Ultrasound Obstet Gynecol 2007; 29: 128–134 Published online 6 December 2006 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/uog.3876

First-trimester maternal serum PP-13, PAPP-A and second-trimester uterine artery Doppler pulsatility index as markers of pre-eclampsia K. SPENCER*, N. J. COWANS*, I. CHEFETZ†, J. TAL‡ and H. MEIRI† *Department of Clinical Biochemistry, Harold Wood Hospital, Romford, UK and †Diagnostic Technologies Ltd., Haifa and ‡Technostat, Kfar Saba, Israel

K E Y W O R D S: hypertension; PAPP-A; pre-eclampsia; pregnancy; PP-13; screening

ABSTRACT Objective To evaluate whether measurement of maternal serum placental protein-13 (PP-13) and pregnancyassociated plasma protein-A (PAPP-A) at 11 + 0 to 13 + 6 weeks of gestation alone or in combination with second-trimester uterine artery pulsitility measured by Doppler velocimetry is useful in predicting those women who will develop pre-eclampsia Methods This was a nested case-control study of preeclampsia cases with controls matched for gestational age and storage time for the maternal serum. Samples were collected as part of a first-trimester prenatal chromosomal anomaly screening program and were routinely tested for PAPP-A. PP-13 was tested using an enzyme linked immunosorbent assay (ELISA) by an examiner who was blinded to pregnancy outcome. All patients also underwent uterine artery Doppler flow velocimetry to measure the mean pulsatility index (PI) at 22–24 weeks’ gestation. Results There were 446 controls and 44 cases with early pre-eclampsia where delivery was induced prior to 35 weeks. In addition there were a further 44 cases with pre-eclampsia in which delivery was not induced before term. Median PP-13 levels for controls, all cases and early pre-eclampsia cases were 176.9, 121.9 and 111.7 pg/mL, with multiples of the median (MoMs) of 1.00, 0.69 and 0.63, respectively (P < 0.001). PAPP-A MoMs were 1.00, 0.89 (P = 0.076) and 0.89 (P = 0.042) and mean PIs were 1.0, 1.6 (P < 0.001) and 1.7 (P < 0.001) for controls, all cases and early cases, respectively. Receiver–operating characteristics (ROC) curve analysis for either all cases or early cases vs. controls yielded areas under the curve for PP-13, PAPP-A and PI respectively of 0.68 (95%

CI, 0.61–0.74; P < 0.001), 0.56 (95% CI, 0.49–0.63; P = 0.076) and 0.79 (95% CI, 0.72–0.87; P < 0.001) for all cases and 0.71 (95% CI, 0.63–0.79; P < 0.001), 0.59 (95% CI, 0.51–0.68; P = 0.076) and 0.86 (95% CI, 0.77–0.94; P < 0.001) for early cases. At a specificity set to 0.80 the sensitivities were 0.50, 0.23 and 0.76 for PP-13, PAPP-A and PI in the early cases and 0.44, 0.24 and 0.73 in all cases. Combining PP-13 and PI using logistic regression analysis yielded an area under the curve of 0.84 (95% CI, 0.78–0.90; P < 0.001) and a sensitivity of 0.74 in all cases, and 0.90 (95% CI, 0.84–0.96; P < 0.001) and a sensitivity of 0.79 for early cases. PAPP-A with PI gave an area under the curve of 0.82 (95% CI, 0.76–0.90; P < 0.001) and a sensitivity of 0.76 in all cases. Combining PAPP-A with PP-13 and PI did not add significantly to the sensitivity. Conclusion First-trimester PP-13 levels may be useful in predicting pre-eclampsia and early pre-eclampsia, and the accuracy of the method increases when coupled with second-trimester Doppler PI measurement. First-trimester PAPP-A provides some prediction for pre-eclamspia when combined with PI but does not add to the prediction of early pre-eclampsia when PP-13 and PI are used together. Further studies are required to establish the real value of PP-13 in first-trimester screening for pre-eclampsia. Copyright  2006 ISUOG. Published by John Wiley & Sons, Ltd.

