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ORIGINAL RESEARCH |
From the Section of Maternal-Fetal Medicine and Department of Obstetrics and Gynecology, Northwestern Memorial Hospital, Northwestern University Medical School, Chicago, Illinois.
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Abstract |
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Methods: Blood samples were collected from patients during the second trimester of prenatal care. We identified patients who subsequently developed preeclampsia and matched them with patients who had no evidence of preeclampsia during their gestation. Matching criteria included gestational age at blood sampling, gestational age at delivery, and birth weight. Assays were then performed to assess the levels of activin A and inhibin A in the control and study groups. A power calculation determined that 12 patients who subsequently developed preeclampsia, if matched with controls in a 1:2 ratio, would allow the detection of differences in analyte levels that were 60% as large as those previously reported between patients already diagnosed with preeclampsia and matched controls.
Results: Twelve patients with preeclampsia were identified and matched with 24 controls. No differences in serum levels of activin A or inhibin A were detected between the two groups. Because of the significant overlap of analyte levels between the two groups, no cutpoint that would allow identification of patients destined to become preeclamptic could be determined.
Conclusion: These data suggest that activin A and inhibin A cannot be used as markers for later development of preeclampsia in a low-risk population.
Recent studies of activin A and inhibin A have raised the possibility that these two proteins could provide insight into the pathophysiology of preeclampsia.1–3 Activin A and inhibin A are dimeric proteins composed of 
ßA and ßAßA peptide chains, respectively, that are produced primarily by the placental trophoblast during pregnancy.4,5 Given that trophoblast dysfunction has been associated with preeclampsia, several investigators have postulated that serum levels of activin A and inhibin A might be altered in women with preeclampsia. Petraglia et al1 first demonstrated that serum activin A was abnormally elevated in preeclamptic patients. Muttukrishna et al2 and Silver et al3 confirmed that finding and also showed that inhibin A was abnormally elevated in the same patients.
No study to date, however, has determined whether both proteins are altered before patients manifest clinical evidence of preeclampsia. These alterations, if found, could aid in the detection of patients at high risk of developing preeclampsia and provide further insight into the pathophysiology of the disease process. One study, that examined only inhibin A, found an elevation of this protein in the second trimester in patients who eventually became preeclamptic.6 This population of preeclamptic women included patients with other complications that by themselves are associated with alterations of inhibin A (eg, abruptio placentae, stillbirth, impaired fetal growth). The present study, therefore, was undertaken to determine whether alterations in the serum levels of both activin A and inhibin A could be detected in patients before they become preeclamptic and whether they could be associated specifically with this disease process.
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Methods |
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For each patient who met the criteria for preeclampsia, potential control subjects who progressed through an uncomplicated gestation without evidence of preeclampsia were identified according to the following criteria: gestational age at blood sampling (within 2 weeks), gestational age at delivery (within 2 weeks), and birth weight at delivery (within 300 g). From this group, two control subjects were matched to each patient who developed preeclampsia. This final matching was done by computer-generated random selection.
Activin A unbound concentrations were measured using a one-step (simultaneous) monoclonal antibody-based enzyme-linked immunosorbent activin assay (ELISA) as described by Wong et al.7 Briefly, microtiter plates were coated overnight with 2F8 antibody at 4 µg/mL. Standard controls or diluted samples (1:5 or 1:8) and freshly diluted horseradish peroxidase-conjugated 6H5 were added in 20% normal human serum and incubated for 4 hours at room temperature. The bound conjugated antibody was measured at 490-nm absorption after addition of orthophenylene diamine and hydrogen peroxide. The assay limit of detection was 250 pg/mL. The ELISA had interassay and intraassay coefficients of variation of 13.8% and 5.9%, respectively. Recombinant human activin A, used as the assay standard, was provided by Dr T. K. Woodruff.
Inhibin A concentrations were measured by a sequential two-site monoclonal antibody-based ELISA (Serotec, Oxford, England) as described by Groome and O’Brien.8 A follicular fluid–based standard and patient samples were prepared with sodium dodecyl sulfate, heating, and addition of hydrogen peroxide. Standards and samples were plated with E4 as the capture antibody at room temperature overnight. Detecting monoclonal antibody (R1), substrate, and amplifier were added sequentially to each well. The ELISA had interassay and intraassay coefficients of variation of 16.6% and 8.2%, respectively. All assays were performed by individuals who were masked to the assignment of control and study subjects.
