-28 WEEKS’ GESTATION Median (pg/mL)

1179.1

1002.4

1045.7

1007.7

3150.2

2829.6

2973.6

313.0-4758.0

1580.0-7941.3

1237.5-9463.2

1291.3-1110.0

845.3-1206.4

2396.3-4492.9

2379.9-3323.9

2200.1-3534.8

.004

.32

.13

2668.0

................................................................................................................................................................................................................................................................................................................................................................................

Range

668.5-3519.5

422.1-2758.3

485.8-4071.4

Interquartile range

948.6-1760.4

849.7-1221.5

843.2-1344.6

P value (vs no PE)

.003

786.9-11740.0

................................................................................................................................................................................................................................................................................................................................................................................

2219.9-3268.5

................................................................................................................................................................................................................................................................................................................................................................................

.98

.41

N/A

N/A

................................................................................................................................................................................................................................................................................................................................................................................

34-38 WEEKS’ GESTATION Median (pg/mL)

—

1139.4

1212.0

Range

—

631.5-3192.5

463.4-2252.8

Interquartile range

—

999.4-1389.4

1014.6-1423.9

P value (vs no PE)

—

1143.0

—

2907.0

2848.5

300.0-3062.5

—

1847.8-5883.2

1187.5-5400.3

997.1-1402.7

—

2374.6-3454.9

2427.6-3635.2

—

.35

.19

2757.4

................................................................................................................................................................................................................................................................................................................................................................................

863.0-6996.0

................................................................................................................................................................................................................................................................................................................................................................................

2336.0-3380.7

................................................................................................................................................................................................................................................................................................................................................................................

.98

.29

N/A

N/A

................................................................................................................................................................................................................................................................................................................................................................................

N/A, not applicable; PE, preeclampsia; TNF, tumor necrosis factor. Sibai. TNF-R2 and preeclampsia. Am J Obstet Gynecol 2009.

C OMMENT This study demonstrated the following: (1) the median plasma concentration of sTNF-R2, but not sTNF-R1, was significantly higher at enrollment in women who subsequently developed preeclampsia at any time in gestation than in women who did not; (2) the median plasma concentration of TNF-R1 and TNF-R2 were also elevated at enrollment and at 24-28 weeks’ gestation in patients subsequently manifesting the disorder at less than 34 week’s gestation; (3) women in the fourth quartile of sTNF-R2 at 24-28 weeks’ gestation had a significantly increased adjusted odds of preeclampsia (OR, 1.63), compared with women in the first quartile, and this association was null among the women assigned to aspirin, and stronger in the women assigned to placebo (OR, 2.76); this finding may be related to the possible antiinflammatory effects of low-dose aspirin, which could have blunted the production of sTNF-R2.; and 4) there was a significantly increased odds of preeclampsia when sTNF-R1 and sTNF-R2 630.e5

increased from enrollment to 24-28 weeks’ gestation (OR, 1.33 per 10 unit increase per day and OR, 1.08 per 10 unit increase per day, respectively). However, diagnostic indices and predictive values derived from these observations do not support use of sTNF-R2 receptor’s circulating concentrations alone as a test to predict preeclampsia. Finally our observations suggest the presence early in pregnancy of an increased systemic inflammatory response, reflected by increased sTNF-R1 and sTNF-R2 concentrations, in patients destined to develop preeclampsia before 34 week’s gestation. Increments in the systemic inflammatory response, a feature of healthy pregnancy, are exaggerated in preeclampsia.24-30 Based on studies conducted with flow cytometry in which the phenotypic and metabolic changes of neutrophils and monocytes have been examined as well as observational studies of the concentrations of maternal proinflammatory cytokines in peripheral blood,16-24 this exaggeration has been offered as a plausible hypothesis

American Journal of Obstetrics & Gynecology JUNE 2009

for the pathogenesis of preeclampsia and/or its phenotypes.28,29 Increased TNF-␣ production prior to and subsequent to development of preeclampsia is probably related to impaired placentation.24 TNF-␣ acts by binding to specific receptors, namely sTNF-R1 and sTNF-R2. The levels of these receptors are known to increase after an increase in TNF-␣ production.30 Each receptor is shed as a soluble protein that specifically binds to TNF-␣. The source of this excessive receptor production in patients with preeclampsia may be activated decidual leukocytes or the placenta itself.30 Soluble TNF receptors are more reliable markers for TNF-␣ activity because they have a longer half-life than TNF-␣, and thus, they serve as the footprints for TNF-␣ activity.30 Because the half-life of both receptors are similar, it is difficult to explain the selective rise in sTNF-R2 in out study. If, as some suggest, the cause of the preeclampsia syndrome is multifactorial, it would be naïve to ascribe the disease in all patients to a single mechanism.

