Preeclampsia is the second largest cause of maternal mortality worldwide. In addition, preeclampsia frequently coexists with intrauterine growth restriction, placental abruption, and the need for iatrogenic preterm delivery, which are other major causes of adverse perinatal outcome.1,2 The severity of this condition and its consequences have demanded a focus on markers capable of predicting preeclampsia. Early detection of this condition allows for planning the appropriate monitoring, management, and early identification of complications. Although trials of prophylactic intervention for preeclampsia from midgestation have been largely unsuccessful, it has been suggested that first-trimester prediction may make early prophylactic strategies feasible.3–6
No single screening test in the prediction of this condition has gained widespread adoption into clinical practice. Uterine artery Doppler, especially in the second trimester, is the best performing of all the available clinical tests to date and is certainly the most widely studied.7–11 Numerous serum markers related to the etiology of preeclampsia have been measured either in maternal serum alone or in combination with uterine artery Doppler to attempt the prediction of preeclampsia.12–15 Combining uterine artery Doppler and maternal serum markers that relate to the proposed etiological pathways may improve the predictive value for preeclampsia compared to using them in isolation. In this study, second-trimester uterine artery Doppler and multiple early-pregnancy maternal serum biomarkers including C-reactive protein (CRP), serum amyloid A, β2-microglobulin, neopterin, and cystatin C were investigated as potential markers for identification of women in whom preeclampsia will subsequently develop.
SUBJECTS AND METHODS
This was a nested case–control study of women who delivered between 2002 and 2005. Gestational age was calculated from the last menstrual period and confirmed by crown–rump length measurement at 11 to 13 6/7 weeks of gestation. Maternal venous serum samples were collected from all women during early pregnancy. In all cases, 5 mL of maternal blood was drawn into a nonheparinized tube, allowed to clot, and centrifuged. Maternal serum samples were stored at −80°C for up to 5 years until analysis, with only one freeze–thaw cycle for allocation of sample aliquots. A second-trimester ultrasound scan was performed as part of routine antenatal care, and uterine artery resistance index (RI) was measured in those that agreed. The ultrasound probe was held in the sagittal axis and moved laterally. B-mode ultrasound examination was used to visualize the vessels lateral to the uterus, and color Doppler was used to identify the uterine artery at the apparent crossover with the internal iliac artery. A pulsed-wave Doppler gate was then placed over the uterine artery with an angle of insonation less than 60°. When three similar consecutive waveforms were obtained, the presence of a protodiastolic notch was recorded and the RI was measured and the mean was calculated. Ultrasound scans were performed only by trained ultrasonographers or fetal medicine specialists.
Patient characteristics including demographics, smoking status, and obstetric and medical history were obtained from women at the first hospital visit and entered into a fetal medicine unit database. Women with medical conditions known to contribute to a preeclampsia risk profile, including diabetes mellitus, connective tissue disease, renal disorders, and essential hypertension, were excluded from the study. All women had a blood pressure less than 140/90 mm Hg and no history of hypertension when requesting maternity care. Pregnancy outcomes were obtained from the delivery suite database or general practitioners and entered into the same database when they became available. Members of the case group were defined as women with preeclampsia diagnosed according to the guidelines of the International Society for the Study of Hypertension in Pregnancy. This requires two recordings of diastolic blood pressure of 90 mm Hg or greater at least 4 hours apart in previously normotensive women and proteinuria of 300 mg or more in 24 hours, or two readings of at least 2+ on dipstick analysis of midstream or catheter urine specimens if a 24-hour collection is not available. The women in the control group were randomly selected and consisted of women with a liveborn neonate delivered beyond 37 weeks of gestation and in the absence of preeclampsia. Wandsworth local research ethics committee approval was obtained for the study, and all women gave written, informed consent. After identification of case and control group members, stored blood samples were retrieved from storage. All analyses of blood samples were performed in a blind fashion. Maternal serum concentrations of cystatin C, β2-microglobulin, serum amyloid A, CRP, and neopterin were measured in case and control group members.
