DOI: 10.1111/j.1471-0528.2008.01902.x

Maternal medicine

www.blackwellpublishing.com/bjog

Placental protein 13 as an early marker for pre-eclampsia: a prospective longitudinal study* R Gonen,a R Shahar,b YI Grimpel,b I Chefetz,b M Sammar,b H Meiri,b Y Giborb a Department of Obstetrics and Gynecology, Bnai Zion Medical Center, Faculty of Medicine, Technion-Israel Institute of Technology, Haifa, Israel b Diagnostic Technologies Ltd., Yokneam, Israel Correspondence: Dr R Gonen, Department of Obstetrics and Gynecology, Bnai Zion Medical Center, 47 Golomb Street, Haifa 31048, Israel. Email [email protected]

Accepted 25 July 2008.

Objective To assess the value of placental protein 13 (PP13) as an

early marker of pre-eclampsia. Design Sequential blood samples were obtained from women with

singleton viable pregnancies at 6–10, 16–20 and 24–28 weeks of gestation. Samples were tested for PP13 using a solid-phase sandwich enzyme-linked immunosorbent assay. Levels were expressed as multiples of the medians (MoM) of the unaffected population. The slope or rate of change in PP13 concentration per week of gestation was also calculated. Setting Thirty-five prenatal care community clinics. Sample In total, 1366 women were recruited, and subsequently,

20 were diagnosed with pre-eclampsia, 41 with gestational hypertension and 1178 were unaffected. Main outcome measures Sensitivity and specificity of screening with PP13 at each gestational period and of PP13

level combined with the slope of PP13 between two testing periods. Results At 6–10 gestational weeks, PP13 levels were significantly lower among the pre-eclampsia group with a median 0.28 MoM (95% CI 0.15–0.39, P < 0.004). Using a cutoff of 0.40 MoM, the sensitivity was 80%, false-positive rate (FPR) was 20% and odds ratio was 16.0 (95% CI 5.3–48.4). Combining MoM of 6–10 weeks and slope between 6–10 and 16–20 weeks, the sensitivity was 78%, the FPR was 6% and odds ratio was 55.5 (95% CI 18.2–169.2). The gestational hypertension group was not different from the normal group. Conclusions PP13 in the first trimester alone or in combination

with the slope between the first and the second trimesters may be a promising marker for assessing the risk of pre-eclampsia. Keywords Placental protein 13, pre-eclampsia, serum markers.

Please cite this paper as: Gonen R, Shahar R, Grimpel Y, Chefetz I, Sammar M, Meiri H, Gibor Y. Placental protein 13 as an early marker for pre-eclampsia: a prospective longitudinal study. BJOG 2008;115:1465–1472.

Introduction Pre-eclampsia affects approximately 2–5% of pregnant women, contributing to fetal, neonatal and maternal morbidity and mortality.1 While the disease manifests during the third trimester, the underlying placental dysfunction begins much earlier in pregnancy. Early maternal or fetal markers reflecting the underlying placental pathology may have the potential to improve clinical management. They could help to identify women who require closer antenatal surveillance, and they could also be used to

*This study was presented at the 26th Annual Meeting of the Society of Maternal Fetal Medicine, Miami Beach, Florida, 2006.

select those most likely to benefit from preventive measures, such as antiplatelet agents2 or dietary calcium supplementation.3 Numerous serum markers that are measured in the maternal circulation have been evaluated in the prediction of pre-eclampsia.4–17 The use of Doppler ultrasound to assess impedance to blood flow in the maternal uterine arteries in the first18 and the second19 trimesters has also been investigated. However, the optimal screening test for pre-eclampsia is yet to be found. Placental protein 13 (PP13) is a 32 kDa dimer protein that is produced only in the placenta and is thought to be involved in normal placentation and maternal artery remodelling.20–22 Studies with real-time polymerase chain reaction revealed a reduced level of PP13 messenger RNA expression in placentas that were obtained from pregnant women with early-onset pre-eclampsia.23 Nicolaides et al.24 found a significant reduction of serum PP13 levels at 11–13 gestational weeks in

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women who subsequently developed early pre-eclampsia. This was subsequently confirmed by Spencer et al.25 and also by Chefetz et al.26 Considering the putative role of PP13 in placentation,20,21 we hypothesised that testing PP13 levels at 6–10 gestational weeks could predict pre-eclampsia better than later testing. We therefore undertook this study to assess PP13 early in the first trimester and aimed to determine if sequential testing would further improve the performance of this test.