Maternal demographic characteristics were compared with the Student t test for continuous variables and Fisher exact test or 
2 analysis for dichotomous variables. The Kolmogorov-Smirnov goodness-of-fit test was used to assess the normality of the distribution of the analytes. Analyte data that fit a normal distribution were analyzed with parametric tests (Student t test, analysis of variance), and analyte levels that fit a nonnormal distribution were evaluated with a nonparametric test (Mann-Whitney U test). A P < .05 was used to define statistical significance. Calculations were done using Minitab 11 (Minitab, Inc., State College, PA). A power calculation based on the serum activin and inhibin values reported by Muttukrishna et al2 (activin levels of 38.1 ± 25.9 ng/mL in preeclamptic women compared with 4.0 ± 2.3 ng/mL in controls and inhibin levels of 3.1 ± 1.8 ng/mL in preeclamptic women compared with 0.36 ± 0.14 ng/mL in controls) was performed with an = .05 and a ß = .2. On the basis of this calculation, with controls matched to patients at a ratio of 2:1, analyte elevations 60% as large as those reported by Muttukrishna et al2 could be detected by studying 36 total subjects (24 controls and 12 patients).
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Results |
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. As expected from the matching criteria, there were no significant differences between groups with respect to birth weight and gestational age at sampling and delivery. All other comparisons between groups were not statistically significant, except for mean arterial pressure at first prenatal visit and mean arterial pressure at presentation for delivery.
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Activin A and inhibin A levels with respect to gestational age at sampling in the control group are shown in Figure 1. To determine whether analyte levels varied during the gestational ages under study, analyte levels from the first half of the second trimester (14–21 weeks) were compared with those from the second half (22–28 weeks). Median activin A levels were not significantly different between the first and second half of the second trimester (117 pg/mL compared with 106 pg/mL, respectively, Mann-Whitney U test, P > .05). Conversely, mean levels of inhibin A were significantly lower at earlier gestational ages (384 ± 179 pg/mL compared with 842 ± 478 pg/mL, Student t test, P < .05).
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) without further segmentation according to gestational age. There were no significant differences between the study and control groups (P > .05). Given the increase in inhibin A levels in the second trimester, two-way analysis of variance was used to compare the means of inhibin A (Table 2) while simultaneously blocking for the gestational age at sampling. As with activin A, there was no significant difference between the control and study groups with respect to inhibin A serum levels. Given the extensive overlap in serum levels of both activin A and inhibin A between the control and study groups (Figure 2, a and b, respectively), no evidence of a discriminatory point could be discerned.
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Discussion |
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Activin A and inhibin A, two proteins with related peptide subunits, are likely to be altered before the development of preeclampsia. During gestation these substances are produced primarily by the placenta.4,5 Levels of activin A and inhibin A have been shown to be abnormal in women who have other pregnancy complications, such as gestational diabetes, preterm delivery, abruptio placentae, and intrauterine fetal death.6,9 Because preeclampsia is associated with placentation abnormalities, serum levels of activin A and inhibin A correspondingly could be altered. These two substances have been shown repeatedly to be altered in women once preeclampsia has already developed.1–3
In this study we attempted to discern whether activin A and inhibin A were also altered before the development of preeclampsia. Using a nested case-control design, we compared serum from patients who eventually developed preeclampsia with serum from patients who remained normotensive throughout pregnancy. Matching and exclusion criteria were used to limit the effects of confounding and to allow an alteration, if it were found, to be specifically associated with preeclampsia. The demographic characteristics of the study and control groups were similar. The control group and study groups had a difference in mean arterial pressures (MAP) at the first visit that was small in magnitude but statistically significant. This finding is consistent with results reported by other investigators who found that an MAP elevation of even 5 mmHg during the second trimester significantly increased the risk of subsequently developing preeclampsia.10
As expected, women who were preeclamptic had significantly higher MAP at delivery. Despite the difference between the two groups with respect to the development of preeclampsia, no evidence could be found that suggested that the study group had altered serum levels of either activin A or inhibin A. Consequently, the ability of activin A and inhibin A to identify patients at high risk of developing preeclampsia and to elucidate any primary etiologic role in this disease process is questionable.
