Prediction of pre-eclampsia by uterine artery Doppler ultrasonography and maternal serum pregnancy-associated plasma protein-A, free β-human chorionic gonadotropin, activin A and inhibin A at 22 + 0 to 24 + 6 weeks' gestation

Authors

  • K. Spencer,

    Corresponding author
    1. Prenatal Screening Unit, Clinical Biochemistry Department, Harold Wood Hospital, Romford, UK
    2. Harris Birthright Research Centre for Fetal Medicine, King's College Hospital Medical School, London, UK
    • Prenatal Screening Unit, Department of Clinical Biochemistry, Harold Wood Hospital, Gubbins Lane, Romford, Essex, RM3 0BE, UK
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  • C. K. H. Yu,

    1. Harris Birthright Research Centre for Fetal Medicine, King's College Hospital Medical School, London, UK
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  • M. Savvidou,

    1. Harris Birthright Research Centre for Fetal Medicine, King's College Hospital Medical School, London, UK
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  • A. T. Papageorghiou,

    1. Harris Birthright Research Centre for Fetal Medicine, King's College Hospital Medical School, London, UK
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  • K. H. Nicolaides

    1. Harris Birthright Research Centre for Fetal Medicine, King's College Hospital Medical School, London, UK
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Abstract

Objective

To investigate the potential value of combining uterine artery Doppler ultrasonography with the measurement of maternal serum pregnancy-associated plasma protein-A (PAPP-A), free β-human chorionic gonadotropin (β-hCG), activin A and inhibin A at 22 + 0 to 24 + 6 weeks' gestation, in the prediction of pregnancies that subsequently develop pre-eclampsia.

Methods

The maternal serum PAPP-A, free β-hCG, activin A and inhibin A concentrations at 22 + 0 to 24 + 6 weeks' gestation were measured in samples obtained from women with singleton pregnancies who participated in a screening study for pre-eclampsia by transvaginal color flow Doppler measurement of the uterine artery pulsatility index (PI). A search was made of the database to identify those who subsequently developed pre-eclampsia (n = 24) and a group of controls with normal outcome (n = 144). Regression analysis was performed to establish any relationship between the biochemical markers themselves and between the biochemical markers and uterine artery mean PI. A multivariate Gaussian model combining various biochemical markers with uterine artery mean PI was developed using standard statistical modeling techniques and the performance of such models in discriminating cases with pre-eclampsia was evaluated by receiver–operating characteristics curve (ROC) analysis.

Results

In the pre-eclampsia group, compared to the controls, the uterine artery mean PI and the maternal serum levels of PAPP-A, free β-hCG, activin A and inhibin A were significantly increased. The predicted detection rates of pre-eclampsia, for a false positive rate of 5%, was 50% by uterine artery mean PI, 5% by PAPP-A, 10% by free β-hCG, 35% by inhibin A and 44% by activin A. Screening by a combination of uterine artery mean PI and maternal serum activin A and inhibin A could detect 75% and 92% of patients who subsequently developed pre-eclampsia, for false positive rates of 5% and 10%, respectively.

Conclusion

Screening for pre-eclampsia by uterine artery PI at 22 + 0 to 24 + 6 weeks' gestation can be improved by measurement of activin A and inhibin A levels. Copyright © 2006 ISUOG. Published by John Wiley & Sons, Ltd.

Introduction

Pre-eclampsia is thought to be a circulatory maladaptation disease that is characterized by defective trophoblastic invasion of the maternal spiral arteries1. The consequent increased resistance in the uteroplacental circulation forms the basis of screening for the condition by uterine artery Doppler ultrasonography2. Thus increased impedance to flow in the uterine arteries, as measured by the pulsatility index (PI) at 20–25 weeks' gestation, can identify, for a 5% false positive rate, about 45% of pregnancies that subsequently develop pre-eclampsia2–4.

There is some evidence that the combination of certain biochemical markers, such as activin A and inhibin A, could potentially enhance the sensitivity of uterine artery Doppler velocimetry for the prediction of pre-eclampsia5, 6. Activin A and inhibin A are dimeric glycoproteins produced by the decidua, placenta and fetal membranes. The maternal serum concentration of these proteins is increased at the time of clinical manifestation of pre-eclampsia or gestational hypertension7–14. Although there is some evidence that activin A and inhibin A are also elevated in the first and second trimesters13, 15, 16, levels appear to decline to normal the further one gets away from the onset of clinical symptoms.