Cystatin C, CRP, β2-microglobulin, and serum amyloid A were measured using a BN ProSpec nephelometer (Siemens Healthcare Diagnostics Ltd, Camberley, Surrey, UK) with a particle-enhanced immunonepholometric assay. Briefly, polystyrene particles coated with appropriate antihuman monoclonal antibodies were mixed with the samples, standards, or quality-control materials. The antibody-coated beads react with their antigens, causing an increase in light scatter that is related to the amount of that specific protein in the sample. The intraassay coefficient of variation for cystatin C, CRP, β2-microglobulin, and serum amyloid A were was than 2.8%, less than 3.0%, less than 4.6%, and less than 9.2%, respectively. The corresponding interassay coefficient of variations was less than 3.1%, less than 2.5%, less than 4.0%, and less than 10.7%, respectively. Neopterin concentrations were measured by an enzyme-linked immunosorbent assay method according to the manufacturer's instructions using the Brahms Neopterin enzyme-linked immunosorbent assay test kit (Brahm's Diagnostica, Berlin, Germany). The intraassay and interassay coefficient of variations were less than 5.7% and less than 6.5%, respectively.
Patients were subdivided into the two groups depending on pregnancy outcome as described. Mean (standard deviation) or median (range) was used to express normally or non-normally distributed data, respectively. For intergroup comparisons, t test, Mann–Whitney test, χ2 test, and Fisher exact test were used, as appropriate. Each of the serum markers and uterine Doppler mean RI were assessed for correlation with gestational age using Pearson correlation. Factors for which a significant correlation was found with gestational age were expressed as multiples of the median. Those that did not change with gestational age were expressed in their original units. After this, the distributions of each of the serum markers and RI were assessed using the Kolmogorov-Smirnov test. Factors for which there was evidence of non-normality were transformed to conform to a Gaussian distribution. Univariable regression analysis was performed to estimate correlations between each serum marker and RI and the development of preeclampsia. Those factors that were significantly correlated at the P=.1 level were entered into a multiple logistic regression model using a backward stepwise approach. Factors were removed from the model using the likelihood ratio statistic with a threshold of P=.05. The odds ratio for uterine artery RI was expressed per 0.1 change in unit to correct for the limited range of possible measurements (0 to 1). Receiver-operating characteristic curves were constructed for each significant marker and area under the curve (AUC) calculated. Likelihood ratios were derived from the overlapping Gaussian distributions of the unaffected and preeclamptic groups. All calculations were performed using the SPSS (release 14; SPSS, Chicago, IL) and StatsDirect (release 2.7.0; StatsDirect, Altrincham, England).
There was a total of 170 patients (45 in the case group, 125 in the control group) included in the analysis. Maternal demographic characteristics and pregnancy outcome data are shown in Table 1. The number of patients who had markers measured is shown in Table 2. A correlation between gestational age and the individual markers was found only with neopterin (r=.203; P=.041), and this was subsequently expressed as multiples of the median. Only uterine artery RI was Gaussian in its raw data form. The remaining markers conformed to a Gaussian distribution after transformation of the data using logarithmic methods.
Univariable analysis of the markers revealed that uterine artery RI, log cystatin, log neopterin multiples of the median, log β2-microglobulin, and log CRP were significantly correlated with the development of preeclampsia (Table 3). Backward stepwise multiple logistic regression revealed that only uterine artery RI, log cystatin C, and log CRP were independent predictors of preeclampsia (Table 4). Of these markers, only log cystatin C and log CRP were weakly correlated (r=.257; P=.004). The areas under the curve for uterine artery RI, log cystatin C, and log CRP as single markers in screening for preeclampsia were 0.728, 0.725, and 0.6343, respectively. The area under the curve for the three markers used in combination was 0.825 (Table 5, Fig. 1). The sensitivity of combined uterine artery RI, cystatin C, and CRP for preeclampsia at screen-positive rates of 10% and 15% were 43.6% and 69.2%, respectively.
Uterine artery Doppler ultrasonograpy is a noninvasive tool that can be used to measure uteroplacental perfusion and thereby indirectly assess trophoblast development.16–18 Uterine artery Doppler has a better detection rate for preterm rather than term preeclampsia and is more sensitive in the second trimester compared with the first trimester.8–11 Cystatin C is an inhibitor of the cysteine proteases, which are thought to play an important role in the degradation of the extracellular matrix that occurs during normal trophoblast invasion. Recent evidence has demonstrated that cystatin C is not only increased in preeclampsia but also elevated in the first trimester in women destined to have preeclampsia develop compared with those having a normal pregnancy.19–21 β2-microglobulin, serum amyloid A, neopterin, and CRP are all markers of cellular or humoral immune activation. All of these markers have been implicated in the pathophysiology of preeclampsia.20,22–30
The current study assessed five early-pregnancy maternal serum markers and second-trimester uterine artery Doppler resistance index in the prediction of preeclampsia. Univariable regression analysis showed that women with preeclampsia do not have increased serum levels of serum amyloid A compared with women with a normal pregnancy outcome. Moreover, when applying multiple regression only cystatin C, CRP, and uterine artery RI remained significantly associated with the subsequent development of preeclampsia. β2-microglobulin and neopterin were too closely correlated with cystatin C to be used in combination. These results are consistent with the hypothesis that preeclampsia has a multifactorial cause and may result from the consequences of a persistent systemic inflammatory response and impaired placentation during early pregnancy. We confirm the hypothesis that by combining independent markers it is possible to improve the detection rate for preeclampsia. The receiver-operating characteristic curve analysis showed that, if we consider the combination of cystatin C, CRP, and mean RI, then the area under the curve is 0.825, which is higher than with each marker in isolation. At a fixed false-positive rate of 10%, prediction of preeclampsia is 38.5% by using uterine artery mean RI; however, if cystatin C and CRP are added, then the detection rate improves to 43.6%. The combined sensitivity of uterine artery RI, log cystatin C, and log CRP for preeclampsia was 69.2% when the screen-positive rate was set at 15%.