Our results are partly at odds with those of Aquilina et al,6 who did not appraise the role of activin A, but found that inhibin A was elevated in patients before the development of preeclampsia. Several factors might account for the different findings. In our study, we sought to determine whether altered serum levels of activin A and inhibin A were specifically associated with the later development of preeclampsia. Therefore, study and control populations excluded women with other complications, such as abruptio placentae or stillbirth, and were matched for birth weight and gestational age at delivery to ensure similar frequency of fetal growth abnormalities. Elevated inhibin A levels have been associated with the above-mentioned complications and with small for gestational age neonates. If those complications, which are also associated with preeclampsia, are not controlled for, alterations of inhibin A preceding preeclampsia might be a manifestation of other placentation abnormalities rather than of preeclampsia specifically.
Additionally, our study population predominantly consisted of women who required delivery for preeclampsia after 37 weeks. Investigators who studied women who have already developed preeclampsia found significantly greater alterations in activin A and inhibin A in women with early-onset preeclampsia. Similarly, Aquilina et al6 found greater alterations of inhibin A in the second trimester in women who developed preeclampsia early in pregnancy. Inhibin A and activin A might be usable as markers to identify women who develop early-onset preeclampsia. Our data can neither support nor refute this possibility. In a low-risk population, few cases of preeclampsia require delivery before 34 weeks, so the adequacy of activin A and inhibin A as a general screening tool for preeclampsia is not readily apparent.
The results of the present investigation could be limited by the sample size. However, studies that have assessed serum levels of inhibin A and activin A once preeclampsia has already developed have found large alterations in those analytes. As noted in the power calculation, Muttukrishna et al2 found that levels of activin A and inhibin A in preeclamptic women were more than eight times higher than levels in controls. The present study had sufficient power to detect significantly smaller differences.
Many biochemicals have been evaluated as markers for and as factors that might cause preeclampsia.11 Unfortunately, the biochemicals studied so far have not elucidated the pathophysiology of preeclampsia or allowed the development of a clinically useful marker. Similarly, the present evaluation of activin A and inhibin A found that these analytes were not specific markers for the development of preeclampsia in a low-risk population of women.
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Footnotes |
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Received November 22, 1999. Received in revised form March 8, 2000. Accepted March 16, 2000.
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References |
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2. Muttukrishna S, Knight PG, Groome NP, Redman CWG, Ledger WL. Activin A and inhibin A as possible endocrine markers for preeclampsia. Lancet 1997;349:1285–8.[Medline]
3. Silver HM, Lambert-Masserlian GM, Star JA, Hogan J, Canick JA. Comparison of maternal serum total activin A and inhibin A in normal, preeclamptic, and nonproteinuric gestationally hypertensive pregnancies. Am J Obstet Gynecol 1999;180:1131–7.[Medline]
4. Wallace EM, Healy DL. Inhibins and activins: Roles in clinical practice. Br J Obstet Gynaecol 1996;103:945–56.[Medline]
5. Qu J, Thomas K. Inhibin and activin production in the human placenta. Endocr Rev 1995;16:485–507.[Medline]
6. Aquilina J, Barnett A, Thompson O, Harrington K. Second-trimester maternal serum inhibin A concentration as an early marker for preeclampsia. Am J Obstet Gynecol 1999;181:131–6.[Medline]
7. Wong W, Garg S, Woodruff T, Bald L, Fendly B, Lofgren J. Monoclonal antibody based ELISAs for measurement of activin in biological fluids. J Immunol Methods 1993;165:1–10.[Medline]
8. Groome NP, O’Brien M. Immunoassays for inhibin and its subunits: Further applications of the synthetic peptide approach. J Immunol Methods 1993;165:167–76.[Medline]
9. Petraglia F, DeVita D, Gallinelli A, Aguzzoli L, Genazzani AR, Romero R, et al. Abnormal concentration of maternal serum activin A in gestational diseases. J Clin Endocrinol 1995;80:558–61.[Abstract]
10. Caritis S, Sibai B, Hauth J, Lindheimer M, VanDorsten P, Klebanoff M, et al. Predictors of preeclampsia in women at high risk. Am J Obstet Gynecol 1998;179:946–51.[Medline]
11. Grunewald C. Biochemical prediction of preeclampsia. Acta Obstet Gynaecol Scand 1997;76:104–7.
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