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TABLE 4

ORs and 95% CIs for preeclampsia by quartiles of sTNF-R2 Quartiles of sTNF-R2 (pg/mL)

Preeclampsia (n ⴝ 209)

No preeclampsia (n ⴝ 777)

Unadjusted OR

95% CI

P value

Adjusted ORa

95% CI

P value

⬍ 2136.9

46 (18.7%)

200 (81.3%)

1.00

(Reference)

—

1.00

(Reference)

—

2136.9-2554.9

45 (18.2%)

202 (81.8%)

0.97

0.61-1.53

.89

1.02

0.65-1.62

.93

2555.0-3089.7

57 (23.2%)

189 (76.8%)

1.31

0.85-2.04

.22

1.39

0.89-2.16

.15

⬎ 3089.7

61 (24.7%)

186 (75.3%)

1.43

0.93-2.20

.11

1.47

0.95-2.28

.09

ENROLLMENT ................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................

24-28 WEEKS’ GESTATION ⬍ 2227.8

48 (19.5%)

198 (80.5%)

1.00

(Reference)

—

1.00

(Reference)

—

2227.8-2697.0

39 (15.8%)

208 (84.2%)

0.77

0.48-1.23

.28

0.80

0.50-1.28

.35

2697.1-3322.3

54 (22.0%)

192 (78.1%)

1.16

0.75-1.80

.50

1.22

0.78-1.90

.38

⬎ 3322.3

68 (27.5%)

179 (72.5%)

1.57

1.03-2.40

.04

1.63

1.06-2.50

.03

................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................

34-38 WEEKS’ GESTATION ⬍ 2343.1

21 (14.6%)

123 (85.4%)

1.00

(Reference)

—

1.00

(Reference)

—

2343.1-2779.3

22 (15.3%)

122 (84.7%)

1.06

0.55-2.03

.87

1.04

0.54-2.01

.91

2779.4-3409.7

26 (18.1%)

118 (81.9%)

1.29

0.69-2.44

.43

1.30

0.69-2.44

.42

⬎ 3409.7

31 (21.4%)

114 (78.6%)

1.59

0.87-2.96

.13

1.62

0.87-3.01

.13

................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................

................................................................................................................................................................................................................................................................................................................................................................................

CI, confidence interval; OR, odds ratio; TNF, tumor necrosis factor. a

Adjusted for body mass index, African American race, and chronic hypertension at baseline. Sibai. TNF-R2 and preeclampsia. Am J Obstet Gynecol 2009.

In addition, multiple causes could result in biochemical markers varying among subjects with different risk factors.31 The patients enrolled in our study included a heterogeneous group of women, although all were at increased risk to develop preeclampsia. Whereas the associations observed in this study were consistent across baseline risk groups, further research may be required to determine whether inflammation is operative in the genesis of preeclampsia in concert with other risk factors. Recently Schipper et al24 studied sTNF-R1 concentrations throughout pregnancy in 68 women with a history of severe preeclampsia, fetal growth retardation, or chronic hypertension. The authors found that serum concentrations of sTNF-R1 were elevated in the second trimester only in those who subsequently developed preeclampsia complicated by fetal growth restriction. The authors suggested that the “increased TNF-␭ production in women with preeclampsia is related to impaired placentation rather than to the maternal syndrome.”24 There is also 1 additional study

that reported that sTNF-R1 levels are increased during the second trimester in patients at low risk for preeclampsia.19 What is the clinical significance of elevated sTNF-R2 concentrations at 13-26 weeks of pregnancy in women who ultimately developed preeclampsia? Using a cutoff value of the 75th percentile, we found that elevated concentrations of this soluble receptor had poor sensitivity (27.3%) and a limited positive predictive value (23.2%) for subsequent diagnosis of preeclampsia. Therefore, our findings indicate that measurement of sTNF-R2 early in pregnancy alone has limitations. The strength of this study is that it provides information about sTNF-R1 and sTNF-R2 plasma concentrations early in pregnancy in a large number of women considered at very high risk for development of preeclampsia. In this respect we could locate no similar data from this large a population. Limitations include the retrospective nature of the measurement of the biomarkers (samples were frozen for longer than 10 years prior to analysis) and failure to obtain samples from all

trial participants as well as the added limitation of restricting the cohort to patients who had at least 2 blood samples available for analysis. The latter resulted in the evaluation of only 39% of the women enrolled in the original trial. Even among these 986 women, we consistently examined only 2 blood samples per woman per pregnancy, precluding repeated measurements in the same patient to search for trends, as might be done in clinical practice. Although our findings at 34-38 weeks’ gestation demonstrated similar trends to those drawn at 24-28 weeks’ gestation, statistical significance was not consistently reached. This may be in part because of the smaller number of samples available for analysis and also because a number of women had delivered because of earlyonset severe preeclampsia before the third sample could be obtained. We did not determine whether there are changes in plasma concentration in sTNF-R1 or sTNF-R2 in women developing gestational hypertension or in pregnancies complicated by fetal growth restriction with or without preeclamp-