INTRODUCTION Hypertensive disorders of pregnancy, particularly preeclampsia, are associated with significant morbidity and mortality, especially when it occurs before 34 weeks’

Correspondence to: Prof. K. Spencer, Department of Clinical Biochemistry, Harold Wood Hospital, Gubbins Lane, Romford, RM3 0BE, UK (e-mail: [email protected]) Accepted: 21 September 2006

Copyright  2006 ISUOG. Published by John Wiley & Sons, Ltd.

ORIGINAL PAPER

PP-13, PAPP-A and PI as markers of pre-eclampsia gestation1,2 . The incidence of pre-eclampsia in the routine pregnancy population is of the order of 5%1,3 . The symptoms of the disorder are well characterized and generally present in the late second to third trimester, however the underlying pathology is present at the early stages of pregnancy and there is evidence that it is associated with a failure of trophoblastic invasion of the maternal spiral arteries4 – 6 . A variety of proteins and hormones have been studied as potential early markers for pre-eclampsia. These include the second-trimester maternal serum markers used for aneuploidy screening such as alpha-fetoprotein (AFP)7,8 , beta-human chorionic gonadotropin (β-hCG)9 – 11 and inhibin-A10 – 13 , some of which have been shown to be altered also in the first trimester14 – 16 along with the first-trimester marker pregnancy-associated plasma protein-A (PAPP-A)14,16 . Other markers that have been proposed include first- or second-trimester measurement of activin17,18 , homocysteine19,20 , soluble fms-like tyrosine kinase (sflt-1)21,22 , placental growth factor22 – 24 and sex hormone binding globulin25 – 27 . However none has achieved sufficiently good discrimination to be used alone in a clinical context, although combinations of secondtrimester biochemical markers28,29 and biochemical and ultrasound markers have been proposed16,30,31 . The use of Doppler ultrasonography to assess increased impedance to blood flow in the maternal uterine arteries is probably the single most effective method of screening women in the second trimester. Studies at 20–24 weeks’ gestation have reported detection rates of 50–70% for a 5% false-positive rate in women developing early preeclampsia32,33 . When this has been investigated in the first trimester at 11–14 weeks34,35 the performance has been much poorer, with only 27% of cases detected at a 5% false-positive rate, rising to 50% among those severe cases requiring delivery before 34 weeks of gestation. Placental protein-13 (PP-13) is a 32-kD dimer and a member of a group of proteins that are highly expressed in the placenta. PP-13 is a galectin36,37 that binds to proteins on the extracellular matrix between the placenta and the endometrium, and through its action as a lysophospholipase-A assists in placental implantation and maternal artery remodelling36 – 38 . Early studies suggest that the gene for PP-13 is down-regulated in women with pre-eclampsia requiring early delivery, and that in early pre-eclampsia there is impaired placental functional responsiveness to PP-13 during the first trimester36,39 . A recent study40 of a small series of pregnancies resulting in delivery before 34 weeks’ gestation owing to early pre-eclampsia has shown some possible benefit of using early first-trimester maternal serum measurement of PP-13 in conjunction with early first-trimester uterine artery Doppler velocimetry as a potential screening tool. In this study we wished to examine further the potential value of first-trimester maternal serum PP-13 measurement alone or in conjunction with maternal serum PAPP-A and/or second-trimester uterine artery Doppler in predicting pre-eclampsia in women requiring delivery

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before 34 weeks’ gestation compared with all women developing pre-eclampsia.