The findings of this study are that second-trimester uterine artery Doppler RI, cystatin C, and CRP are independent predictors of preeclampsia, and that combining the markers improves the detection of disease. We have also shown that careful analysis of multiple marker models is necessary because many exhibit a close correlation. Our data support the hypothesized etiological pathways related to preeclampsia, namely maternal systemic inflammation coupled with incomplete trophoblastic invasion and the dysregulation of placental proteases. Further prospective studies of larger populations are required to develop a panel of multiple predictors to cover all possible pathogenetic mechanisms. Combining independent markers gives the possibility of establishing a screening test with a high detection rate and low false-positive rate for the definitive prediction of preeclampsia.
1. Papageorghiou AT. Predicting and preventing preeclampsia-where to next? Ultrasound Obstet Gynecol 2008;31:367–70.
2. Sibai B, Dekker G, Kupferminc M. Pre-eclampsia. Lancet 2005;365:785–99.
3. Villar J, Abdel-Aleem H, Merialdi M, Mathai M, Ali MM, Zavaleta N, et al. World health organization calcium supplementation for the prevention of preeclampsia. Am J Obstet Gynecol 2006;194:639–49.
4. Rumbold AR, Crowther CA, Haslam RR, Dekker GA, Robinson GA. ACTS Study Group. Vitamins C and E and the risks of preeclampsia and perinatal complications. N Engl J Med 2006;354:1796–806.
5. Poston L, Briley AL, Seed PT, Kelly FJ, Shennan AH. Vitamins in Pre-eclampsia (VIP) Trial Consortium. Vitamins in preeclampsia (VIP) Trial Consortium. Lancet 2006;367:1145–54.
6. Askie LM, Duley L, Henderson-Smart DJ, Stewart LA. PARIS Collaborative Group. Antiplatelet agents prevention for preeclampsia: a meta-analysis of individual patient data. Lancet 2007;369:1791–8.
7. Albaiges G, Missfelder-Lobos H, Lees C, Parra M, Nicolaides KH. One-stage screening for pregnancy complications by color Doppler assessment of the uterine arteries at 23 weeks' gestation. Obstet Gynecol 2000;96:559–64.
8. Papageorghiou AT, Yu CK, Bindra R, Pandis G, Nicolaides KH. Fetal Medicine Foundation Second Trimester Screening Group. Multicenter screening for preeclampsia and fetal growth restriction by transvaginal uterine artery Doppler at 23 weeks of gestation. Ultrasound Obstet Gynecol 2001;18:441–9.
9. Martin AM, Bindra R, Curcio P, Cicero S, Nicolaides KH. Screening for preeclampsia and fetal growth restriction by uterine artery Doppler at 11–14 weeks of gestation. Ultrasound Obstet Gynecol 2001;18:583–6.
10. Papageorghiou AT, Leslie K. Uterine artery Doppler in the prediction of adverse pregnancy outcome. Curr Opin Obstet Gynecol 2007;19:103–9.
11. Melchiorre K, Wormald B, Leslie K, Bhide A, Thilaganathan B. First-trimester uterine artery Doppler indices in term and pre-term preeclampsia. Ultrasound Obstet Gynecol 2008;32:133–7.
12. Aquilina J, Thompson O, Thilaganathan B, Harrington K. Improved early prediction of pre-eclampsia by combining second-trimester maternal serum inhibin-A and uterine artery Doppler. Ultrasound Obstet Gynecol 2001;17:477–84.