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630.e6

Research

Obstetrics

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TABLE 5

ORs and 95% CIs for preeclampsia by quartiles of sTNF-R2, by treatment assignment Aspirin Quartiles of sTNF-R2 (pg/mL)

Placebo

Adjusted ORa

95% CI

P value

Adjusted ORa

95% CI

P value

⬍ 2136.9

1.00

(Reference)

—

1.00

(Reference)

—

2136.9-2554.9

1.00

0.52-1.93

1.00

1.03

0.54-1.99

.92

2555.0-3089.7

1.52

0.81-2.84

.19

1.31

0.69-2.48

.40

⬎ 3089.7

1.18

0.62-2.24

.61

1.79

0.97-3.33

.06

⬍ 2227.8

1.00

(Reference)

1.00

(Reference)

—

2227.8-2697.0

0.49

0.25-0.95

.04

1.35

0.68-2.69

.39

2697.1-3322.3

0.64

0.34-1.20

.17

2.37

1.23-4.58

.01

⬎ 3322.3

1.02

0.56-1.86

.95

2.76

1.45-5.27

.002

⬍ 2343.1

1.00

(Reference)

1.00

(Reference)

—

2343.1-2779.3

0.68

0.27-1.71

.41

1.59

0.61-4.15

.35

2779.4-3409.7

0.92

0.39-2.14

.84

1.91

0.72-5.06

.19

⬎ 3409.7

0.45

0.16-1.31

.14

3.88

1.64-9.17

.002

ENROLLMENT ................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................

24-28 WEEKS’ GESTATION —

................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................

34-38 WEEKS’ GESTATION —

................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................ ................................................................................................................................................................................................................................................................................................................................................................................

................................................................................................................................................................................................................................................................................................................................................................................

CI, confidence interval; OR, odds ratio; TNF, tumor necrosis factor. a

Adjusted for body mass index, African American race, and chronic hypertension at baseline. Sibai. TNF-R2 and preeclampsia. Am J Obstet Gynecol 2009.

sia. Finally, one must be circumspect in interpreting changes occurring before overt disease in relation to causation. Preeclampsia has a preclinical phase, and there is a substantial literature describing small but discernible differences among populations developing the disease, and those remaining normotensive, that are present long before the disorder becomes obvious. These findings should be verified in a prospectively designed study with serial measurements of sTNF-R1 and sTNF-R2 concentrations. In summary, our results reveal that among women at high risk for preeclampsia, the plasma concentrations of sTNF-R2 at 13-26 or 24-28 weeks’ gestation are higher in women later diagnosed with preeclampsia than in women not diagnosed with preeclampsia. In addition, we found that plasma concentrations of sTNF-R1 at 13-26 or at 24-28 week’s gestation are higher in women later diagnosed with preeclampsia at less than 34 week’s gestation. This finding pro630.e7

vides support for the hypothesis that intravascular inflammation is a mechf anism of preeclampsia. ACKNOWLEDGMENTS The following subcommittee members participated in protocol development and coordination between clinical research centers (M. Cotroneo, RN, and B. Collins, PhD) and protocol/ data management and statistical analysis (Elizabeth Thom, PhD, and Cora MacPherson, PhD). In addition to the authors, other members of the Eunice Kennedy Shriver National Institute of Child Health and Human Development Maternal-Fetal Medicine Units Network are as follows: University of Pittsburgh, J. Kuller, M. Cotroneo, and T. Kamon; University of Tennessee, B. Mercer and R. Ramsey; University of Southern California, Y. Rabello, D. McCart, and E. Mueller; University of Alabama at Birmingham, R. Goldenberg and R. Copper; Wayne State University, Y. Sorokin, G. Norman, and A. Millinder; Medical College of Virginia, J. T. Christmas, S. McCoy, and S. Elder; University of Cincinnati, N. Elder, B. Carter, and V. Pemberton; University of Oklahoma, A. Meier and V. Minton; Wake Forest University, M. Swain; University of Chicago, A. H. Moawad and P. Jones; Ohio State University, J. D. Iams, S. Meadows, and S. Brenner; Medical University of South Caro-

American Journal of Obstetrics & Gynecology JUNE 2009

lina, B. Collins, R. B. Newman, and S. G. Carter; The George Washington University Biostatistics Center, E. A. Thom, M. McNellis, C. MacPherson, D. Johnson, and M. L. Fischer; and the Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, D. McNellis, C. Spong, C. Catz, and S. Yaffe.

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