1 Nuffield Department of Obstetrics and Gynaecology, University of Oxford, John Radcliffe Hospital, Headington, Oxford OX3 9DU, UK, 2 Research Centre for Reproductive Medicine, Department of Obstetrics and Gynaecology, National Women's Hospital, Auckland, New Zealand and 3 School of Biological and Molecular Sciences, Oxford Brookes University, Headington, Oxford OX3 0BH, UK
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Abstract |
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Key words: activin A/gestational hypertension/inhibin A/pre-eclampsia/pregnancy
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Introduction |
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Inhibins (-ß dimers) and activins (ß-ß dimers) are glycoprotein hormones belonging to the transforming growth factor ß superfamily. Concentrations of circulating dimeric inhibin A rise in early pregnancy, fall after 12 weeks gestation, and remain low until 24 weeks (Muttukrishna et al., 1995
; Birdsall et al., 1997
). Thereafter, concentrations increase gradually but with a marked rise in the third trimester (Muttukrishna et al., 1995
). Circulating concentrations of activin A are similar during the first and second trimesters and rise progressively in the third trimester with a steep increase at term (Muttukrishna et al., 1996
). The fetoplacental unit is an important source of inhibin A and activin A in pregnancy (Birdsall et al., 1997
; Muttukrishna et al., 1997a
). It has been shown that maternal serum concentrations of inhibin A and activin A are 10-fold higher in women with severe pre-eclampsia compared to gestational age matched controls (Muttukrishna et al., 1997b
). Other investigators have also reported elevated inhibin A and activin A concentrations in pre-eclampsia (Petraglia et al., 1995
; Fraser et al., 1998
). In women who subsequently developed pre-eclampsia, inhibin A concentrations were found to be elevated at 1318 weeks in a retrospective analysis of a Down's screening programme (Cuckle et al., 1998
; Aquilina et al., 1999
). Neither of these studies analysed the time courses of the changes in serum measurements or how such changes may vary depending on when pre-eclampsia develops. These studies were not designed to test the ability of activin A and inhibin A to predict pre-eclampsia. In a nested case-control study, elevated inhibin A concentrations were found at 1522 weeks in women who later developed pre-eclampsia, particularly in those delivering preterm (King et al., 1998
).
This study attempted to confirm and extend the results of the earlier reports. There were two objectives. The first was to establish in a longitudinal study whether serum concentrations of inhibin A and activin A were raised prior to the clinical onset of pre-eclampsia and gestational hypertension and, if so, with what time course in relation to the time when clinical pre-eclampsia became manifest. The second was to determine, in a nested case-control study, whether measurements of serum inhibin A or activin A at 1518 weeks and 2124 weeks gestation could be clinically effective predictors of pre-eclampsia, in particular of early-onset pre-eclampsia.
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Materials and methods |
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Gestational hypertension was defined as systolic blood pressure >140 mm Hg with a rise >30 mm Hg or diastolic blood pressure >90 mm Hg with a rise >15 mm Hg on two or more occasions after 20 weeks gestation, but before the onset of labour. Pre-eclampsia was defined as gestational hypertension with proteinuria (>300 mg/24 h or if no formal quantitation available >1 g/l on spot urinalysis).
Study 1: time course of changes
Samples from five groups of women were studied. The method of selection from the total serum bank was as follows. (i) 223 women were identified by random number selection (Paradox 4.5, Borland, Scott Valley, California, USA) from the total cohort of normotensive women who delivered after 37 weeks. The first 25 of these women with complete sets of specimens were selected (normal pregnant control women). (ii) The first 25 women, with the most complete sets of specimens, of the 46, who developed pre-eclampsia and delivered at >37 weeks were taken (term pre-eclampsia). Samples from (iii) all women with pre-eclampsia who delivered at 3436 weeks (n = 14) (pre-term pre-eclampsia) and (iv) all women with pre-eclampsia who delivered before 34 weeks (early onset pre-eclampsia; n = 11) were selected. (v) The first 25 of the 117 women who developed gestational hypertension, who delivered after 37 weeks and who had the most complete sets of specimens were included.
Study 2: clinical prediction
Serum samples taken at 1519 weeks and 2125 weeks were analysed using a nested case control design. A control group (n = 244) was randomly selected by computer from women remaining in the cohort after exclusion of women with pre-eclampsia. A serum sample was not available at one time point in some women either because they had failed to present for sampling or because insufficient volume was stored. Forty-two of the 244 women in the control group developed other pregnancy complications. Twenty (8.2%) developed gestational hypertension (one with iatrogenic preterm birth), nine (3.7%) had spontaneous preterm birth and 13 (5.3%) had idiopathic fetal growth retardation (birthweight <5th centile). These women were excluded when the reference ranges were established, but included when the predictive characteristics of the tests were calculated.
