Objective
To determine if early pregnancy serum biomarkers in high-risk women who develop preeclampsia vary according to risk factor.
Study Design
We performed a secondary analysis of the Maternal-Fetal Medicine Units Network randomized controlled trial of low-dose aspirin for the prevention of preeclampsia in high-risk women. Serum biomarker levels at enrollment (before initiation of aspirin or placebo) were compared between women who did and did not develop preeclampsia, both for the group as a whole and within each of 4 high-risk groups (insulin-dependent diabetes, hypertension, multiple gestation, and previous preeclampsia) using a regression model adjusting for gestational age at collection and prepregnancy body mass index.
Results
1258 women were included (233 with insulin-dependent diabetes, 387 with chronic hypertension, 315 with a multiple gestation, 323 with previous preeclampsia). Multiple early pregnancy serum biomarkers differed between women who did and did not develop preeclampsia. Each high-risk group had a unique and largely nonoverlapping pattern of biomarker abnormality. Differences between those who did and did not develop preeclampsia were noted in vascular cell adhesion molecule in the diabetes group; human chorionic gonadotropin, soluble tumor necrosis factor receptor-2, tumor necrosis factor-alpha, selectin and angiogenin in the chronic hypertension group; interleukin-6, placental growth factor, soluble fms-like tyrosine kinase plus endoglin to placental growth factor ratio in the multiple gestation group; and angiogenin in the previous preeclampsia group.
Conclusion
Patterns of serum biomarkers vary by high-risk group. These data support the hypothesis that multiple pathogenic pathways lead to the disease recognized clinically as preeclampsia.
Hypertensive disorders of pregnancy, including preeclampsia and chronic hypertension, continue to be a significant source of maternal and fetal morbidity and mortality. Although the ability to identify biomarkers or clinical attributes to predict the development of preeclampsia has had some success, translating these findings into effective preventative or treatment modalities to reduce the burden of disease has been decidedly mixed.
The Maternal-Fetal Medicine Units (MFMU) Network randomized controlled trial to assess the ability of low-dose aspirin (LDA) to prevent preeclampsia in women at high-risk for developing preeclampsia (MFMU High Risk Aspirin trial) tested the explicit assumption that a prostacyclin/thromboxane (TXA) imbalance is pathogenic in preeclampsia. This study enrolled specifically women with risk factors for preeclampsia including: insulin-dependent diabetes, chronic hypertension, multiple gestation or previous preeclampsia. This study demonstrated that LDA was ineffective in the prevention of preeclampsia in the study population as a whole as well as in each high-risk group. In an ancillary study, neither TXA levels at enrollment nor reductions in TXA levels in women receiving LDA were predictive of preeclampsia suggesting that preeclampsia occurs in a TXA-independent fashion at least in some women.
It is increasingly becoming recognized that preeclampsia may be a clinical syndrome resulting from many possible antecedents. We therefore hypothesized that the ability of biomarkers to predict the occurrence of preeclampsia would vary among high-risk subgroups (insulin-dependent diabetes, hypertension, multiple gestation. or previous preeclampsia). As an initial test of this hypothesis, we undertook a secondary analysis of the MFMU High Risk Aspirin dataset to determine whether the levels of multiple serum biomarkers before the initiation of LDA or development of preeclampsia differed in high-risk women who subsequently did vs did not develop preeclampsia. Further, we asked whether such differences varied by high-risk subgroup.
Materials and Methods
Our study is a secondary analysis of the MFMU High Risk Aspirin trial of LDA to prevent preeclampsia. Patients were enrolled between 1991 and 1995 at 13 centers, with collection of serum samples starting in 1992. Included patients were at least 12 weeks’ gestational age with at least one risk factor for preeclampsia; namely, insulin-dependent diabetes, chronic hypertension, multiple gestation, or preeclampsia in a previous pregnancy. Full diagnostic criteria for the high-risk subsets and the clinical criteria to define hypertension, proteinuria, and the diagnosis of preeclampsia have been previously published. The MFMU study was approved by institutional review boards at each of the study sites with each subject providing written informed consent. This secondary analysis was considered exempt by the Colorado Multiple Institutional Review Board.