Methods We performed a prospective, longitudinal, observational, multicentre study in the second largest health management organisation in Israel (Maccabi Healthcare Services), enrolling women attending 35 prenatal community clinics throughout the country between May 2002 and September 2004. An initial blood sample was obtained at 6–10 weeks of gestation (all enrollees), and whenever possible, additional samples were obtained at 16–20 weeks (93% of enrollees) when routine biochemical screening is performed and at 24–28 weeks (96% of enrollees) when the glucose challenge test is performed. The study was approved by the Maccabi Institutional Review Board, and each woman signed an informed consent. Only singleton viable pregnancies were included. Gestational age was determined by the last menstrual period and verified by ultrasound crown–rump length. Demographic, medical and obstetric data were obtained by the attending physician at enrolment. Physicians and subjects were blinded to the results of PP13 testing. Delivery records were obtained from 22 hospitals across Israel. Pre-eclampsia, hypertension (blood pressure [BP] >140/90 mmHg), that first develops after 20 weeks of gestation in a previously normotensive woman, and proteinuria exceeding 300 mg in a 24-hour collection or ‡2+ by dipstick on a spot urinalysis as well as gestational hypertension and superimposed pre-eclampsia were defined according to the International Society for the Study of Hypertension in Pregnancy.27 The medical records for each woman were reviewed, and all affected cases were verified by the principal investigator (R.G.) together with 10% of the normal women selected at random. Samples were kept for 1–2 hours at room temperature and centrifuged for 10 minutes at 10 000 · g; the serum was collected and stored at 2–8
C for up to 48 hours until transferred in ice boxes to the Maccabi Central Laboratory. The samples were then divided into five aliquots and stored at –20
C until tested. The stability of frozen samples was assessed throughout the period and verified to be the same for the entire study period and for a minimum of 2 years. Maternal serum concentration of PP13 was measured in ‘blinded’ samples using a solid-phase sandwich enzyme-linked immunosorbent assay with a pair of PP13-specific monoclonal antibodies, marked with amplified biotin–extravidin–horseradish peroxidase

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complex and developed with tetramethylbenzidine substrate, as previously described.20,24 Optical density was measured at 450 nm and converted to pg PP13/ml according to a calibration curve derived from PP13 standards.20,21 The lower limit of detection was 3 pg/ml, and the proportion of samples with undetectable values was 5.8, 3.8 and 1.1% in the three gestational periods, respectively. Intra-assay and interassay variations were 6.5 and 9.4%, respectively. PP13 levels increase with gestational age (P < 0.001), and to allow for this, levels were expressed as multiples of the medians (MoM) of the unaffected population based on weighted regression of the observed medians at each completed week, an approach that is standard for Down syndrome screening markers28 and has previously been used for PP13.24 We adjusted PP13 for gestational week, maternal body mass index (negative association, P = 0.10), maternal age (P = 0.70), parity (P = 0.85) and ethnicity (P = 0.14) in the adjusted MoM analyses. To evaluate the use of sequential PP13 testing, the slope or PP13 change between test periods was calculated. For example, if for an individual subject the concentrations of PP13 in two periods were X1 and X2 at gestational weeks W1 and W2, then slope = (X2–X1)/(W2–W1).

Statistical analysis Baseline characteristics were compared among all unaffected, pre-eclampsia and gestational hypertension groups using the Fisher’s exact test for categorical variables and independent t tests for continuous variables. Since PP13 levels in MoMs were not normally distributed and transforming them to logarithms did not help, we compared groups using the nonparametric Wilcoxon rank-sum test. Slopes were compared using the t test. A probability less than 0.05 was considered statistically significant. Assuming a 2% prevalence of pre-eclampsia in our population, a 7% miscarriage rate after enrolment at 6–10 weeks, a sensitivity of 0.80–0.87 and specificity of 0.8–0.9,24 a sample size of 1192 was required to achieve 0.8 power by a one-sided exact binomial distribution. To calculate the sensitivity and specificity, we compared the pre-eclampsia and gestational hypertension groups with the unaffected group using the receiver operating characteristic (ROC) curve analysis and estimating the area under the curve (AUC) calculated from MoMs or slopes. Two strategies were evaluated: sequential testing whereby all women were tested twice (6–10 and 16–20 gestational weeks) and contingent screening where the decision to take the second sample is dependent on the initial MoM result. A priori risk was evaluated using multivariate logistic regression with the stepwise parameter selection method based on individual risk factors that are considered to be associated with pre-eclampsia. All values were used as categorical values. To combine a priori risk with firsttrimester PP13 MoM or first- to second-trimester PP13 slope,