Other potentially useful biochemical markers of impaired placentation are pregnancy-associated plasma protein-A (PAPP-A) and free β-human chorionic gonadotropin (β-hCG), which are now increasingly being used, in combination with fetal nuchal translucency thickness, in first-trimester screening for chromosomal defects17. Recently, PAPP-A has been shown to be an insulin-like growth factor (IGF) binding protein (IGFBP) protease18. Since IGFBPs have a key role in modulating IGF activity, PAPP-A could be important in regulating fetal growth and development19 and in trophoblastic invasion of the decidua20. The maternal serum concentration of PAPP-A is increased soon after the onset of pre-eclampsia21, but at 11–14 weeks' gestation the levels are decreased in those pregnancies that subsequently develop pre-eclampsia15, 22.

The aim of this study was to investigate the potential value of combining uterine artery Doppler ultrasonography with the measurement of maternal serum PAPP-A, free β-hCG, activin A and inhibin A at 22 + 0 to 24 + 6 weeks' gestation in the prediction of pregnancies that subsequently develop pre-eclampsia.

Patients and Methods

This was a case-control study in which uterine artery PI and maternal serum PAPP-A, free β-hCG, activin A and inhibin A concentrations were measured at 22 + 0 to 24 + 6 weeks' gestation in 24 singleton pregnancies that subsequently developed pre-eclampsia and 144 normal controls, matched for gestation and duration of storage of samples. The patients were recruited from a screening study for pre-eclampsia by transvaginal color flow Doppler measurement of the uterine artery PI3. Blood samples were also collected and the serum stored at −20 °C. A search was made of the database to identify all singleton pregnancies that had maternal serum collected and that had also had 22 + 0 to 24 + 6 week Doppler investigations. The hospital notes and delivery suite records were then searched for each one of these patients to identify any pregnancy complications and obtain delivery details. Specifically pregnancies with pre-eclampsia were identified in which pregnancy-induced hypertension was defined by a diastolic blood pressure of ≥ 110 mmHg on any one occasion or a diastolic blood pressure of ≥ 90 mmHg on two consecutive occasions 4 h apart in women with no pre-existing hypertensive or renal disease, and the presence of either more than 300 mg of total protein in a 24-hour urine collection or a 1 + albumin on reagent strip23.

Free β-hCG and PAPP-A were measured by the Kryptor Immunoassay system (Brahms AG, Berlin, Germany), the performance of which has been described previously17. Activin A and inhibin A were measured in duplicate by commercial quantitative enzyme linked immunosorbent assay (ELISA) techniques (Serotec, Kidlington, UK), the performance of both of which has also been previously described15, 24, 25.

Statistical analysis

Biochemical marker results were expressed as a multiple of the median (MoM) value in normal pregnancies using the values obtained in this study. Comparison of the biochemical data in the pre-eclampsia group with the normal group was by means of unpaired t-test of unequal variance after log10 transformation of the MoM values to achieve a Gaussian distribution of the data. Regression analysis was performed to establish any relationship between the biochemical markers themselves and between the biochemical markers and uterine artery mean PI. The detection rate for pre-eclampsia and false positive rate for different cut-offs in marker levels were calculated and receiver–operating characteristics (ROC) curves were constructed to establish clinical utility. A multivariate Gaussian model combining various biochemical markers with uterine artery mean PI (after log10 transformation to achieve Gaussianarity) was developed using standard statistical modeling techniques, and the performance of such models in discriminating cases with pre-eclampsia was assessed by ROC analysis. All statistical analysis was performed using Analyse-It (Smart Software, Leeds) and Microsoft Excel or with SPSS 12 (SPSS, Woking).

Results

The uterine artery mean PI was significantly higher in the pre-eclampsia group than in the controls [1.70 vs. 0.908, P < 0.001; log10 mean PI (SD) 0.1982 (0.1195) vs. −0.0342 (0.1681)]. Similarly, the maternal serum levels of PAPP-A, free β-hCG, activin A and inhibin A were significantly higher in the pre-eclampsia group than in the controls (Table 1, Figure 1). In the pre-eclampsia group there was no significant correlation between uterine artery mean PI and any of the biochemical markers, and in the controls there was only a small but significant correlation with activin levels (Table 2). A significant correlation was seen between free β-hCG and PAPP-A and inhibin A in the pre-eclampsia group and between inhibin A and PAPP-A and free β-hCG in the control group.

Figure 1.