PATIENTS AND METHODS All women booked for maternity care at Harold Wood Hospital, Essex, between October 1999 and January 2003 were offered screening for trisomy 21 by a combination of measurement of fetal nuchal translucency thickness and maternal serum free β-hCG and PAPP-A at 11 to 13 + 6 weeks’ gestation in a one-stop clinic for assessment of risk (OSCAR)41,42 . Serum free β-hCG and PAPPA were measured using the Kryptor analyzer (Brahms AG, Berlin, Germany), and the ultrasound scans were carried out by sonographers who had obtained The Fetal Medicine Foundation Certificate of Competence in the 11 to 13 + 6-week scan (www.fetalmedicine.com). A second ultrasound examination was routinely performed at 22–24 weeks for measurement of fetal growth and examination for fetal defects. In the cases where no major fetal defect was detected, women were offered the option of participating in a screening study for pre-eclampsia by Doppler measurement of the uterine artery pulsatility index (PI)32 . Each uterine artery was identified using color-flow mapping, and pulsed wave Doppler was then used to obtain three similar consecutive waveforms. The PI was measured and the mean PI of the two uterine arteries was calculated. The Doppler studies were performed by sonographers who had received The Fetal Medicine Foundation Certificate of Competence in Placental and Fetal Doppler (www.fetalmedicine.com). Demographic characteristics, ultrasound findings and the results of biochemical testing were entered into a computer database at the time of assessment. Data on pregnancy outcome were obtained from examination of individual patient notes and labor ward records. The main outcome measures were pre-eclampsia and delivery week. Pre-eclampsia was defined according to the guidelines of the International Society for the Study of Hypertension in Pregnancy. This requires two recordings of systolic/diastolic blood pressure (at least one of which is > 140/90 mmHg) measured at least 4 h apart in previously normotensive women, and proteinuria of 300 mg or more in 24 h, or two readings of at least 2+ on dipstick analysis of midstream or catheter urine specimens if no 24-h collection is available43 .

Blood sampling and measurement of PP-13 Blood samples were centrifuged at room temperature for 10 min at 4000 rpm and the serum was separated and analyzed immediately for PAPP-A and free β-hCG; the samples were then stored at −20◦ C. Samples from women developing pre-eclampsia (n = 88) were retrieved from frozen storage, and 446 samples from unaffected pregnancies, covering the same gestational age and storage period, were used as controls. All the samples were shipped to Israel for analysis of PP-13 using solid-phase sandwich enzyme linked immunosorbent assay (ELISA) as

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described previously36,40 . All samples were analyzed by an examiner blinded to the clinical outcome. The betweenday precision at PP-13 concentrations in the range 10–500 pg/mL was less than 10% and the limit of detection was 5 pg/mL.

Statistical analysis PAPP-A results were expressed as a multiple of the median (MoM) in normal pregnancies, using previously derived parameters44 and adjusted for maternal weight45 , smoking status46 and ethnicity44,47 . PP-13 results and mean PI were also expressed as MoM for normal pregnancies of the same gestational age. The significance of differences in serum free PP-13, PAPP-A and uterine artery mean PI between the pregnancies with a normal outcome (pregnancies without pre-eclampsia resulting in delivery after 37 weeks of a liveborn infant with birth weight above the 5th centile) and all pre-eclamptic pregnancies and those with early pre-eclampsia (requiring delivery before 35 weeks) was determined by unpaired t-test of unequal variance. The detection rates of each adverse pregnancy outcome and false-positive rates for different cut-offs in PP-13, PAPP-A and mean PI were calculated and receiver–operating characteristics (ROC) curves were constructed. Multiple logistic regression analysis was used to model combining PAPP-A, PP13 and mean PI using standard statistical techniques. Logistic regression yielded a predicted score for each subject and the performance in discriminating cases with pre-eclampsia compared to either measure alone using the predicted score was also assessed by ROC analysis. All statistical analysis was performed using Analyse-It (Smart Software, Leeds, UK) and Microsoft Excel or SPSS 12 (SPSS, Woking, UK).