Hormone assays
Samples from women who developed pre-eclampsia and from controls were included in each assay plate.
Inhibin A
Serum concentrations of dimeric inhibin A were measured in duplicate 5 µl aliquots as described elsewhere (Groome et al., 1994). Samples were oxidized with 6% hydrogen peroxide and plated on to 96 well plates coated with a mouse monoclonal antibody (E4) against the ßA subunit. After overnight incubation, the plates were washed and incubated for 2 h with a second mouse monoclonal antibody (against the alpha subunit) conjugated to alkaline phosphatase. Bound alkaline phosphatase was quantified using a commercially available enzyme-linked immunosorbent assay (ELISA) amplification kit (ImmunoSelect ELISA amplification system, Life Technologies, Uxbridge, UK). Minimum detection limit of the assay for human recombinant inhibin A (National Institute for Biological Standards and Controls, South Mimms, UK) was 2 pg/ml. The mean intra- and inter-assay coefficients of variation were 4.3 and 5.1% respectively. The intra- and inter-assay variations were calculated at three points on the standard curve [low concentration (0.41.0 pg/well), middle concentration (3.136.25 pg/well) and high concentration (12.525 pg/well)] using three quality controls (n = 12 assays).
Activin A
Serum concentrations of total activin A were measured in duplicate 10 µl aliquots using an enzyme immunoassay (EIA) specific for total activin A, as described previously (Knight et al., 1996). Samples were treated with 1:1 volume of 15% (w/v) sodium dodecyl sulphate (SDS) and incubated in a boiling water bath for 10 min. SDS treated samples were oxidized with 2.5% (v/v final concentration) hydrogen peroxide and 100 µl/well transferred to 96 well plates coated with E4 antibodies. After 10 min incubation, biotinylated E4 antibody (25 µl/well) was added and the plates were incubated overnight. On day 2, they were washed and 50 µl/well of extravidin (Sigma Chemical Co., St Louis, MO, USA) conjugated with alkaline phosphatase (1:10 000 v/v) was added to all wells. After 2 h, the plates were washed and bound alkaline phosphatase was quantified as described above. The minimum detection limit of the assay for human recombinant activin A (Genentech, San Francisco, CA, USA) was 50 pg/ml. The mean intra- and inter-assay coefficients of variation were 6.5 and 7.7% respectively. The intra and interassay variations were calculated at three points on the standard curve [low concentration (510 pg/well), middle concentration (62.5125 pg/well) and high concentration (250500 pg/well)] using three quality controls (n = 12 assays).
Statistics
Analyses of time courses of changes
As the raw data were not normally distributed, they were log transformed (base 10) to obtain normal distribution. General estimating equations (GEE) statistical analysis was used to analyse the data (STATA statistical software, STATA Corporation, Houston, Texas, USA).
Analyses of predictive efficacy
The reference ranges at 1519 and 2125 weeks gestation were generated from women (n = 202) who remained normotensive and delivered a normally grown (birthweight >5th centile) infant at term. The 90th and 95th centiles for activin A and inhibin A are shown in Table I. These threshold values were then used to calculate the sensitivity, specificity, likelihood ratio and post-test probability (post-test odds = pre-test oddsxlikelihood ratio; probability = odds/1 + odds) (Deeks and Morris, 1996
). The test characteristics were then calculated separately for early onset pre-eclampsia (delivered before 34 weeks) and compared with those of the control group.