Box–whisker plots for various markers in controls compared with those in cases with pre-eclampsia (PET). The diamonds indicate means and their 95% confidence intervals. The notched boxes show the medians and their 95% confidence intervals and the upper/lower interquartile ranges. hCG, human chorionic gonadotropin; MoM, multiple of the median; PAPP-A, pregnancy-associated plasma protein-A; PI, pulsatility index.

Table 1. Biochemical marker levels in cases with pre-eclampsia and controls
 Pre-eclampsia (n = 24)Controls (n = 144)P
  1. hCG, human chorionic gonadotropin; MoM, multiple of the median; PAPP-A, pregnancy-associated plasma protein-A; SD, standard deviation.

Free β-hCG 
 Median MoM1.981.000.003
 Mean log10 MoM0.19760.0270 
 SD log10 MoM0.23850.2879 
PAPP-A 
 Median MoM1.621.00 
 Mean log10 MoM0.0864−0.03730.02
 SD log10 MoM0.22750.2691 
Inhibin A 
 Median MoM2.031.05<0.001
 Mean log10 MoM0.39570.0530 
 SD log10 MoM0.35070.1707 
Activin A 
 Median MoM2.141.02<0.001
 Mean log10 MoM0.33510.0017 
 SD log10 MoM0.23070.2181 
Table 2. Marker correlations in pre-eclampsia cases and controls
ControlsPre-eclampsia
Mean PIFree β-hCGPAPP-AInhibin AActivin A
  1. hCG, human chorionic gonadotropin; PAPP-A, pregnancy-associated plasma protein-A; PI, pulsatility index.

Mean PI0.22 (P = 0.499)0.35 (P = 0.298)0.24 (P = 0.457)−0.13 (P = 0.697)
Free β-hCG0.02 (P = 0.896)0.11 (P = 0.198)0.26 (P = 0.002)0.03 (P = 0.678)
PAPP-A0.02 (P = 0.860)0.52 (P = 0.009)0.18 (P = 0.034)−0.03 (P = 0.752)
Inhibin A0.18 (P = 0.129)0.48 (P = 0.017)0.23 (P = 0.278)0.03 (P = 0.688)
Activin A0.23 (P = 0.046)−0.17 (P = 0.433)0.10 (P = 0.626)−0.32 (P = 0.125)

The predicted detection rates of pre-eclampsia, for different false positive rates, by uterine artery mean PI and maternal serum biochemical markers are shown in Table 3 and Figure 2. Screening by a combination of uterine artery mean PI and maternal serum activin A and inhibin A could identify 75% of patients who subsequently developed pre-eclampsia for a false positive rate of 5%. Alternatively, the detection rate was 92% at a false positive rate of 10%.

Figure 2.

Receiver–operating characteristics plot for screening for pre-eclampsia by uterine artery mean pulsatility index (PI) (------) and uterine artery mean PI combined with maternal serum activin A and inhibin A (------).

Table 3. Area under the receiver–operating characteristics curve (AUC) and detection rates at 5% and 10% false positive rates (FPR) when screening for pre-eclampsia by uterine artery Doppler velocimetry and maternal serum levels of free β-hCG, PAPP-A, inhibin A and activin A
Method of screeningAUCDetection rate (%)
FPR 5%FPR 10%
  1. hCG, human chorionic gonadotropin; PAPP-A, pregnancy-associated plasma protein-A; PI, pulsatility index.

Uterine artery mean PI0.8725066
Maternal serum free β-hCG0.6881015
Maternal serum PAPP-A0.630510
Maternal serum inhibin A0.8193553
Maternal serum activin A0.8504460
Maternal serum inhibin A and activin A0.9496783
Uterine artery mean PI and maternal serum inhibin A0.9136775
Uterine artery mean PI and maternal serum activin A0.9355775
Uterine artery mean PI, and maternal serum activin A and inhibin A0.9707592

In women in which the pre-eclampsia was severe enough to warrant delivery prior to 35 weeks' gestation (n = 10) compared with those who delivered at or after 35 weeks (n = 14), the median marker MoMs were 1.92 vs. 1.11 (P = 0.28) for PAPP-A, 2.07 vs. 1.16 (P = 0.10) for free β-hCG, 3.20 vs. 1.78 (P = 0.23) for inhibin A and 2.09 vs. 2.20 (P = 0.10) for activin A. The difference in mean PI was 1.99 vs. 1.31 (P < 0.001). The number of cases that delivered before 35 weeks' gestation was insufficient to allow a full ROC analysis.