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or a combination of the two, we used logistic regression with either the sequential or the contingent approach. All logistic regressions were carried out without selection (either stepwise or any other) of explanatory variables in the model, using the equation of: logit(p/(1–p) = MoM + slope (or a priori risk + MoM or a priori risk + slope etc.) To calculate the odds ratio, we used:

OR = ðSensitivity=ð1 – SensitivityÞÞ= ðð1 – SpecificityÞ=SpecificityÞ

Results Of the 1366 enrollees, 20 had pre-eclampsia, 41 had gestational hypertension, 1178 were unaffected by hypertensive disorders and 127 were excluded (miscarriage, 95; noncompliance with

protocol, 32). Table 1 presents the demographic and obstetric characteristics of the study groups. The median gestational age at enrolment was 8 weeks and 38% were nulliparous. An unexpected finding was the lower proportion of nulliparous women among the pre-eclampsia group (4/20). This is probably due to the relatively small number of subjects in this group. BP at enrolment was similar in women who subsequently experienced pre-eclampsia to that of the unaffected subjects; conversely, the BP of women who subsequently had gestational hypertension was significantly higher. Compared with unaffected subjects, women with pre-eclampsia were significantly more likely to deliver smaller fetuses by caesarean delivery at a younger gestational age, with subsequent longer hospitalisation for both mothers and newborns

A priori risk A priori risk was calculated for individual medical and demographic variables that have been reported to be relevant to the

Table 1. Demographic and obstetrics characteristics Variable

Unaffected (n 5 1178)

Age (years), median (range) 30 (18–43) BMI (kg/m2), median (range) 22.5 (15.6–42.3) Nulliparous, n (%) 402 (34) Ethnicity, n (%) Jews 1121 (95) Non-Jew 57 (5) Major risk factors for pre-eclampsia, n (%) 49 (4) Chronic hypertension 6 (1) Previous pregnancy hypertensive disorders 43 (4) Teenagers (,18 years), n (%) 1 (0) GA at enrolment, median (range) 7.0 (6.0–10.0) BP (mmHg) at enrolment, median (range) Systolic 110 (60–162) Diastolic 68 (40–97) Highest pregnancy BP (mmHg), median (range) Systolic 110 (60–162) Diastolic 68 (40.0–97) Proteinuria at delivery, median (range) 0 (0–2) GA at delivery, median (range) 39.4 (20.0–43.0) Newborn Birthweight (g), median (range) 3300 (778–4610) IUGR, n (%) 53 (4) Preterm delivery, n (%) 87 (7) Fetal death, n (%) 8 (1) Caesarean delivery, n (%) 209 (18) Hospital days, median (range) Mother 3.0 (0.0–28.0) Newborn 3.0 (0.0–137.0)

Pre-eclampsia (n 5 20)

Gestational hypertension (n 5 41)

28 (20–44) 23.6 (17.9–33.8) 4 (20)

30 (22–42) 23.9 (17.7–33.6) 15 (37)

15 (75)** 5 (25)** 4 (20)* 1 (5) 3 (15)* 0 7.0 (6.0–8.0)

41 (100) 0 8 (20)*** 1 (2) 7 (17)** 0 7.0 (6.0–10.0)

100 (90–140) 70 (50–80)

118 (90–140)*** 75 (60–98)***

150 (140–186)*** 90 (80–120)*** 2 (2–4)*** 38.0 (25.6–41.7)***

140 (103–170)*** 90 (67–107)*** 0 (0–2)*** 39.1 (34.4–42.1)***

2795 (450–4600)*** 5 (25)** 6 (30)** 1 (5)* 7 (35)

3225 (1970–4685)* 1 (2) 8 (20)** 0 5 (12)

4.0 (2.0–14.0)*** 4.0 (2.0–19.0)*

3.0 (2.0–7.0) 3.0 (2.0–49.0)

BMI, body mass index; GA, gestational age; IUGR, intrauterine growth restriction.Proteinuria by dipstick. Values at enrolment were below 1 for all groups. All continuous parameters were compared by t test, categorical parameters by Fisher’s exact test and Wilcoxon rank-sum for noncontinuous parameters (comparison with unaffected). For major risk factors, a woman could have more than one. *P , 0.05, **P , 0.01, ***P , 0.001.