RESULTS The study population comprised 5867 singleton pregnancies that had both measurement of free β-hCG and PAPP-A at 11 to 13 + 6 (median 12) weeks’ gestation and measurement of uterine artery mean PI at 22–24 (median 23) weeks. The median maternal age of the study population was 30 (16–47) years, with no difference in age between pre-eclamptic women and controls. In the study population 5312 (90.5%) of the women were Caucasian, 142 (2.4%) were of Afro-Caribbean origin and 115 (1.9%) were Asian, with the rest of unknown origin. Among women with pre-eclampsia, 13% of the entire group – and 18% of those with early onset preeclampsia – were non-Caucasian. While in the study population 2376 (40.5%) were nulliparous, the proportion of nulliparous women in the whole pre-eclampsia group and in those with early-onset pre-eclampsia was 49% and 50%, respectively. Cigarette smoking was declared in 880 women (15.0%). In 5339 (91.0%) pregnancies there was no preeclampsia and the outcome was delivery after 37 weeks of a liveborn infant with birth weight above the 5th centile (normal group). The frequency of pre-eclampsia in the study population was at the low end of the expected range – 88 (1.5%) of pregnancies with live births, from which 44 (50%) were delivered before 35 weeks (early pre-eclampsia group). From the normal group we selected five controls who were matched to each pre-eclampsia case by taking the next five normal cases who entered the study after the pre-eclampsia case – in this way the PP-13 tests were matched for length of sample storage. Table 1 describes the clinical features of the control group and the total pre-eclamptic group and those with early preeclampsia. The pre-eclamptic women had very high peak blood pressure values of 159/106 and 172/109 mmHg for

Table 1 Clinical descriptors of the three study populations

Variable Age, mean (SD) (years) BMI, mean (SD) (kg/m2 ) Nulliparity, n (%) Ethnicity, n (%) Caucasian Asian African Other GA at enrolment, mean (SD) (days) BP at onset of PE, mean (SD) (mmHg) Systolic Diastolic Peak BP, mean (SD) (mmHg) Systolic Diastolic GA at delivery (weeks) Birth weight (g) Fetal death Cesarean delivery (%)

Controls (n = 446)

All pre-eclampsia (n = 88)

Early-onset pre-eclampsia (n = 44)

30.2 (4.8) 25.4 (5.1) 143 (32)

29.5 (5.9) 27.1 (6.2) 43 (49)*

28.9 (6.3) 27.2 (5.4) 22 (50)*

418 (93.7) 13 (2.9) 13 (2.9) 2 (0.4) 87 (4.4)

76 (86.4) 3 (3.4) 9 (10.2) — 86 (4.5)

36 (81.1) 2 (4.5) 6 (13.6) — 87 (4.9)

No pathological findings

149 (14.3) 100 (11.5)

149 (12.3) 102 (14.5)

No pathological findings

159 (18.2) 106 (13.2) 36.2* 2025* None 72*

172 (20.5) 109 (14.2) 30.9* 1361* None 97*

39.1 3418 None 16

*P < 0.001 compared with controls. BMI, body mass index; BP, blood pressure; GA, gestational age, PE, pre-eclampsia.

Copyright  2006 ISUOG. Published by John Wiley & Sons, Ltd.

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PP-13, PAPP-A and PI as markers of pre-eclampsia

131 (a) 100

2.0 1.8 1.6

80 Detection rate (%)

Median MoM

1.4 1.2 1.0 0.8 0.6 0.4

60

40

20

0.2 0.0 PAPP-A

0

PI

Figure 1 First-trimester marker level expressed as median multiples of the median (MoM) and second-trimester uterine artery pulsatility index (PI) for controls (
), all cases of pre-eclampsia ( ) and early-onset pre-eclampsia cases ( ). Values are mean ± 95% CI. PAPP-A, pregnancy-associated plasma protein-A; PP-13, placental protein-13.