The original study investigators selected a panel of serum biomarkers that have been associated with preeclampsia. These biomarkers were measured shortly after study enrollment at 13-26 weeks (mean, 19.6 weeks) and serially during the pregnancy in 2000 of the 2503 patients who agreed to additional blood draws using methods that have been previously published. Specimens were collected at each of the sites, divided into aliquots and frozen to −80 ° C for transport to a central laboratory for analysis. For our data analysis, we focused only on the initial serum biomarker levels, before initiation of LDA or the development of preeclampsia, as our primary interest is in early preeclampsia pathogenesis and not abnormalities that may occur with disease development or with LDA treatment. To this end, we compared levels of biomarkers between women who did and did not develop preeclampsia for the overall group and within each high-risk subset. We did not compare biomarker levels between women who did and did not receive LDA within each high-risk subgroup as there was no difference in the outcome of preeclampsia between groups with or without this intervention in the primary MFMU trial. Specifically, the following biomarkers were analyzed: angiogenin, cotinine, endoglin, estriol, estriol/progesterone ratio, human chorionic gonadotropin (hCG), interleukin-2 (IL-2), interleukin-6 (IL-6), placental growth factor (PlGF), progesterone, selectin, soluble fms-like tyrosine kinase (sFlt-1), soluble tumor necrosis factor receptor-1 (sTNFr-1), soluble tumor necrosis factor receptor 2 (sTNFr-2), tumor necrosis factor-alpha, thrombin/antithrombin III complex, TXA, vascular cell adhesion molecule, and the ratio of sFlt-1+endoglin/PlGF. Women were included if they had at least one of these biomarkers available, as the number of women with available results varied and is indicated in the footnotes for each table.
The original trial contains details of the patients included and the study endpoint definitions. Briefly, in the original trial, and for our analysis, preeclampsia was defined as hypertension with a systolic and/or diastolic blood pressure greater than 140/90 in association with proteinuria (≥300 mg in 24 hours, or 2 urine dipstick results of 2+ or greater at least 4 hours apart). In women with chronic hypertension, the development of preeclampsia was established with new-onset proteinuria or thrombocytopenia (platelet count <100,000). In women who had documented proteinuria at the time of enrollment, proteinuria needed to worsen to at least 5 times the baseline value to be considered worsening and indicative of preeclampsia. Charts were reviewed prospectively by at least three physicians to ensure that women met these strict diagnostic criteria for preeclampsia.
Comparison of baseline demographics between groups were made using analysis of variance for continuous variables and χ 2 for dichotomous variables. Comparisons of levels of serum biomarkers were made between groups using a multiple regression model that included gestational age at the time of study enrollment and maternal body mass index (BMI) as covariates. For continuous measures better summarized by a measure less sensitive to a skewed distribution (such as BMI), analysis was performed on the log-scale, back transformed and presented as geometric means with 95% CI. A P value < .05 was considered statistically significant. All analyses were performed in SAS (SAS Institute, Cary, NC).
Results
A total of 1258 women were included in the analysis: 233 women with insulin-dependent diabetes, 387 with chronic hypertension, 315 with a multiple gestation, and 323 with previous preeclampsia. Demographic characteristics of the study population are detailed in Table 1 . Women with diabetes were more often white and had the lowest parity. The chronic hypertension group tended to be older, have a higher BMI and be African American. Women with multiple gestations were enrolled in the study at a slightly later gestational age and delivered at an earlier gestational age. Higher parity and African American race were more common in the group with previous preeclampsia.