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Table 2. Sensitivity and specificity by a priori risk Risk factor

Chronic hypertension (yes/no) Previous pre-eclampsia or gestational hypertension (yes/no) BMI . 27 kg/m2 Age ,18 or 40 years Non-Jewish Nulliparous (yes/no) Smoking (yes/no) Systolic BP . 120 Diastolic BP . 80 At least one risk factor

Specificity Sensitivity (%) (%)

OR

99.4

5.0

9.2 (1.1–80.0)

96.0

15.0

4.2 (1.2–15.0)

86.3 99.3 93.9 66.6 95.2 89.7 95.2 95.6

25.0 5.0 29.4 26.7 0.0 9.1 0.0 42.9

2.1 (0.8–5.9) 7.8 (0.9–66.6) 6.4 (2.4–17.1) 0.7 (0.3–2.0) NA 0.9 (0.2–3.8) NA 16.3 (3.5–76.1)

BMI, body mass index; NA, not applicable.

risk of develop of pre-eclampsia (Table 2). Each factor by itself was not an effective predictor. The presence of at least one risk factor had a sensitivity of 43% and a specificity of 96% for the development of pre-eclampsia (OR 16.7, 95% CI 3.6–77.8).

PP13 median and MoM Among unaffected subjects, PP13 median values (Figure 1) and median MoM at 6–10 weeks were 115 pg/ml (95% CI 109–124) or 1.01 MoM (95% CI 0.92–1.09). The levels remained steady at 16–20 weeks (124 pg/ml [95% CI 112–132] or 0.98 MoM [95% CI 0.93–1.08]) and increased at 24–28 weeks (220.5 pg/ml [95% CI 200–240] or 1 MoM [95% CI 0.91–1.09]) (Figure 1). For women who subsequently developed pre-eclampsia, the corresponding median PP13 values were 30.5 pg/ml (95% CI 19–39) or 0.30 MoM (95% CI 0.15–0.40) at 6–10

Figure 1. Median serum PP13 in unaffected women according to gestational age (GA).

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gestational weeks, 212.5 pg/ml (95% CI 173–265) or 1.78 MoM (95% CI 1.36–1.94) at 16–20 gestational weeks and 394 pg/ml (95% CI 344–730) or 1.97 (95% CI 1.55–4.32) at 24–28 gestational weeks. Compared with the unaffected group, women who subsequently developed pre-eclampsia had significantly lower levels of PP13 at 6–10 weeks (P < 0.001) and higher at the other two time periods (P < 0.001) (Figure 2). For women who subsequently developed gestational hypertension, the respective median PP13 values were not significantly different from those of the unaffected groups at any of the examined periods: 136.5 (95% CI 90–269) or 1.08 MoM (95% CI 0.67–2.44) at 6–10 weeks, 146 (95% CI 89–232) or 1.2 (95% CI 0.74–2.01) at 16–20 weeks and 271 (95% CI 200–391) or 1.44 (95% CI 1.06–1.89) at 24–28 weeks (Figure 2). Figure 3 shows the ROC curves generated from the MoM PP13 values for the development of the two disorders. The AUC for the group with pre-eclampsia at 6–10 gestational weeks was 0.80 (95% CI 0.71–0.89; P < 0.0001). Given 80% specificity (20% false-positive rate [FPR]), the MoM cutoff was 0.40, the sensitivity was 80% (OR 16.0, 95% CI 5.3–48.4). It should be noted that all five subjects with preterm preeclampsia necessitating delivery before 37 gestational weeks, as well as five of six subjects with term severe pre-eclampsia, had PP13 levels below the cutoff level and were thus identified by the test. The respective AUCs for the differences between the pre-eclampsia and the normal groups for the 16–20 and 24–28 weeks were 0.72 (95% CI 0.54–0.86; P < 0.001) and 0.68 (95% CI 0.57–0.8; P < 0.001), respectively. Given a specificity of 80%, the sensitivities were 33 and 38%, respectively.