all cases and early-onset cases, respectively. Gestational age at delivery was 36.2 and 30.9 weeks for all preeclamptics and early onset pre-eclamptics compared to 39.1 weeks in normal deliveries, with a birth weight of 2025 and 1361 g vs. 3418 g for pre-eclamptic, early-onset pre-eclamptic vs. normal pregnancies. The frequency of Cesarean section was 72% in pre-eclamptic, and 97% in early-onset cases compared with 16% in the normal population. The median PP-13 level for controls was 176.9 pg/mL and for the total pre-eclampsia group it was 121.9 pg/mL being 111.7 pg/mL in the early pre-eclampsia group and 130.1 in the late (i.e. those in whom pre-eclampsia was not severe enough to warrant delivery before 35 weeks) preeclampsia group. In MoM terms (Figure 1) this was 1.00, 0.69 (P < 0.001), 0.63 (P < 0.001) and 0.74 (P < 0.001), respectively. For PAPP-A and mean PI the corresponding median MoMs were 1.00, 0.89 (P = 0.076), 0.89 (P = 0.042), 0.98 (P = 0.557) and 1.00, 1.60 (P < 0.001), 1.70 (P < 0.001) and 1.36 (P < 0.001) respectively. Figure 1 shows the distributions and the 95% confidence intervals in the median MoM. The sensitivity of each of the markers alone and in various combinations for the detection of pre-eclampsia of any severity or early pre-eclampsia or late pre-eclampsia are shown in Figure 2 and in Tables 2–4. At 80% specificity, it appears that first-trimester serum testing using PP-13 could identify 50% of cases with early pre-eclampsia, which is an improvement over PAPP-A alone (23%) but not as good as the 76% achievable using uterine artery Doppler velocimetry in the second trimester. Combining first-trimester PP-13 with secondtrimester uterine artery Doppler increases the level of detection of early onset pre-eclampsia to 79% compared with 76% with Doppler alone. The area under the curve was significantly higher for the combination PI + PP-13

Copyright  2006 ISUOG. Published by John Wiley & Sons, Ltd.

0

20

40

60

80

100

80

100

False-positive rate (%) (b) 100

80 Detection rate (%)

PP-13

60

40

20

0

0

20

40 60 False-positive rate (%)

Figure 2 Receiver–operating characteristics curves showing the clinical discrimination of different markers alone and in combination at detecting early-onset pre-eclampsia. (a) Pulsatility ), placental protein-13 (PP-13) ( ) and PI plus index (PI) ( ); (b) PI ( ), pregnancy-associated plasma PP-13 ( protein-A (PAPP-A) ( ), and PI plus PAPP-A ( ).

compared to PI alone for the all-pre-eclampsia group (P < 0.001) as well as for the early-onset pre-eclampsia group (P < 0.001). There was no additive effect of using both serum markers, and the use of all three markers had no advantage over using just two of them. Detection was on average 6–12% less for all cases of pre-eclampsia than it was for early-onset pre-eclampsia (Table 2).

DISCUSSION This study is the first to measure in parallel PP-13 and PAPP-A in the first trimester of pregnancy as potential serum markers for the prediction of pre-eclampsia. The findings of this study demonstrate that in pregnancies with pre-eclampsia first-trimester maternal serum levels of the protein PP-13 and of PAPP-A are reduced and that with more severe or early pre-eclampsia the levels may be further reduced. It is quite interesting that the results

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132 Table 2 Area under the curve (AUC) and sensitivity for each marker and combination of markers for the prediction of all cases of pre-eclampsia using logistic regression analysis with a specificity set to 0.80 Marker PP-13 PAPP-A Mean PI PP-13 + mean PI PAPP-A + mean PI PP-13 + PAPP-A PP-13 + PAPP-A + mean PI

AUC (95% CI)

P*

Sensitivity

0.68 (0.61–0.74) 0.56 (0.49–0.63) 0.79 (0.72–0.87) 0.84 (0.78–0.90) 0.82 (0.76–0.90) 0.71 (0.63–0.78) 0.84 (0.74–0.94)

< 0.001 0.076 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001

0.44 0.24 0.73 0.74 0.76 0.40 0.74

*In comparison with controls. PAPP-A, pregnancy-associated plasma protein-A; PI, pulsatility index; PP-13, placental protein-13.

Table 3 Area under the curve (AUC) and sensitivity for each marker and combination of markers for the detection of early pre-eclampsia using logistic regression analysis with a specificity set to 0.80 Marker PP-13 PAPP-A Mean PI PP-13 + mean PI PAPP-A + mean PI PP-13 + PAPP-A PP-13 + PAPP-A + mean PI

AUC (95% CI)

P*

Sensitivity

0.71 (0.63–0.79) 0.59 (0.51–0.68) 0.86 (0.77–0.94) 0.90 (0.84–0.96) 0.86 (0.77–0.94) 0.71 (0.63–0.78) 0.85 (0.77–0.94)

< 0.001 0.076 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001

0.50 0.23 0.76 0.79 0.76 0.40 0.70

*In comparison with controls. PAPP-A, pregnancy-associated plasma protein-A; PI, pulsatility index; PP-13, placental protein-13.