13. Spencer K, Yu CK, Savvidou M, Papageorghiou AT, Nicolaides KH. Prediction of preeclampsia by uterine artery Doppler ultrasonography and maternal serum pregnancy-associated plasma protein A, free beta-human chorionic gonadotropin, activin A and inhibin A at 22+0 to 24+6 weeks' gestation. Ultrasound Obstet Gynecol 2006;27:658–63.
14. Prefumo F, Canini S, Casagrande V, Pastorino D, Venturini PL, De Biasio P. Correlation between first-trimester uterine artery Doppler indices and maternal serum free beta-human chorionic gonadotropin and pregnancy-associated plasma protein A. Fertil Steril 2006;86:977–80.
15. Cnossen JS, ter Riet G, Mol BW, van der Post JA, Leeflang MM, Meads CA, et al. Are tests for predicting pre-eclampsia good enough to make screening viable? A review of reviews and critical appraisal. Acta Obstet Gynecol Scand. 2009;88:758–65.
16. Prefumo F, Sebire NJ, Thilaganathan B. Decreased endovascular trophoblast invasion in first trimester pregnancies with high-resistance uterine artery Doppler indices. Hum Reprod 2004;19:206–9.
17. Lin S, Shimizu I, Suehara N, Nakayama M, Aono T. Uterine artery Doppler velocimetry in relation to trophoblast migration into the myometrium of the placental bed. Obstet Gynecol 1995;85(5 Pt 1):760–5.
18. Aardema MW, Oosterhof H, Timmer A, van Rooy I, Aarnoudse JG. Uterine artery Doppler flow and uteroplacental vascular pathology in normal pregnancies and pregnancies complicated by pre-eclampsia and small for gestational age fetuses. Placenta 2001;22:405–11.
19. Kristensen K, Larsson I, Hansson SR. Increased cystatin C expression in the pre-eclamptic placenta. Mol Hum Reprod 2007;13:189–95.
20. Kristensen K, Wide-Swensson D, Schmidt C, Blirup-Jensen S, Lindström V, Strevens H, et al. Cystatin C, beta-2-microglobulin and beta-trace protein in preeclampsia. Acta Obstet Gynecol Scand 2007;86:921–6.
21. Thilaganathan B, Ralph E, Papageorghiou AT, Melchiorre K, Sheldon J. Raised maternal serum cystatin C: an early pregnancy marker for preeclampsia. Reprod Sci 2009;16:788–93.
22. Haddad B, Desvaux D, Livingston JC, Barranger E, Paniel BJ, Sibai BM. Failure of serum beta2-microglobulin levels as an early marker of preeclampsia. Am J Obstet Gynecol 2000;182:595–8.
23. Engin-Ustün Y, Ustün Y, Karabulut AB, Ozkaplan E, Meydanli MM, Kafkasli A. Serum amyloid A levels are increased in preeclampsia. Gynecol Obstet Invest 2007;64:117–20.
24. Kronborg CS, Knudsen UB, Moestrup SK, Allen J, Vittinghus E, Møller HJ. Serum markers of macrophage activation in preeclampsia: no predictive value of soluble CD163 and neopterin. Acta Obstet Gynecol Scand 2007;86:1041–6.
25. Kaleli I, Kaleli B, Demir M, Yildirim B, Cevahir N, Demir S. Serum Levels of Neopterin and Interleukin-2 Receptor in Women With Severe Preeclampsia. J Clin Lab Anal 2005;19:36–9.
26. Ustün Y, Engin-Ustün Y, Kamaci M. Association of fibrinogen and C-reactive protein with severity of preeclampsia. Eur J Obstet Gynecol Reprod Biol 2005;121:154–8.
27. Djurovic S, Clausen T, Wergeland R, Brosstad F, Berg K, Henriksen T. Absence of enhanced systemic inflammatory response at 18 weeks of gestation in women with subsequent preeclampsia. BJOG 2002;109:759–64.
28. Qiu C, Luthy DA, Zhang C, Walsh SW, Leisenring WM, Williams MA. A prospective study of maternal serum C-reactive protein concentrations and risk of preeclampsia. Am J Hypertension 2004;17:154–60.
29. Tjoa ML, Van Vugt JM, Go AT, Blankenstein MA, Oudejans CB, van Wijk IJ. Elevated C-reactive protein levels during first trimester of pregnancy are indicative of preeclampsia and intrauterine growth restriction. J Reprod Immunol 2003;59:29–37.
30. Garcia RG, Celedon J, Sierra-Laquado J, Alarcon MA, Luengas C, Silva F, et al. Raised C-reactive protein and impaired flow-mediated vasodilatation precede the development of preeclampsia. Am J Hypert 2007;20:98–103.