PP13 slope Figure 4 shows that in 19/20 (all except subject number 20) women with pre-eclampsia, PP13 increases over the three test periods with slope for the three time periods ranging between 8.44 (subject number 4) and 42.862 (subject number 19).

Figure 2. Median MoM PP13 for the three testing periods. **P < 0.01.

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Sensitivity

Sequential testing

1.0

Sequential testing involves performing two blood tests, at 6–10 and 16–20 gestational weeks, in all women. A logistic regression, combining PP13 MoM of 6–10 gestational weeks and PP13 slope between 6–10 and 16–20 gestational weeks, yielded AUC of 0.90 (95% CI 0.85–0.95; P < 0.001). Given 94% specificity (6% FPR), sensitivity was 78% (OR 55.5, 95% CI 18.2–169.2).

0.9 0.8 0.7 0.6 0.5

Contingent screening

0.4 0.3 0.2

Pre-eclampsia

0.1

Gestational hypertension

0.0 0.0

0.1

2.0

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

1 – specificity Figure 3. ROC curve generated from MoM PP13 values for the development of pre-eclampsia and gestational hypertension.

In individual subjects who developed pre-eclampsia, the lower the 6–10 gestational week PP13 MoM, the higher the 16–20 gestational week value (correlation coefficient of 0.6 for the individual women’s PP13 slope between 6–10 gestational weeks and 16–20 gestational weeks), yielding a median slope of 14.48 (95% CI 10.41–18.55) compared with 3.30 (95% CI 1.0–5.6) among individual unaffected subjects (P < 0.001). The respective slope in the group with gestational hypertension was 1.25 (95% CI –5.09 to –7.59; P = 0.744 compared with unaffected). Between 6–10 and 16–20 weeks, the ROC curve for the PP13 slope for the group with pre-eclampsia compared with unaffected subjects yielded AUC of 0.83 (95% CI 0.74–0.93; P < 0.001). At 80% specificity, the slope cutoff was 10.00, sensitivity was 89% and odds ratio was 32.1 (95% CI 7.3–141.1). The corresponding slope for the group with gestational hypertension had AUC of 0.51 (95% CI 0.45–0.59), indicating that the slope is not different between the gestational hypertension group and the unaffected group. The median slope between 6–10 and 24–28 weeks was 4.6 (95% CI 3.9–5.3) for the unaffected group and 20.0 (95% CI 17.8–31.9; P < 0.001) for the group with pre-eclampsia. The PP13 slope for the gestational hypertension group was 6.2 (95% CI 0.2–12.8) and was not significantly different from the slope of the unaffected group. In ROC analysis, a slope cutoff of 13.4 yielded 80% specificity and 83% sensitivity. The median second-trimester slope between 16–20 and 24–28 weeks was 9.0 (95% CI 8.0–11.0) for the unaffected group, 22.0 (95% CI 16.0–68.0; P < 0.004) for pre-eclampsia and 12.0 (95% CI 8.0–32.0) for gestational hypertension. In ROC analysis, a slope cutoff of 28.0 yielded 80% specificity and 36% sensitivity.

In contingent screening, the evaluation of PP13 slope as a second test was reserved only for subjects screened positive by a low PP13 MoM. Analysed by a parametric combined regression, the AUC was 0.89 (95% CI 0.86–0.92; P < 0.001). At 83% sensitivity, the specificity was 91% (OR 48.4, 95% CI 13.9–168.2). With contingent screening, only 16% of the subjects required a second test, corresponding to an approximately 40% reduction in the total number of tests performed.

A priori risk and PP13 testing In sequential analysis that included a priori risk and PP13, the presence of a priori risk and abnormal PP13 yielded a sensitivity of 71% and specificity of 80% (OR 10.1, 95% CI 1.9–52.8). This held true regardless of the approach used for PP13 assessment (first-trimester PP13 MoM, first- to secondtrimester PP13 slope or the two combined).