Table 4 Area under the curve (AUC) and sensitivity for each marker and combination of markers for the detection of late pre-eclampsia using logistic regression analysis with a specificity set to 0.80 Marker PP-13 PAPP-A Mean PI PP-13 + mean PI PAPP-A + mean PI PP-13 + PAPP-A PP-13 + PAPP-A + mean PI

AUC (95% CI)

P*

Sensitivity

0.64 (0.55–0.72) 0.47 (0.37–0.58) 0.73 (0.61–0.85) 0.79 (0.69–0.88) 0.81 (0.72–0.90) 0.64 (0.55–0.72) 0.82 (0.75–0.88)

0.003 0.556 < 0.001 < 0.001 < 0.001 0.003 < 0.001

0.39 0.26 0.70 0.70 0.70 0.37 0.73

*In comparison with controls. PAPP-A, pregnancy-associated plasma protein-A; PI, pulsatility index; PP-13, placental protein-13.

imply different levels of detection and no additive value. As such, they point towards the potential future use of PP13 and PAPP-A as useful tools for identifying sub-types of pre-eclampsia and also enable the refinement of follow-up strategies of surveillance and treatment. As has previously been reported, second-trimester mean PI is increased in pregnancies developing pre-eclampsia and that with more severe or early pre-eclampsia the mean PI is further increased. As has previously been shown, this prediction could be improved by adding demographic

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and medical history information. While overall secondtrimester mean PI was a more significant predictor of preeclampsia than any of the first-trimester serum markers, it is a late predictor on its own, and the ability of the serum marker to identify a significant proportion of cases 10–12 weeks earlier could be beneficial for the further development of strategies for combining prediction with prevention and patient follow-up. This study yields a lower prediction of pre-eclampsia by PP-13 as compared with the study of Nicolaides et al.40 , which looked at a very small number of first-trimester cases with early pre-eclampsia and measured PP-13 and first-trimester mean PI. In that study of 10 cases, levels of PP-13 were found to be very low in all but one case, with eight cases having a MoM of less than 0.1 and two cases with a reported MoM of 0.03. The study of Nicolaides et al. predicted – for a 10% false-positive rate – a detection rate of 80% for PP-13, 40% for PI and 90% when the two were combined. In the present study, for a 10% false-positive rate, second-trimester mean PI alone achieved a 70% detection for early pre-eclampsia, whereas PP-13 alone gave 36% and combining the two yielded no additional gain in detection. Comparing the two studies by other parameters such as the severity of the cases judged by mean blood pressure or by birth weight, the two are similar. However, the two studies differ in two major population characteristics relevant to the disease. First, ethnicity – in this study there were approximately 85–88% Caucasians among cases compared with 30% in the study of Nicolaides et al.40 . Second, the frequency of nulliparity is lower here than the 60% in the study of Nicolaides et al.40 . Our data, while confirming that reduced levels of PP-13 are a useful predictor, are thus somewhat different in the level of prediction, a finding presumably related to potential variation in the detection rate as a result of ethnicity and parity. Further studies are required in larger populations to establish the real potential of PP-13, PAPP-A and Doppler velocimetry as individual markers and the proper combination for combined, sequential and multiple testing. Certainly both PP-13 and PAPP-A, when coupled with second-trimester mean PI as measured by Doppler velocimetry, do improve prediction over the use of Doppler ultrasonography alone, and a larger study will be able to reveal the anticipated detection rate and right method of using each marker alone or as a part of a panel of serum and sonographic markers.

ACKNOWLEDGMENT This study was supported in part by a grant from NHS R&D (RF4:Risk Assessment in Pregnancy) to Professor Kevin Spencer.

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