Discussion

The findings of this study, that measurement of the uterine artery mean PI at 22 + 0 to 24 + 6 weeks' gestation can, for a 5% false positive rate, identify about 50% of pregnancies that subsequently develop pre-eclampsia, are compatible with the results of previous large-scale prospective screening studies3. In addition, our findings suggest that the performance of screening can be improved by combining uterine artery Doppler scan with maternal serum biochemical analysis. Thus, a combination of uterine artery mean PI and maternal serum activin A and inhibin A could detect 75 and 92%, respectively, of patients who subsequently develop pre-eclampsia, with false positive rates of 5 and 10%.

Two previous studies have also reported that the detection rate of pre-eclampsia was higher by combining uterine artery Doppler sonography and maternal serum biochemistry than with either method alone. Thus, Aquilina et al.5, in a study that included 35 patients who subsequently developed pre-eclampsia, reported that for a false positive rate of about 7% the detection rate with uterine artery Doppler at 18–22 weeks' gestation was 60% and this increased to 71% by combining Doppler scan with maternal serum inhibin A levels at 15–19 weeks. Florio et al.6 measured activin A and inhibin A levels in 58 asymptomatic pregnant women at 24 weeks' gestation with Doppler evidence of increased impedance to flow in the uterine arteries. The probability of developing pre-eclampsia was calculated for different combinations of results. Activin A achieved a detection rate of 60% at a false positive rate of 10%, while inhibin A had a detection rate of 41% with a false positive rate of 10%. This compares well with our results of 60% for activin A and 53% for inhibin A in an unselected population at a 10% false positive rate.

The findings of maternal serum biochemical markers at 22 + 0 to 24 + 6 weeks, together with the results of previous studies, suggest that the pattern of serum concentration changes across pregnancy in women developing pre-eclamsia.

The maternal serum PAPP-A levels in pregnancies that subsequently develop pre-eclampsia are reduced at 11–14 weeks' gestation15, 22, 26–31 and mildly increased at 22 + 0 to 24 + 6 weeks. In women with pre-eclampsia in the third trimester, the levels are increased21, 31, 32. Similarly, tissue PAPP-A levels in placental extracts of pre-eclamptic patients at term are higher than normal33.

The maternal serum free β-hCG levels in pregnancies that subsequently develop pre-eclampsia may be mildly reduced at 11–14 weeks15, 16, 22. At 15–20 weeks' gestation the median free β-hCG in patients who developed pre-eclampsia was 1.41 MoM for intact or total hCG14, 34–36 and 1.17 MoM for free β-hCG9, 37, 38. At 30–40 weeks the level in those who developed severe pre-eclampsia was 4.26 MoM39. In established pre-eclampsia the levels of hCG are increased40–42.

We found that at 22 + 0 to 24 + 6 weeks' gestation the maternal serum levels of activin A and inhibin A in women who subsequently develop pre-eclampsia are on average twice as high as in normal pregnancies. Previous studies reported that at 11–14 weeks the levels in those who subsequently develop pre-eclampsia are about 1.5 MoM for both activin A and inhibin A16, 43, whereas in women with pre-eclampsia, at 25–41 weeks, the median levels of activin A and inhibin A ranged from 2.59 to 9.64 MoM and 1.92 to 8.47 MoM, respectively7, 8, 10, 32, 44. A longitudinal study of 71 women who subsequently developed pre-eclampsia reported that the median MoM levels of both inhibin A and activin A increased with gestation from 8 to 30 weeks13. Studies of women at 15–20 weeks' gestation reported that in those who subsequently developed pre-eclampsia the median values of inhibin A were increased and ranged from 1.09 to 2.01 MoM9, 36, 38, 45, whereas the levels of activin A were not significantly different from normal34.

Conclusions

In this study we used standard statistical analysis, as used in screening for trisomy 21, to establish a method of screening for pre-eclampsia by a combination of uterine artery Doppler ultrasonography and maternal serum biochemical testing at 22 + 0 to 24 + 6 weeks' gestation. Such an approach can improve the detection rate, for a false positive rate of 10%, from 66% with uterine artery Doppler alone to more than 90% by combining Doppler velocimetry with maternal serum activin A and inhibin A levels. The results also suggest that for PAPP-A, free β-hCG and inhibin, levels of these markers are even further elevated in cases of severe pre-eclampsia.

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