Discussion In this prospective observational study, maternal blood testing at 6–10 gestational weeks demonstrated significantly decreased serum levels of PP13 in women who experienced pre-eclampsia several months later. The use of PP13 testing at the first prenatal visit achieved 80% sensitivity with a 20% FPR for the prediction of pre-eclampsia. Performing a second test (concomitant with the routine second-trimester biochemical screening), calculating the PP13 slope between the two tests, and thus performing sequential testing, enhanced the accuracy by decreasing the FPR to 6% with only a slight decrease in sensitivity (78%). A contingent screening strategy would be cost-saving. In this study, there was a 40% reduction in the number of tests, while the sensitivity was slightly elevated from 80 to 83% and the FPR was reduced from 20 to 9%. The results of our study corroborate the results of three previously published studies.24–26 Nicolaides et al.24 reported that for a 90% detection rate of pre-eclampsia requiring delivery before 34 weeks, the FPR of screening by PP13 was 12%, by uterine artery pulsatility index (PI) it was 32%, and by a combination of the two methods it was 9%. Spencer et al.25 concluded that measurement of PP13 at 11–14 weeks of gestation may be useful in predicting pre-eclampsia, and the accuracy of the method increases when coupled with second-trimester uterine artery Doppler PI measurements.

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Figure 4. Longitudinal change in serum PP13 levels in women with pre-eclampsia. GA, gestational age.

Chefetz et al.26 evaluated first-trimester PP13 as a screening tool for pre-eclampsia in a case–control study that was conducted in the USA. They found that at a 90% specificity rate, the sensitivity was 79%. The authors concluded that maternal PP13 levels in the first trimester are a promising diagnostic tool for the prediction of pre-eclampsia. Of the two approaches, testing twice with PP13 or combining a single PP13 with Doppler,24,25 two blood tests offers simplicity and accessibility over Doppler studies. However, further studies are necessary to compare these two approaches. In line with previous reports, analysis of a priori risk by risk factors yielded a poor prediction for pre-eclampsia. Compared with the measurement of PP13 alone, the combination of a priori risk with PP13 reduced the accuracy of the prediction of pre-eclampsia. The combination of PP13 with some other putative first-25 or second-trimester29 biochemical markers also appears not to be beneficial. Spencer et al.25 failed to show any benefit from combining PP13 with pregnancy-associated plasma protein A (PAPP-A) for first-trimester testing, whereas the assessment of the risk to develop pre-eclampsia by each biomarker on its own was enhanced when combined with second-trimester uterine artery PI. In another study, Spencer et al.29 concluded that combining PP13 with either PAPP-A, activin, inhibin or free beta-hCG did not enhance the detection of pre-eclampsia. Whether early assessment of risk for pre-eclampsia with PP13 alone or in combination with other biomarkers can lead to an improvement in pregnancy outcome or a reduction in healthcare costs remains unclear. Nevertheless, women who are found to be at increased risk could benefit from closer antenatal surveillance as well as from preventive measures, such as low-dose aspirin or dietary calcium supplementation.

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Indeed, a meta-analysis on the use of antiplatelet agents for prevention of pre-eclampsia2 and a systematic review on dietary calcium supplementation for prevention of preeclampsia3 concluded that both strategies appear to reduce the risk for pre-eclampsia as well as associated adverse outcome measures. Moreover, some studies have indicated that starting treatment with low-dose aspirin in the first trimester generates a better outcome than the same treatment given in the second trimester.30,31 The molecular biological composition of PP13 provides clues to explain its potential role in the pathogenesis of preeclampsia, a disorder that arises as a consequence of abnormal placental development. PP13 shares high-sequence homology makeup with the galectin family; 8 of the 16 invariant residues that comprise the carbohydrate recognition/binding domains in galectins are conserved in PP13.20,21 PP13 is produced in the trophoblast and is packaged into endosomes that are released through ‘coated pits’, through a calcium mobilisation process.20 The secreted PP13 binds to sugar residues of extracellular matrix molecules (such as annexin II), which creates a ‘molecular bridge’ for placental implantation in the endometrium.21 PP13 also demonstrates mild lysophospholipaseA activity that leads to the liberation of fatty acid constituents of the plasma membrane, particularly linoleic acid, which might contribute to normal implantation. Furthermore, PP13 increases liberation of prostaglandins, particularly prostacyclin, which are important for trophoblast-stimulated vascular remodelling in the maternal spiral arteries in early placental development.20,21,23,32 Finally, PP13 binds to betaand gamma-actin within trophoblasts21 and thus is involved in the migration of trophoblasts towards the placental bed.32 Although these effects require further study, it is possible that