| Characteristic | Diabetes n = 233 | Chronic HTN n = 387 | Multiple gestation n = 315 | Previous preeclampsia n = 323 | P value a |
|---|---|---|---|---|---|
| Maternal age at randomization, mean ± SD | 26.0 (6.04) | 29.5 (6.48) | 25.0 (5.88) | 24.4 (5.40) | < .001 |
| Race, n (%) | |||||
| White | 139 (59.66) | 96 (24.81) | 109 (34.60) | 71 (21.98) | < .001 |
| Hispanic | 15 (6.44) | 36 (9.30) | 37 (11.75) | 8 (2.48) | |
| African American | 77 (33.05) | 254 (65.63) | 168 (53.33) | 244 (75.54) | |
| Other | 2 (0.86) | 1 (0.26) | 1 (0.32) | 0 (0.00) | |
| Parity (deliveries >20 wks), mean ± SD | 0.82 (1.20) | 1.62 (1.60) | 1.11 (1.21) | 1.77 (1.14) | < .001 |
| Body mass index (kg/m 2 ), geometric mean (95% CI) | 27.1 (26.23–27.96) | 31.9 (31.08–32.78) | 25.7 (24.99–26.36) | 27.4 (26.57–28.16) | < .001 |
| Gestational age at enrollment (wks), mean ± SD | 18.0 (3.69) | 19.8 (3.79) | 21.1 (3.60) | 20.0 (3.96) | < .001 |
| Gestational age at delivery (wks), mean ± SD | 36.0 (4.10) | 36.9 (3.97) | 34.4 (3.59) | 37.5 (3.63) | < .001 |
| Infant death, n (%) | 1 (0.43) | 3 (0.78) | 9 (2.86) | 3 (0.93) | .06 |
| Maternal death, n (%) | 0 (0.00) | 0 (0.00) | 0 (0.00) | 1 (0.17) | .69 |
a Comparisons between groups were made using analysis of variance for continuous variables, χ 2 tests for categorical measures, and Fisher exact test for infant and maternal death.
Table 2 shows the comparison of biomarkers for all patients with and without the diagnosis of preeclampsia. The levels of cotinine, endoglin, estriol, estriol/progesterone, IL-2, IL-6, PlGF, selectin, sFlt-1, sTNF-1, thrombin/antithrombin III complex, TXA, and vascular cell adhesion molecule did not differ between women who did vs did not develop preeclampsia. However, angiogenin, hCG, progesterone, sTNFr-2, and tumor necrosis factor-alpha levels were greater in the women who developed preeclampsia than those without preeclampsia with a trend existing for the (sFlt-1+endoglin)/PlGF ratio to be elevated as well ( Table 2 ).
| Serum biomarker a | Preeclampsia n = 263 c | No preeclampsia n = 995 c | P value b |
|---|---|---|---|
| Angiogenin, pg/mL | 265 (253.3–276.9) | 240 (234.3–246.6) | < .001 |
| hCG, IU/mL d | 23,952 (20,993–27,328) | 18,141 (17,052–19,299) | < .001 |
| Progesterone, ng/mL d | 48.92 (45.41–52.71) | 44.65 (43.12–46.24) | .03 |
| sTNFr-2, pg/mL | 2707 (2595–2823) | 2572 (2517–2629) | .035 |
| TNF-α, pg/mL | 3.03 (2.87–3.19) | 2.74 (2.60–2.89) | .008 |
| (sFlt-1+endoglin)/PlGF | 42.95 (33.25–55.47) | 32.50 (28.49–37.08) | .06 |
a All reported means are geometric means with 95% CI with the exception of angiogenin, which is reported as arithmetic means with 95% CI
b Comparisons between groups were made using a regression model adjusting for gestational age at the time the serum biomarker was drawn and prepregnancy maternal BMI
c Available sample size for each biomarker ranged as follows: angiogenin (214–790), hCG (108–491), progesterone (108–494), sTNFr-2 (214–790), TNF-α (261–259), (sFlt-1+endoglin)/PlGF (38–138) for preeclampsia and no preeclampsia groups, respectively
Comparisons of biomarkers between women who did vs those who did not develop preeclampsia within each high-risk subgroup showed considerable heterogeneity ( Table 3 ). Angiogenin levels were higher in women with chronic hypertension and previous preeclampsia who developed preeclampsia than in those who did not ( Table 3 ). Women with multiple gestation or chronic hypertension who developed preeclampsia had higher levels of hCG than those who did not develop preeclampsia ( Table 3 ). There were no biomarkers that were associated with the development of preeclampsia across all high-risk subgroups ( Figure ).