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Results |
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Discussion |
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As far as is known, trophoblast dysfunction is the primary problem in pre-eclampsia (Redman, 1991). It is thought that the maternal syndrome in some way may be caused by oxidative stress in the placenta (Wang and Walsh, 1998
) associated with spiral artery disease. It is not known why inhibin A and activin A are increased and whether they contribute to the aetiology of the disease. Inhibin
subunit, inhibin/activin ß subunit and immunoreactive inhibin (ir-inhibin) are synthesized in trophoblast throughout pregnancy (McLachlan et al., 1986
; de Kretser et al., 1994
; Qu and Thomas, 1995
; Petraglia, 1997
). Other endocrine products of trophoblast, namely human chorionic gonadotrophin (HCG) (Ashour et al., 1997
) and corticotrophin releasing hormone (Perkins et al., 1995
), are also increased during or before the onset of pre-eclampsia, indicating dysfunction that affects several secreted syncytiotrophoblast hormones. Activin A stimulates production of HCG by first-trimester trophoblast (Caniggia et al., 1997
; Song et al., 1996
) and HCG increases secretion of ir-inhibin production by cultured placental cells (Qu and Thomas, 1995
). This may be an explanation for the concomitant increase in activin A, HCG and inhibin A (Keelan et al., 1998
). Inflammatory cells (Yu et al., 1996
) also produce activin A. Since the maternal syndrome of pre-eclampsia is characterized by an intense systemic inflammatory response (Redman et al., 1999
), circulating activin A may be derived from circulating inflammatory cells rather than, or as well as, from trophoblast. This may explain why circulating activin A is a more sensitive marker of the disease than inhibin A. Follistatin is a high affinity binding protein for activin A. At 3839 weeks gestation, activin A and follistatin concentrations both rise to a peak and their concentrations are highly correlated. The parallel increase throughout pregnancy probably reflects fetoplacental secretion (O'Connor et al., 1999
). There are different molecular forms of follistatin in the circulation and FS315 is the predominant form in humans. The assay used in the above stated study measures FS285 and has a 10% cross-reaction with FS315. Therefore, the concentrations measured by using this ELISA do not give an accurate measure of `total' follistatin, making it difficult to evaluate precisely the follistatin:activin ratio. This ratio would indicate the availability of `free' activin A that is biologically active.
It is possible that elevated serum concentrations of activin A and inhibin A in pre-eclampsia result from reduced clearance. In the first trimester, inhibin A and activin A clear from the circulation within 6 h of pregnancy termination (Muttukrishna et al., 1997a). The rates and modes of clearance in pre-eclampsia are not known. However, the early rise before the onset of pre-eclampsia cannot be attributed to impaired renal function, which is not established before 20 weeks in patients who later develop pre-eclampsia.
Although activin A was better than inhibin A for predicting pre-eclampsia, 4175% of women who subsequently developed pre-eclampsia would still not be identified. This low risk nulliparous population had an incidence of pre-eclampsia of 4.8%. The post-test probabilities of disease for activin A were 22.1 and 19.7% at 1519 weeks and 2125 weeks respectively. These are comparable to probabilities of 2030% in women identified as at high risk on clinical criteria alone (Caritis et al., 1998).
The majority of women with early onset pre-eclampsia, the subset at greatest risk of adverse maternal and fetal outcome, were detected by measures of serum inhibin A and activin A at 2125 weeks. However, the incidence of early onset disease was only 0.74%. Consequently, despite likelihood ratios of 5.610.9 for inhibin A and 4.111.9 for activin A, the post-test probabilities ranged from 4.07.6 and 38.2% respectively (Table IV). The interpretation of these low values needs to take account of the rarity of the endpoint. Hence a serum activin A or inhibin A concentration above the 95th centile increases the odds of developing early onset pre-eclampsia from 1 in 135 to 1 in 10 or 11. The study was deliberately underpowered to estimate accurately the test characteristics for early onset pre-eclampsia, and larger studies are necessary.
We (Redman et al., 1999) and others (Ness and Roberts, 1996
) have emphasized the heterogeneity of pre-eclampsia in relation to the interaction between multiple factors specific to the pregnancy or to the mother. From such considerations, it was predicted that the search for a single predictive test would never succeed, but that some tests might predict some subtypes of pre-eclampsia (Redman et al., 1999
). The results of this study are entirely consistent with this viewpoint, as are those of all previous searches for early biochemical or haematological markers.
These results are only applicable to healthy nulliparous women. Studies in high risk women are required to test if activin A or inhibin A are useful predictors in these populations. It will be important to assess whether the measurement of other indices combined with activin A or inhibin A can produce a test with greater sensitivity for pre-eclampsia as occurs with triple or quadruple blood tests for Down's syndrome. If this was possible, an effective predictive test before 20 weeks would allow antenatal care to be tailored appropriately to the individual's level of risk. It would also identify women suitable for recruitment into randomized trials testing prophylactic therapy.
In conclusion, serum concentrations of activin A and inhibin A were elevated prior to the onset of pre-eclampsia and may have a clinical application in identifying women at risk of early onset pre-eclampsia, although larger studies are needed to confirm this point. The time course of changes is consistent with the view that there is placental dysfunction prior to the appearance of the clinical signs of pre-eclampsia.
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Acknowledgments |
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Notes |
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References |
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Submitted on January 26, 2000; accepted on March 17, 2000.