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a shortage of PP13 in the first trimester of pregnancy, which is marked by decreased circulating levels, either because of decreased transcription or increased catabolism, may impair several critical functions that are required for normal implantation and maternal vascular remodelling. It is not clear why in the women who developed preeclampsia, PP13 levels increased above normal values at the second and third sampling points. Our study indicates that the first- to second-trimester increase in PP13 level correlates with the severity of the disease. The time of the increase corresponds to the onset of the release of syncytiotrophoblast microparticles (STBM) deported to the maternal blood, as described previously by Huppertz and Kingdom.33 Huppertz34 showed by immunohistochemistry that the STBM are rich in PP13 and that in pre-eclampsia, they detach from the placenta and enter the maternal circulation. Accordingly, the STBM could be the vehicle carrying large quantities of PP13 that was previously caged inside impaired trophoblasts, and this could account for the later increase of PP13 in preeclampsia as reported in this study. This hypothesis is now being tested in placental explants in culture. This study has several limitations. (1) Although the vast majority of our population closely resembles the Caucasian population in Europe and the USA, our results need to be verified in a larger low-risk and high-risk cohorts in different populations. (2) Despite the relatively large cohort, the number of affected women is relatively small, underscoring the need for large-scale studies. (3) The current PP13 kit is unable to detect PP13 levels below 3–5 pg/ml, and further development of detection amplification may lead to better accuracy. (4) In the absence of Doppler PI measurements in this study, it is not possible to assess the value of combining first-24 and/or second-trimester24,25 Doppler with PP13, particularly in the context of sequential and contingent testing. (5) The sensitivities and specificities were derived from the test population and for a true estimate of the sensitivity, specificity and predictive values, a prospective evaluation with prespecified cutoffs would be necessary. In conclusion, this prospective longitudinal study of a lowrisk population demonstrates that PP13 in the first trimester alone or in combination with the slope between the first and the second trimesters may be a promising and effective marker for the assessment of the risk of pre-eclampsia. Continuing studies in Israel, Europe and the USA will provide additional data on the potential benefit of screening for preeclampsia with prespecified cutoffs.

Disclosure of interest R.G. was paid for consultation and for time spent on organising the project and auditing medical records. His fee was covered by grants from Diagnostic Technologies. The company also covered a part of Prof Gonen’s travel expenses to

present the initial results at the 26th Annual Meeting of the American Society of Maternal Fetal Medicine in Miami, 2006. R.S., Y.I.G., I.C. and M.S. were full- or part-time employees of Diagnostic Technology, the company, which own the PP13 kits, and their salary was covered by the funding grants specified above. I.C. and M.S. also hold options to 0.3% of the issued shares of the company. Y.G. is a consultant for regulatory affairs of Diagnostic Technologies and his consultation fees were covered by the grants specified above. He holds options to 0.2% of the issued shares of the company. H.M. is the CEO and CTO of Diagnostic Technologies and her salary is fully paid by the company, partially from the grants mentioned above. H.M. holds options to shares of the company in an amount of ;5% of all issued shares.

Contribution to authorship R.G. designed the study and the data collection forms, reviewed the clinical records to verify the outcome and wrote the manuscript. R.S. built the electronic database and performed the analysis together with H.M. who also participated in writing the manuscript. M.S. prepared the recombinant PP13 for the study. Y.I.G. and I.C. were in charge of PP13 testing. Y.G. was in charge of the study protocol, regulatory approval and regulatory monitoring of the study.

Details of ethics approval The study was approved by the Maccabi Institutional Review Board on 12 May 2002, Ref. No. 003-02-972. This approval was extended every 12 months, until completion of recruitment and deliveries in December 2005.

Funding This study was supported in part by the Office of the Chief Scientist, Israel Government grant numbers 31851, 34472 and 41913 to H.M., PhD, and by EU Pregenesys Program grant number 037244 to H.M., PhD.

Acknowledgements The authors thank Maccabi Healthcare Services for their contribution to the study. The authors are grateful to Judith Zwickel, PhD, for her involvement in the design of the study and to Jossef Azuri, MD, Unit of Clinical Research, Maccabi Healthcare Services, for his dedication and insightful involvement in carrying out this study throughout. We thank Ido Kunreich and Tal Otiker from TechnoSTAT, Kfar Saba, Israel for reviewing and editing the statistical analysis. j

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